Pancreatic cancer has an abysmal 5-year survival rate of 8%, making it a deadly disease with a need for novel therapies. Here we describe a multitargeting heparin-based mimetic, necuparanib, and its antitumor activity in both in vitro and in vivo models of pancreatic cancer. Necuparanib reduced tumor cell proliferation and invasion in a three-dimensional (3D) culture model; in vivo, it extended survival and reduced metastasis. Furthermore, proteomic analysis demonstrated that necuparanib altered the expression levels of multiple proteins involved in cancer-driving pathways including organ development, angiogenesis, proliferation, genomic stability, cellular energetics, and invasion and metastasis. One protein family known to be involved in invasion and metastasis and altered by necuparanib treatment was the matrix metalloprotease (MMP) family. Necuparanib reduced metalloproteinase 1 (MMP1) and increased tissue inhibitor of metalloproteinase 3 (TIMP3) protein levels and was found to increase RNA expression of TIMP3. MMP enzymatic activity was also found to be reduced in the 3D model. Finally, we confirmed necuparanib's in vivo activity by analyzing plasma samples of patients enrolled in a phase I/II study in patients with metastatic pancreatic cancer; treatment with necuparanib plus standard of care significantly increased TIMP3 plasma protein levels. Together, these results demonstrate necuparanib acts as a broad multitargeting therapeutic with in vitro and in vivo antiinvasive and antimetastatic activity.

Introduction: Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers, still most patients do not respond to PD-1 or CTLA-4 inhibitors. Thus, new Immuno-Oncology (IO) therapies that could potentially benefit non-responding patients are greatly needed. Jounce has generated cell type-specific gene signatures as a means of probing The Cancer Genome Atlas and other large datasets for novel IO targets. Regulatory T cells (Tregs) are one attractive cell type for targeting as they may contribute to resistance to ICB. While Tregs are critical for immune homeostasis and preventing tissue damage, they are often present in large numbers within tumors where they may suppress anti-tumor immunity. Therapeutic strategies that specifically deplete tumor-infiltrating Tregs (TITRs) while sparing peripheral and normal tissue Tregs are highly desirable. Using a Treg gene signature, we have a found a strong correlation with TITRs and CCR8 (C-C motif chemokine receptor 8) across multiple tumor types. CCR8 may be differentiated from other known Treg targets in this regard, as its expression was found to be highly selective to TITRs. Methods and Results: We first assessed CCR8 levels on TITRs across multiple tumor types and compared expression to Tregs in normal colon tissue or peripheral blood. On average, peripheral blood Tregs had nearly undetectable CCR8 expression and normal colon tissue Tregs showed 4 to 5-fold lower levels of CCR8 than TITRs. We then generated a panel of monoclonal antibodies (mAbs) that bind specifically to CCR8, but not other family members, and block CCR8 signaling induced by its ligand CCL1. The ability of these mAbs to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells expressing CCR8 was tested. When target cells expressed CCR8 at levels equivalent to normal tissue Tregs no ADCC activity was observed. In contrast, when cells expressed CCR8 at levels equivalent to TITRs, robust ADCC was observed, but only using antibodies in which the human IgG1 Fc was afucosylated. Thus, afucosylated anti-CCR8 antibodies demonstrated a therapeutic window whereby TITRs but not normal tissue Tregs could be depleted. An Fc competent, mouse-specific, anti-CCR8 antibody showed single agent tumor growth inhibition across several murine tumor models - including models in which anti-PD-1 was ineffective. Anti-CCR8 was a potent combination partner with anti-PD-1 resulting in 50% complete tumor regressions in PD-1 resistant models. Conclusions: Based on these pre-clinical data JTX-1811, a high affinity CCR8-specific humanized monoclonal antibody with enhanced ADCC activity, is being developed for the selective depletion of tumor-infiltrating Tregs. JTX-1811 may be useful in PD-1 resistant settings and may restore the activity of PD-1 inhibitors in the setting of primary or acquired resistance to ICB. Citation Format: Fabien Dépis, Changyun Hu, Jessica Weaver, Lara McGrath, Boris Klebanov, Joshua Buggé, Ben Umiker, Christine Fregeau, Dhruvkumar Upadhyay, Anirudh Singh, Chang-Ai Xu, Vikki Spaulding, Michelle Priess, Masie Wong, Seema Naheed, Yan Zhang, Kristin Legendre, Edward C. Stack, Alessandro Mora, Margaret Willer, Kristan Meetze, Monica Gostissa, Michael A. Meehl, Donald R. Shaffer. Preclinical evaluation of JTX-1811, an anti-CCR8 antibody with enhanced ADCC activity, for preferential depletion of tumor-infiltrating regulatory T cells [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 4532.

BackgroundLeukocyte immunoglobulin-like receptor B2 (LILRB2; ILT4) is an immunoinhibitory protein expressed on the surface of myeloid cells that has been increasingly recognized as a therapeutic target of interest in immuno-oncology (IO). Upon binding its ligands, MHC I molecules (e.g. HLA-G/HLA-A), LILRB2 inhibits myeloid cell activation and promotes an M2-like (anti-inflammatory) state. LILRB2 was the first target prioritized from a macrophage discovery effort leading to the development of JTX-8064, a humanized monoclonal antibody that specifically binds to and antagonizes LILRB2. JTX-8064 has been shown to induce an M1-like (pro-inflammatory; anti-tumor) functional state in macrophages. Rodents do not express LILRB proteins limiting their usefulness as a model for preclinical study of JTX-8064. To overcome this limitation, we conducted an ex vivo human tumor histoculture study to assess the pharmacodynamic effects of LILRB2 antagonism. Protein and/or gene expression analysis of matched tumor samples enabled the discovery of predictive biomarkers associated with the induction of specific pharmacodynamic signatures in ex vivo-cultured human tumors in response to JTX-8064. Finally, tumor types were identified that had a high prevalence of these predictive biomarkers suggesting they may be priority indications for JTX-8064 therapy.MethodsMore than 100 fresh treatment-naïve human tumor samples obtained post-surgery from kidney, lung, and head and neck cancer were treated with JTX-8064 or isotype control antibody for 24 hrs in the histoculture system. RNA was isolated from tumors prior to any treatment as well as from JTX-8064 and isotype control treated samples. Gene expression was analyzed using the NanoString nCounter® and qPCR assays. Additional IHC analyses were performed on baseline untreated tumor samples.ResultsJTX-8064 was shown to induce pharmacodynamic responses to treatment significantly above isotype control indicative of macrophage polarization, IFNg-signaling, and T cell inflammation. To identify predictive biomarkers of pharmacodynamic response to JTX-8064, matched untreated samples were characterized by gene expression analysis and by IHC (CD8, CD163, and HLA-G proteins). Numerous LILRB2 pathway-related molecules (e.g. HLA-A, HLA-B, CD163, LILRB2) and gene signatures were found to be statistically significantly higher in the untreated kidney, head and neck, and lung cancer samples of matched pharmacodynamic responders compared to non-responders. Further bioinformatics analysis revealed additional cancer subtypes where these biomarkers are enriched.ConclusionsThese data will inform indication selection and combination strategies for JTX-8064 to maximize potential therapeutic benefit for patients with solid tumor malignancies.

Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer. Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.