Julia Piasecki

Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis

In bone metastases, tumor cells interact with the bone microenvironment to induce osteoclastogenesis, leading to bone destruction and the growth factor release. RANK ligand (RANKL) is essential for osteoclast formation, function, and survival. Tumor cell-mediated osteolysis is thought to occur ultimately via induction of RANKL within the bone stroma, and inhibition of RANKL in models of breast cancer bone metastases blocks tumor-induced osteolysis and reduces skeletal tumor burden. In addition, the skeleton is co-opted by tumor cells and functions as a supportive tumor microenvironment. Inhibition of RANKL, by reducing tumor-induced osteoclastogenesis, may reduce the local release of growth factors and calcium which may potentially enhance the anti-tumor activity of cytotoxic or direct tumor apoptotic agents. Recombinant human Apo2 ligand/ TNF-related apoptosis-inducing ligand (rhApo2L/TRAIL/dulanermin) is a dual pro-apoptotic receptor agonist that preferentially induces apoptosis in cancer cells versus normal cells. We therefore examined RANKL inhibition (using RANK-Fc) in combination with rhApo2L/TRAIL on tumor-induced osteolysis and skeletal tumor burden in a murine intracardiac injection model of MDA-MB-231 breast carcinoma bone metastasis. rhApo2L/TRAIL treatment resulted in a rapid reduction of skeletal tumor burden. Treatment with RANK-Fc prevented osteolytic lesions and reduced skeletal tumor burden. Combining RANK-Fc with rhApo2L/TRAIL was superior to either rhApo2L/TRAIL or RANK-Fc alone at reducing skeletal tumor burden in the bone metastasis model. Our findings show that RANKL inhibition effectively inhibits pathologic osteolysis induced by human breast adenocarcinoma MDA-MB-231 cells in animals with established tumors, and also enhances the ability of rhApo2L/TRAIL to reduce skeletal tumor burden in vivo.

Local Delivery of OncoVEXmGM-CSF Generates Systemic Antitumor Immune Responses Enhanced by Cytotoxic T-Lymphocyte–Associated Protein Blockade

Purpose: Talimogene laherparepvec, a new oncolytic immunotherapy, has been recently approved for the treatment of melanoma. Using a murine version of the virus, we characterized local and systemic antitumor immune responses driving efficacy in murine syngeneic models. Experimental Design: The activity of talimogene laherparepvec was characterized against melanoma cell lines using an in vitro viability assay. Efficacy of OncoVEXmGM-CSF (talimogene laherparepvec with the mouse granulocyte-macrophage colony-stimulating factor transgene) alone or in combination with checkpoint blockade was characterized in A20 and CT-26 contralateral murine tumor models. CD8+ depletion, adoptive T-cell transfers, and Enzyme-Linked ImmunoSpot assays were used to study the mechanism of action (MOA) of systemic immune responses. Results: Treatment with OncoVEXmGM-CSF cured all injected A20 tumors and half of contralateral tumors. Viral presence was limited to injected tumors and was not responsible for systemic efficacy. A significant increase in T cells (CD3+/CD8+) was observed in injected and contralateral tumors at 168 hours. Ex vivo analyses showed these cytotoxic T lymphocytes were tumor-specific. Increased neutrophils, monocytes, and chemokines were observed in injected tumors only. Importantly, depletion of CD8+ T cells abolished all systemic efficacy and significantly decreased local efficacy. In addition, immune cell transfer from OncoVEXmGM-CSF-cured mice significantly protected from tumor challenge. Finally, combination of OncoVEXmGM-CSF and checkpoint blockade resulted in increased tumor-specific CD8+ anti-AH1 T cells and systemic efficacy. Conclusions: The data support a dual MOA for OncoVEXmGM-CSF that involves direct oncolysis of injected tumors and activation of a CD8+-dependent systemic response that clears injected and contralateral tumors when combined with checkpoint inhibition. Clin Cancer Res; 23(20); 6190–202. ©2017 AACR.

