Eric Humke

A NOD2–NALP1 complex mediates caspase-1-dependent IL-1β secretion in response to Bacillus anthracis infection and muramyl dipeptide

NOD2, a NOD-like receptor (NLR), is an intracellular sensor of bacterial muramyl dipeptide (MDP) that was suggested to promote secretion of the proinflammatory cytokine IL-1β. Yet, the molecular mechanism by which NOD2 can stimulate IL-1β secretion, and its biological significance were heretofore unknown. We found that NOD2 through its N-terminal caspase recruitment domain directly binds and activates caspase-1 to trigger IL-1β processing and secretion in MDP-stimulated macrophages, whereas the C-terminal leucine-rich repeats of NOD2 prevent caspase-1 activation in nonstimulated cells. MDP challenge induces the association of NOD2 with another NLR protein, NALP1, and gel filtration analysis revealed the formation of a complex consisting of NOD2, NALP1, and caspase-1. Importantly, Bacillus anthracis infection induces IL-1β secretion in a manner that depended on caspase-1 and NOD2. In vitro, Anthrax lethal toxin strongly potentiated IL-1β secretion, and that response was NOD2 and caspase-1-dependent. Thus, NOD2 plays a key role in the B. anthracis-induced inflammatory response by being a critical mediator of IL-1β secretion.

Abstract LB-290: Targeting MUC16 with the antibody-drug conjugate (ADC) DMUC5754A in patients with platinum-resistant ovarian cancer: A phase I study of safety and pharmacokinetics.

Background: MUC16 is a large transmembrane protein overexpressed by the majority of ovarian cancers (OC), compared with normal tissues. The role of MUC16 in the pathogenesis of ovarian cancer is unknown; however, MUC16 may facilitate the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity, and may inhibit natural killer cell-mediated anti-tumor cytotoxic responses. Conjugation of a highly potent cytotoxic drug to a MUC16-specific monoclonal antibody represents a novel approach to treatment of MUC16-expressing tumors. DMUC5754A is an ADC containing the humanized IgG1 anti-MUC16 monoclonal antibody linked to the potent anti-mitotic agent MMAE and demonstrates anti-tumor activity in MUC16-expressing tumor xenograft models. Methods: This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamic (PD) activity of DMUC5754A (0.3-3.2 mg/kg) given every 3 weeks (q3w) to patients with advanced recurrent platinum-resistant OC. A standard 3+3 design was used to determine the maximum-tolerated dose, followed by cohort expansion. Tumor tissue was assessed for expression of MUC16 and other relevant markers. Clinical activity was evaluated per RECIST. Results: Forty-four patients (22 escalation, 22 expansion at 2.4 mg/kg), median age 62 (44-79), ECOG PS 0-1, received a median of 4 doses (range 1-20) of DMUC5754A. Two DLTs, 1 Grade 4 neutropenia and 1 Grade 4 uric acid increase, occurred at the maximally administered dose of 3.2 mg/kg. Grade ≥ 3 related adverse events (AE) occurring in ≥ 5% of patients included fatigue (4 at 2.4 mg/kg; 9% total) and neutropenia (1 at 3.2 mg/kg, 3 at 2.4 mg/kg; 9% total). The most common related AEs over all dose levels were fatigue (57%), nausea (34%), vomiting (27%), decreased appetite (25%), peripheral neuropathy (25%), and diarrhea (23%). Serious drug-related AEs (SAE) were small intestine obstruction (2 patients), hypocalcemia (1 patient), and neutropenia (1 patient). Total antibody and conjugated MMAE were not impacted by circulating CA125 and displayed dose-dependent PK with clearance decreasing as dose increased. Accumulation of total antibody, conjugated MMAE and free MMAE was not observed due to their short half-lives (≤5 days). Tumor MUC16 expression was evaluable in 42 patients, showing 20% IHC 0, 16% IHC 2+, and 64% IHC 3+. All confirmed responses (1 CR and 4 PRs) occurred in patients treated at 2.4 mg/kg and whose tumors were MUC16 IHC 2+ or 3+. Six additional patients had minor responses (3 at 2.4 mg/kg). Sixteen of the twenty-nine patients at 2.4 mg/kg were on study ≥ 105 days. Human epididymis protein 4 was a potential surrogate marker for serologic response measures in the presence of anti-MUC16/CA125-binding therapy. Conclusions: DMUC5754A at 2.4 mg/kg q3w has an encouraging safety profile and evidence of anti-tumor activity in MUC16-expressing OC. Further studies are planned. Citation Format: Joyce Liu, Kathleen Moore, Michael Birrer, Suzanne Berlin, Ursula Matulonis, Jeffrey Infante, Jian Xi, Robert Kahn, Yulei Wang, Katie Wood, Daniel Coleman, Daniel Maslyar, Eric Humke, Howard Burris. Targeting MUC16 with the antibody-drug conjugate (ADC) DMUC5754A in patients with platinum-resistant ovarian cancer: A phase I study of safety and pharmacokinetics. [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 LB-290. doi:10.1158/1538-7445.AM2013-LB-290

