Carl Uli Bialucha

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Combination of Antibody That Inhibits Ligand-Independent HER3 Dimerization and a p110α Inhibitor Potently Blocks PI3K Signaling and Growth of HER2+ Breast Cancers

We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

ABSTRACT Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.

Discovery and Optimization of HKT288, a Cadherin-6 Targeting ADC for the Treatment of Ovarian and Renal Cancer.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Utilizing panels of patient derived xenografts to aid the development of antibody drug conjugates

ABSTRACT Despite numerous endeavors in clinical trials there are few clinically approved Antibody Drug Conjugate (ADC) therapies. Here we comment on our recent publication demonstrating the power of using panels of patient-derived xenografts (PDX) prior to Phase 1, to assess the potential heterogeneity of response a clinical candidate may show across a population. Furthermore we discuss how the same approach has been used in an additional ADC program.

Abstract 4261: Targeting HER3 and PI3K in head and neck squamous cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide, with more than 40,000 new cases developing annually in the US alone. 80-90% of HNSCC cases display elevated EGFR expression. Cetuximab an anti-EGFR monoclonal is an approved treatment of HNSCC. However, patient response to cetuximab is variable. Activation of the HER2/3 and/or the down stream PI3K pathway is a potential mechanism leading to cetuximab resistance in HNSCC. LJM716 is a fully human IgG1 ati-HER3 monoclonal antibody. BYL719 is a PI3Ka-selective small molecule inhibitor. Here, we profiled a panel of HNSCC cell lines for 1) the activation status of the EGFR/HER2/3/PI3K pathway; 2) their response to LJM716 and/or to BYL719 in combination with cetuximab. Methods: In this study, we evaluated LJM716 or BYL719 anti-tumor activity either alone, combined, or their respective combination with cetuximab in head and neck squamous cell cancer. LJM716, cetuximab and BYL719 single agent as well as combination activity of LJM716/cetuximab or LJM716/BYL719 were assessed in 33 head and neck cell lines using the CellTiter-Glo® Luminescent Cell Viability Assay. Single agent and combination anti-tumor activities of LJM716 were also tested in selected xenograft mouse models. To explore predictive markers for LJM716 and BYL719, baseline EGFR/HER2/3 and PI3K activation status was further analyzed. NRG1 level was assessed based on Affymetrix U133 plus2 genechip array. Results: Both BYL719 and LJM716 displayed anti-tumor activity in HNSCC cell lines in vitro, with BYL719 superior in most of the lines tested. There was minimum combination benefit observed with the combination of LJM716 and BYL719. Both reagents further enhanced cetuximab activity and expanded the number of cell lines sensitive to cetuximab. Anti-tumor activity was also observed with LJM716 or BYL719 in mouse xenograft models either as single agent or in combination with cetuximab. There was no correlation between baseline NRG1 or HER3 phosphorylation level with in vitro LJM716 or BYL719 efficacy. Conclusion: HNSCC is a disease highly dependent on PI3K activity. Mechanisms leading to sustained PI3K activity in HNSCC include genomic changes of PIK3CA/PTEN, activation of HER3 and additional undefined molecular events. Combining HER3/PI3K targeted therapies with cetuximab may overcome some of the resistance to cetuximab therapy in HNSCC. Future work is underway to identify biomarkers that would predict response to these combination regimens. Citation Format: Qing Sheng, HuiQin Wang, Rita Das, Yan Chen, Jinsheng Liang, Alex Cao, Carl Uli Bialucha, Seth Ettenberg, Alan Huang. Targeting HER3 and PI3K in head and neck squamous cancer cells. [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 4261. doi:10.1158/1538-7445.AM2013-4261

Nicotinamide Phosphoribosyltransferase Inhibitor as a Novel Payload for Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) are a novel modality that allows targeted delivery of potent therapeutic agents to the desired site. Herein we report our discovery of NAMPT inhibitors as a novel nonantimitotic payload for ADCs. The resulting anti-c-Kit conjugates (ADC-3 and ADC-4) demonstrated in vivo efficacy in the c-Kit positive gastrointestinal stromal tumor GIST-T1 xenograft model in a target-dependent manner.