Abstract 258: Talilmogene laherparepvec increases the anti-tumor efficacy of the anti-PD-1 immune checkpoint blockade

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells that have metastasized. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative viral replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. Recently, in a retrospective analysis of a Phase III melanoma trial investigators found that about two-thirds of the lesions injected with talimogene laherparepvec shrank 50% or more. And the same effect was seen in about a third of all uninjected tumors in the skin and lymph nodes and about a sixth of uninjected visceral lesions providing an indication that the treatment is triggering the desired immune system effect. Ongoing clinical trials are investigating T-VEC in combination with the immune checkpoint inhibiting antibodies ipilimumab and pembrolizumab in advanced melanoma. We sought to determine if the combination of intratumoral injection of talimogene laherparepvec and the systemic delivery of the checkpoint anti-PD1 antibody could increase the anti-tumor efficacy in a preclinical syngeneic contralateral tumor model. Using established MC-38 colon carcinoma tumors in C57Bl/6 mice, we delivered three intratumoral injections three days apart of the talimogene laherparepvec surrogate OncoVEXmuGM-CSF and twice weekly systemic injections of an antagonistic anti-PD-1 antibody. Either OncoVEXmuGM-CSF or anti-PD-1 alone induced modest tumor growth inhibition/tumor regressions of contralateral tumors. In combination, the OncoVEXmuGM-CSF injected tumor displayed 8/10 complete regressions and the distant tumors had 2/10 complete regressions. Peripheral blood was analyzed at 4 days and 10 days post the initial OncoVEXmuGM-CSF and anti-PD-1 antibody injections. OncoVEXmuGM-CSF increased the percent of PD-L1+ CD4 and CD8 T cells and the combination increased the percent of activated CD8+ T cells. Our findings demonstrate that localized therapy with talimogene laherparepvec augments the systemic anti-tumor immune response seen with anti-PD-1 therapy, providing a strong rationale for continued investigation of such combinations in the clinic. Citation Format: Julia Piasecki, Tiep le, Rafael Ponce, Courtney Beers. Talilmogene laherparepvec increases the anti-tumor efficacy of the anti-PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2015-258

Abstract 578: Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor

TIGIT is a recently identified coinhibitory immune checkpoint receptor expressed on NK, effector T, and regulatory T cells. In the oncology setting, TIGIT is upregulated on tumor infiltrating immune cells and is co-expressed with exhaustion markers including PD-1, TIM-3 and LAG-3 on infiltrates. TIGIT binds at least two ligands, CD155 and CD112, which are expressed on antigen presenting cells and other tissues, including tumor cells. These ligands also bind the activating receptor CD226, often co-expressed with TIGIT, creating a network that modulates adaptive and innate immune response in a manner analogous to the CD28-CTLA4-CD80-CD86 network. The absence of TIGIT signaling, resulting from genetic deficiency or blockade, enhances anti-tumor immunity in murine models, suggesting that disruption of TIGIT signaling may have clinical utility. To explore this concept, yeast antibody display was used to identify fully human, anti-TIGIT antibodies that block binding to ligands. Multiple rounds of selection with human and mouse TIGIT protein were performed to promote species cross-reactivity, diversity and affinity. A pool of 695 unique clones were screened for binding to TIGIT protein; 65 clones were then selected for further evaluation. Of the 65, 63 competed with CD155 for binding to TIGIT in a ForteBio screen. Fifty-three clones bound cyno TIGIT and 25 bound TIGIT from all three species (human, cyno, mouse). Antibodies bound endogenous TIGIT on primary T cells and blocked binding of ligands to cell surface expressed TIGIT in a dose dependent manner. Twelve clones showed functional activity in a TIGIT blockade bioassay and showed synergy with anti-PD-1 antibody in a PD-1/TIGIT combination bioassay. Activity in the bioassays correlated with affinity for recombinant and cell surface expressed TIGIT. Based on species cross-reactivity, binding affinity and activity in the bioassays, a lead candidate antibody was selected and produced as mouse IgG1 and IgG2a chimeras for testing in mouse tumor models. The chimeric antibodies behaved similarly to the parent clone in vitro exhibiting high affinity for TIGIT, competition with ligand for binding to TIGIT, and functional blockade of CD155-TIGIT interaction. Evaluation of the chimeric anti-TIGIT candidates alone and in combination with anti-PD-1 antibody in mouse syngeneic tumor models is ongoing, and results will be reported at the meeting. Antibody mediated blockade of coinhibitory immunoreceptors has proven clinically efficacious and supports the development of antibodies that target TIGIT. The unique human, non-human primate, and murine cross-reactive TIGIT-specific antibodies described here offer a simplified preclinical development path and the functional activity of these molecules supports their consideration as candidates for therapeutic development. Citation Format: Julia C. Piasecki, Kenneth Brasel, Robert Rosler, Kevin M. Klucher, Scott R. Peterson. Discovery and characterization of novel antagonistic antibodies that bind with high affinity to human, cynomolgus, and murine TIGIT, an immune checkpoint receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2017-578