Development of a robust flow cytometry-based pharmacodynamic assay to detect phospho- protein signals for phosphatidylinositol 3-kinase inhibitors in multiple myeloma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients.MethodsWe conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application.ResultsThe phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells.ConclusionsWe developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.

Abstract POSTER-THER-1441: Biomarker evaluation of phase 1 clinical trials of antibody-drug conjugates (ADCs) in platinum resistant ovarian cancer

Purpose: DNIB0600A and DMUC5754A are two ADCs that conjugate the anti-mitotic agent MMAE with anti-NaPi2b and anti-MUC16 monoclonal antibodies, respectively. Both ADCs have shown promising anti-tumor activity in patients with platinum resistant ovarian cancer. Here we report biomarker analysis in patient samples collected from these phase 1 studies. The main goal of this study is to evaluate tissue-based biomarkers that can predict response or resistance to these ADCs. We also explored the utility of serum protein biomarkers and circulating tumor cells (CTCs) as potential surrogates for monitoring treatment response to ADCs and disease progression. Methods: Biomarker analysis was done on 55 ovarian cancer patients treated with clinically relevant doses (1.8-3.2mg/kg) from DNIB0600A and DMUC5754A Phase 1 studies. Protein and mRNA expression levels of NaPi2b and MUC16 targets were assessed in archival tumor specimen by immunohistochemistry (IHC) and qRT-PCR respectively. Serum collected at baseline and post-treatment were analyzed by CA125 and HE4 ELISA assays as well as by the OLINK 96-plex PEA protein biomarker panel. CTCs at baseline and post-treatment were analyzed using the Veridex CellSearch System. Results: Target expression in tumor tissues for both NaPi2b and MUC16 measured by IHC and qRT-PCR are concordant. High NaPi2b or MUC16 expression (IHC 2+/3+) was identified in all responders by RECIST criteria (11 from DNIB0600A and 5 from DMUC5754A) for respective target, while no patient from either study with IHC 0 showed RECIST response. In patients treated with DNIB0600A, longitudinal changes in serum CA125 level correlated with RECIST response. Additionally, CTC was detected in 60% of patients at baseline in the DNIB0600A trial, and decreased CTC counts was observed after 1-2 cycles of treatment for two-third of patients. In patients treated with DMUC5754A, circulating CA125 (i.e. extra-cellular domain of MUC16 shed in circulation) is cleared after initial dosing; therefore other ovarian cancer biomarkers including HE4 were assessed. Baseline serum HE4 level correlates well with the tumor burden at pre-treatment in DMUC5754A trial, and showed excellent correlation with RECIST response post-treatment. Conclusions: Target expression in archival tumor tissues is predictive to clinical response to ADCs. CTC enumeration as well as serum HE4 could be used as potential surrogate biomarkers for monitoring treatment response in ovarian cancer. Further validation of these findings is required. Citation Format: Yulei Wang, Ron Firestein, Lisa Ryner, Walter Darbonne, Yinghui Guan, Shan Lu, YJ Choi, Yuanyuan Xiao, Paul Polakis, Becky Suttmann, Rupal Desai, Ling Fu, Ola Saad, Kirsten Achilles Poon, Mitch Denker, Vincent Leveque, Teiko Sumiyoshi, Mark Lackner, David Shames, Eric Humke, Daniel Mayslar. Biomarker evaluation of phase 1 clinical trials of antibody-drug conjugates (ADCs) in platinum resistant ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1441.