Abstract 872: In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial

The Cadherin-6 (CDH6) gene was found to be frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CHD6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the tubulin-binding maytansinoid payload DM4. CDH6-ADC was evaluated across multiple linker-payload combinations with the sulfo-SPDB-DM4 format being selected based on a superior combined profile pertaining to activity, selectivity and tolerability. To gain a broader understanding of CDH6-ADC activity in vivo we profiled the lead candidate against a panel of 31 unselected patient derived ovarian xenograft (PDX) models in a 1×1×1 PDX clinical trial, similar to that described in Gao et al., 2015. In this unbiased high throughput in vivo screen, CDH6-ADC demonstrated robust antitumor activity, with an overall response rate of 39%. Responses were generally durable beyond 150 days and were achieved at doses yielding exposures anticipated to be achievable in humans and observed in PDX models featuring a range of CDH6 expression level and degree of tumor heterogeneity. Retrospective analysis of individual PDX responses and molecular profiling data demonstrate that sensitivity to CDH6-ADC is highly correlated to CDH6 transcript and protein levels. These findings suggest an ability to prospectively identify patients most likely to benefit from this novel targeted therapy. Furthermore, CDH6-ADC demonstrated robust tumor regressions in a representative PDX xenograft model that was refractory to carboplatin/paclitaxel standard of care therapy. These data suggest that CDH6-ADC may benefit both treatment naive patients and patients that have progressed on prior therapy containing tubulin-targeting anti-mitotics. Extending beyond ovarian cancer, we found CDH6 to be frequently overexpressed in renal cancer. CDH6-ADC was active against RCC PDX models featuring patient relevant levels of CDH6 expression. Data described herein suggest that this novel ADC may be an effective treatment for patients with CDH6 expressing tumors, including ovarian and renal cancer - both indications with a high unmet medical need. Citation Format: Carl U. Bialucha, Scott D. Collins, Yeonju Shim, Xiamei Zhang, Roberto Velazquez, Colleen Kowal, Caroline Bullock, Hongbo Cai, Stacy M. Rivera, Julie M. Goldovitz, Esther Kurth, Alice T. Loo, Guizhi Yang, John Green, Lance Ostrom, Matthew J. Meyer, Rebecca Mosher, Hui Gao, Juliet Williams, Emma Lees. In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 872.

Abstract 2974: Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer

In an attempt to mine tumor versus normal mRNA expression datasets for novel tumor antigens, we identified the Cadherin-6 (CDH6) gene as frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. CDH6, also known as K-(kidney)-cadherin, is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CDH6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the maytansinoid payload DM4. The antibody component of CDH6-ADC was selected from a panel of anti-CDH6 antibodies based on a multi-factorial lead selection campaign incorporating readouts of internalization propensity, in vitro cytotoxicity, as well as in vivo PK and efficacy across multiple linker-payload formats. CDH6-ADC features potent, target-dependent in vivo activity consistent with the mechanism of the anti-mitotic, tubulin-targeting sulfo-SPDB-DM4 linker-payload combination used. Specifically, treatment of CDH6-expressing ovarian cancer xenograft models with CDH6-ADC results in the time-dependent generation of intra-tumoral ADC catabolites and concomitant induction of phospho-histone H3 and cleaved caspase-3 - markers of G2/M arrest and apoptosis, respectively. CDH6-ADC induces durable tumor regressions at clinically relevant exposures in multiple human patient-derived tumor xenografts (PDX) across both ovarian and renal cancer lineages. To gain a more thorough understanding of CDH6-ADC activity in pre-clinical models of human ovarian cancer and identify potential molecular correlates for patient stratification, we profiled CDH6-ADC in a PDX clinical trial or PCT comprising 31 individual PDX models. In this unselected population, treatment with CDH6-ADC resulted in robust anti-tumor activity. Integration of PDX response data with CDH6 target expression in both the PDX models and human clinical samples indicate CDH6 expression patterns consistent with in vivo activity are found in a substantial fraction of ovarian, renal and cholangiocarcinoma patients. Together, the encouraging pre-clinical efficacy and tolerability data support the clinical evaluation of CDH6-ADC. Citation Format: Scott D. Collins, Parmita Saxena, Xiao Y. Li, Yeonju Shim, Lance Ostrom, Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Xiuxia Sun, Sanela Bilic, William R. Tschantz, Meghan Flaherty, Keith Mansfield, Tiancen Hu, Vladimir Capka, Markus Kurz, Ivana Liric Rajlic, Anne Serdakowski London, Duc Nguyen, Rebecca Mosher, Matthew J. Meyer, Aaron Bourret, Jamal Saeh, Scott Cameron, Emma Lees, Carl U. Bialucha. Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2974.