Abstract 295: CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function. Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug. Citation Format: Alex Vo, Janelle Taylor, Robert Rosler, Julia Piasecki, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Bob Boyle, Rich Boyce, Scott Peterson. CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2017-295

Abstract 4287: Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) immunotherapy designed to selectively replicate in tumors and to initiate an anti-tumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. The talimogene laherparepvec-induced immune mediated mechanism of action in both the virus-injected and distant tumors have yet to be fully understood therefore, we evaluated viral replication, tumor cell lysis, and the changes in immune cell populations in both the injected and distant tumors using syngeneic contralateral tumor models. Animals received a single intratumoral dose of the murine surrogate of talimogene laherparepvec (OncoVEXmuGM-CSF), which induced regression in the majority of virus-injected tumors and tumor growth inhibition/regression in the contralateral (uninjected) tumors. HSV-1 antigen was only detected by IHC in the virus-injected tumor and not the contralateral tumor (and no other tissues). Virally-mediated tumor destruction (oncolysis) was also localized to only the virus-injected tumor. In preliminary experiments, morphometric analysis of the tumor tissue revealed that the percent area of CD3+ and CD8+ lymphocytes were significantly increased in both the virus-injected and contralateral tumors compared to the formulation control mice. In addition, the percent area occupied by CD103+ cells (a marker found on potent cytotoxic T cell stimulating dendritic cells) was increased in the virus-injected tumor compared to the contralateral tumor and the tumors in the control animals. Although cellular infiltration was increased in both virus-injected and contralateral tumors and was inversely correlated with tumor volume, the decreased volume of injected tumors was attributed to viral oncolysis. Taken together these data supports that talimogene laheparepvec activates a systemic T cell mediated anti-tumor immune response. Citation Format: Julia Piasecki, Jim Rottman, Tiep Le, Rafael Ponce, Courtney Beers. Talimogene laherparepvec activates systemic T-cell-mediated anti-tumor immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2015-4287

Talilmogene Iaherparepvec generates systemic T-cell-mediated anti-tumor immunity

Talimogene laherparepvec is an investigational oncolytic immunotherapy based on a modified herpes simplex virus type-1 (HSV-1) that is designed to selectively replicate in tumor tissue and to stimulate a systemic antitumor immune response. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors. Iterative viral replication of virus within permissive tumor tissue results in lytic cell destruction and local release of progeny virus and cellular derived antigens. GM-CSF, a product of the viral transgene, is also produced locally such that it can recruit and stimulate antigen presenting cells which, in addition to relevant tumor-derived antigens, are required for the initiation of a systemic antitumor immune response. A phase 3 clinical trial of talimogene laherparepvec in regionally or distantly metastatic melanoma has recently reported a 26% ORR objective response rate (6% for GM-CSF alone){(J Clin Oncol 31, 2013 (suppl; abstr LBA9008)}. Since the immune mediated anti-tumor mechanisms have yet to be fully understood, we sought to identify immune-specific changes post talimogene laherparepvec administration in the A20 syngeneic contralateral tumor model. We analyzed immune populations in both injected and non-injected tumors and in peripheral hematopoietic tissues at 2, 5 and 10 days post injection. Animals received one 5x106 PFU intratumoral dose of talimogene laherparepvec which induces complete regression in approximately 70-100% of the injected tumors and approximately 50-60% of the contralateral non-injected tumors. Injection of virus resulted in marked increases in injected tumor draining lymph node and spleen size compared to control animals. Upon further flow cytometric analysis, changes in T-cell populations were observed in the injected and non-injected tumor draining lymph nodes, spleen and peripheral blood and T-cells expressed higher levels of cell surface activation markers as early as 2 days post-treatment in talimogene laherparepvec treated animals as compared to vehicle control animals. To functionally examine the T-cell-mediated anti-tumor immunity, we evaluated the memory response by re-challenging mice that completely regressed their primary A20 tumors with either A20 or three other Balb/c-derived tumor cell lines; 4T1, CT26 and RENCA. Interestingly, the mice displayed broad immunity to Balb/c tumor cells and rejected the A20, CT26 and RENCA but not 4T1 tumor cells whereas all the tumor cell lines grew robustly in control naive animals. In addition, splenic T-cells from talimogene laherparepvec-treated mice induced A20 tumor cell killing ex vivo while T-cells from vehicle control-treated mice did not. Intratumoral injection of talimogene laherparepvec systemically activates T cells and generates anti-tumor immunity in mice.