Anti-NaPi2b antibody–drug conjugate lifastuzumab vedotin (DNIB0600A) compared with pegylated liposomal doxorubicin in patients with platinum-resistant ovarian cancer in a randomized, open-label, phase II study

Background Lifastuzumab vedotin (LIFA) is a humanized anti-NaPi2b monoclonal antibody conjugated to a potent antimitotic agent, monomethyl auristatin E, which inhibits cell division by blocking the polymerization of tubulin. This study is the first to compare an antibody-drug conjugate (ADC) to standard-of-care in ovarian cancer (OC) patients. Patients and methods Platinum-resistant OC patients were randomized to receive LIFA [2.4 mg/kg, intravenously, every 3 weeks (Q3W)] or pegylated liposomal doxorubicin (PLD) (40 mg/m2, intravenously, Q4W). NaPi2b expression and serum CA-125 and HE4 levels were assessed. The primary end point was progression-free survival (PFS) in intent-to-treat (ITT) and NaPi2b-high patients. Results Ninety-five patients were randomized (47 LIFA; 48 PLD). The stratified PFS hazard ratio was 0.78 [95% confidence interval (95% CI), 0.46-1.31; P = 0.34] with a median PFS of 5.3 versus 3.1 months (LIFA versus PLD arm, respectively) in the ITT population, and 0.71 (95% CI, 0.40-1.26; P = 0.24) with a median PFS of 5.3 months versus 3.4 months (LIFA versus PLD arm, respectively) in NaPi2b-high patients. The objective response rate was 34% (95% CI, 22% to 49%, LIFA) versus 15% (95% CI, 7% to 28%, PLD) in the ITT population (P = 0.03), and 36% (95% CI, 22% to 52%, LIFA) versus 14% (95% CI, 6% to 27%, PLD) in NaPi2b-high patients (P = 0.02). Toxicities included grade ≥3 adverse events (AEs) (46% LIFA; 51% PLD), serious AEs (30% both arms), and AEs leading to discontinuation of drug (9% LIFA; 8% PLD). Five (11%) LIFA versus 2 (4%) PLD patients had grade ≥2 neuropathy. Conclusion LIFA Q3W was well tolerated and improved objective response rate with a modest, nonstatistically significant improvement of PFS compared with PLD in platinum-resistant OC. While the response rate for the monomethyl auristatin E-containing ADC was promising, response durations were relatively short, thereby highlighting the importance of evaluating both response rates and duration of response when evaluating ADCs in OC. Clinical trials.gov NCT01991210.

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic-Based Multiplex PCR and Next-Generation Sequencing

Circulating tumor DNA (ctDNA) has potential to serve as a biomarker for noninvasive monitoring of treatment response and disease progression. However, broad clinical applicability of ctDNA has been limited by the low sensitivity, throughput, and patient coverage offered by existing ctDNA detection methods. Herein, we report the adaptation and characterization of the microfluidics multiplex PCR sequencing technology for high-throughput and sensitive quantitation of ctDNA. A multiplex PCR preamplification step was developed and incorporated into the microfluidics multiplex PCR sequencing work flow to enable low-input ctDNA analysis with enhanced sensitivity. An empirical bayesian model was developed to characterize both position and substitution-associated system errors specific to this platform and provided a tailored approach to greatly enhance the confidence and accuracy of variant calling for ctDNA analysis. Clinical validation of this platform for ctDNA mutation detection demonstrated an overall sensitivity of 92% and specificity of 100% when using mutation calls in the matched tumor tissues as a benchmark. Finally, we established an early proof of concept of clinical utility of this ctDNA work flow for monitoring disease progression using clinical trial samples. Our novel ctDNA work flow provides a high-throughput and sensitive platform that can be implemented in clinical trials for mutation detection and disease monitoring from plasma ctDNA.