Abstract 5442: Microscale methods for preparation and screening of antibody-drug conjugates

Antibody-drug conjugates (ADCs) are hybrid biotherapeutics that combine the targeting specificity of monoclonal antibodies with chemically conjugated, highly potent small molecule chemotherapeutics. Using established research scale approaches, the amount of antibody material needed to prepare candidate ADCs far exceeds the quantities required for initial in vitro screening. The need to scale up production across many antibodies slows down early lead selection efforts and wastes material. We have therefore developed methods for conjugating multiple antibodies with ADC payloads in parallel at the 50-150 μg scale in 96-well plates. Pilot reactions show that antibodies can be titrated to different final drug:antibody ratios (DARs) with different payloads, and that differences in pH can alter the reaction kinetics with useful effects. We show that 96-well centrifugal ultrafiltration enables highly parallel ADC purification while maintaining the rigorous removal of residual cytotoxic impurities observed with more established methods such as gel filtration chromatography. In addition, we describe characterization of microscale ADCs using a single chromatographic assay requiring ∼5 μg of material. The resulting platform reduces the required input quantity of antibody required for in vitro ADC screening by at least 5-10 fold. It also enables much higher conjugation throughput with concomitant decrease in time needed to generate and characterize ADCs. To assess the utility of the platform for ranking candidate antibodies, we compare in vitro cytotoxicity results for a panel of ADCs produced by both microscale and research scale methods. We also present three example screens in which antibody libraries of 10-85 members against different targets were conjugated using microscale methods and the resulting ADCs ranked by in vitro potency. For each antibody library, the screen size, conjugation conditions, and target DAR range were adjusted to suit the target biology, antibody type,and payload class. For example, we present evidence suggesting that, for targets in which functional antibody activity is not observed, normalization of DAR to the 2-6 range is adequate for screening. Across the screens, the success rates for producing ADC in quantity and quality suitable for screening were in the 75-90% range, using 200-600 μg of input antibody. Cytotoxic potencies ranging over 2-3 orders of magnitude were observed in the resulting ADC libraries, suggesting that microscale conjugation can rapidly focus ADC discoverycampaigns on high potency molecules. For early stage antibody and ADC screening efforts, we find that microscale conjugation methods yield ADCs that can substitute for traditionally prepared conjugates. We expect these methods will be applicable across many different ADC targets and payloads, and possibly applicable more generally to conjugated macromolecule therapeutic or diagnostic reagents. Citation Format: Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Carl Uli Bialucha, Parmita Saxena, Chen Bai, Kathy L. Miller, Thomas G. Gesner, Mikias Woldegiorgis, Stuart W. Hicks, Megan E. Lewis, Michael S. Fleming, Hans K. Erickson, Seth E. Ettenberg, Thomas A. Keating. Microscale methods for preparation and screening of antibody-drug conjugates. [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 5442. doi:10.1158/1538-7445.AM2015-5442