Abstract CT009: Targeting MUC16 with the THIOMABTM-drug conjugate DMUC4064A in patients with platinum-resistant ovarian cancer: a Phase I escalation study

Background: MUC16 is a transmembrane protein that is overexpressed by ovarian cancer. The role of MUC16 in the pathogenesis of ovarian cancer is unknown; however, MUC16 may facilitate binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Traditional antibody drug conjugates, with conjugation through inter-chain disulfides, are heterogeneous mixtures of drug antibody ratios (DAR) ranging from 0-8, resulting in complex pharmacokinetics (PK) and potentially unfavorable safety and efficacy. In contrast, THIOMABTM drug conjugates (TDCs) use novel technology that achieves site-directed drug conjugation yielding a homogeneous DAR. DMUC4064A is a cysteine-engineered TDC comprising a humanized anti-MUC16 IgG1 and 2 potent anti-mitotic monomethyl auristatin E (MMAE) molecules. Methods: The dose-escalation component of the Phase I study evaluated safety, tolerability, PK, pharmacodynamics, and early activity of DMUC4064A given Q3W to patients with platinum-resistant ovarian cancer. A standard 3+3 design was used to determine the maximum-tolerated dose. Archival tumor tissue was used to assess expression of MUC16 and other markers. Clinical activity was evaluated per RECIST v4.0 criteria. Results: Forty-four female patients, median age 63 (35-84), ECOG PS 0-1, received a median of 4 doses (range 1-12) of DMUC4064A at 1.0-5.6 mg/kg. At 5.6 mg/kg, one patient experienced three adverse events (AE) each qualifying as a DLT (colitis, hyperglycemia, and hypokalemia; all Grade 3). At 5.2 mg/kg, another patient experienced a DLT of Grade 5 septic shock. Grade ≥ 3 AE occurring in ≥ 5% of patients included hyponatremia (1 each at 1.8, 3.2, and 5.2 mg/kg; 2 at 2.4 mg/kg; 11% total), ascites (2 at 2.4 mg/kg and 1 at 3.2 mg/kg; 7% total) and hyperglycemia (3 at 5.6 mg/kg; 7% total), all unrelated except hyperglycemia. The most common (≥20%) related AEs for all dose levels were fatigue (34%), nausea (32%), diarrhea (23%) and abdominal pain (21%). Ocular toxicities (related, Grade ≥ 2) included keratitis (Grade 3, n=2), blurred vision (Grade 3, n=1; Grade 2, n=3), and dry eye (Grade 2, n=1). Total antibody and conjugated MMAE showed dose-dependent PK; neither were impacted by circulating CA125. Total antibody, conjugated and free MMAE accumulation was minimal. At doses ≥2.4 mg/kg, DMUC4064A had decreased clearance and achieved higher exposures versus MUC16 MMAE ADC. Confirmed responses (1 CR and 6 PRs) occurred at ≥ 3.2 mg/kg (n=30). Tumor MUC16 IHC scores were 2+/3+ in responders for whom data were available (n=5). Conclusions: These data are the first reported for an MMAE-containing TDC in a clinical trial (clinicaltrials.gov NCT02146313). DMUC4064A has an acceptable safety profile with improved stability compared to MUC16 MMAE ADC and shows evidence of anti-tumor activity, warranting further evaluation in ovarian cancer. Citation Format: Joyce F. Liu, Kathleen N. Moore, Judy S. Wang, Manish Patel, Michael J. Birrer, Erika Hamilton, Lisa Barroilhet, William M. Flanagan, Yulei Wang, Amit Garg, Xuyang Lu, Anjali Vaze, Dilip Amin, Doug Leipold, S Renee Commerford, Eric W. Humke, Harold A. Burris. Targeting MUC16 with the THIOMABTM-drug conjugate DMUC4064A in patients with platinum-resistant ovarian cancer: a Phase I escalation study [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 CT009. doi:10.1158/1538-7445.AM2017-CT009

Abstract CT092: Pharmacokinetics of a THIOMABTMantibody drug conjugate (TDC): DMUC4064A in a phase 1 study with platinum-resistant ovarian cancer

BACKGROUND: DMUC4064A is a TDC comprising a humanized anti-MUC16 IgG1 and a potent anti-mitotic agent, monomethyl auristatin E (MMAE) through a protease-labile linker through engineered cysteine at a Drug Antibody Ratio (DAR) of 2. The use of engineered cysteines is intended to allow more homogenous conjugation, which in turn can improve the overall performance of the TDC (i.e. slower clearance), compared to the conventional ADC (prepared by stochastic conjugation to interchain disulfide bond cysteines). MUC16 is a transmembrane glycoprotein overexpressed on ovarian cancer cells, which in its soluble form is known as Carbohydrate antigen 125 (CA125). A phase I study in PROC patients is ongoing to assess the safety, efficacy and PK of DMUC4064A given every 3 weeks (Q3W). METHODS: Serum/plasma samples at multiple time points were quantified for three analytes (unconjugated MMAE, DMUC4064A total antibody (tAb), and DMUC4064A conjugate (measured as antibody-conjugated MMAE, acMMAE)). The cycle 1 PK was assessed by standard non-compartmental analysis. The impact of serum CA125 on PK was investigated. RESULTS: Within the tested dose range (1.0-5.6 mg/kg), acMMAE and tAb showed nonlinear PK with more than dose-proportional exposure at the higher doses (4.8 mg/kg and 5.6 mg/kg). A clear correlation of acMMAE with tAb exposure was observed. The PK of DMUC4064A at the highest dose tested (5.6 mg/kg), as measured by acMMAE, showed a volume of distribution of approximately 52 mL/kg and a clearance of ~7 mL/day/kg with a terminal half-life of approximately 5.5 days. Unconjugated MMAE exposure was consistently lower than acMMAE with a mean Cmax approximately 150-300 fold lower than that of acMMAE. At higher doses (>4.0 mg/kg), unconjugated MMAE exposure was variable and overlapping across doses. Accumulation of all analytes was minimal after repeated dosing (Q3W). Based on preliminary data from this ongoing study, baseline CA125 levels have not shown any apparent impact on cycle 1 exposure. Compared to the conventional anti-MUC16 ADC (DMUC5754A, DAR 3.5) at 2.4 mg/kg (MTD), the TDC DMUC4604A at 5.6 mg/kg demonstrates a greater than 3 fold slower acMMAE clearance1. CONCLUSION: In this ongoing study of DMUC4604A, nonlinear PK of acMMAE and tAb were observed over the dose range studied, and exposures of acMMAE and tAb were highly correlated. This is the first report of clinical PK data of a TDC demonstrating slower clearance compared to a conventional ADC format. 1Liu JF, et al. Annals of Oncology 27: 2124–2130, 2016 Citation Format: Amit Garg, Gaohong She, Ian Donatello, Matts Kagedal, Douglas D. Leipold, Ola Saad, Joyce Liu, Kathleen Moore, Erika Hamilton, Howard Burris, Judy Wang, Michael Birrer, Eric Humke, Sandhya Girish. Pharmacokinetics of a THIOMABTM antibody drug conjugate (TDC): DMUC4064A in a phase 1 study with platinum-resistant ovarian cancer [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 CT092. doi:10.1158/1538-7445.AM2017-CT092