Abstract LB-72: An immunoassay detecting Bcl-2:Bim and Mcl-1:Bim complexes in the phase 1 monotherapy study of the Bcl-2 inhibitor, ABT-199/GDC-0199, in chronic lymphocytic leukemia patients
Authors: Li Jessica , Chen Jun , Kadel Ward , Chyla Brenda , McKeegan evelyn , Punnoose Elizabeth , Darbonne Walter 
Published: May 2015

Introduction

Proteins in the Bcl-2 family are key regulators of the apoptotic process. This family comprises of pro- and anti-apoptotic proteins. Overexpression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bim, can inhibit pro-apoptotic proteins’ functions. Bcl-2 and Bim are highly expressed in CLL patients and a Bcl-2:Bim complex is formed at mitochondria in these patients.

ABT-199 (GDC-0199) is a potent, selective, small molecule inhibitor of Bcl-2. Preclinical data shows Bcl-2:Bim disruption can serve as a proximal PD biomarker for this treatment. Conversely, an increase of Mcl-1:Bim complex could indicate an inhibition of Mcl-1's anti-apoptotic function thereby blocking a mechanism of escape from Bcl-2 inhibitor treatment. Here we describe the development of an immunoassay for detecting Bcl-2:Bim and Mcl-1:Bim complexes in CLL patient blood. This assay is used to evaluate these two complexes in patient PBMC samples from the Phase 1 monotherapy study of GDC-0199 in CLL/SLL (M12-175).

Method

Bcl-2:Bim or Mcl-1:Bim complexes from PBMC lysates were captured in solution using anti-Bcl-2 or anti-Mcl-1 antibody, followed by incubation with anti-Bim antibody, which was immobilized on a MSD plate. Bcl-2:Bim or Mcl-1:Bim concentration was calculated using a standard curve. The standard curve was generated using Bcl-2:Bim or Mcl-1:Bim complexes formed from recombinant human Bcl-2 or Mcl-1 protein mixed with recombinant human Bim protein at equal molar concentration. In pre-clinical studies, PBMCs from healthy donor treated with ABT-0199 ex-vivo, or from the SUDHL4 tumor xenograft model was run. In the M12-175 clinical study, PBMCs were collected from patients once prior to GDC-0199 treatment, 6 hour and 24 hours post first dose.

Results

Our data from pre-clinical studies showed a dose dependent decrease of Bcl-2:Bim association in PBMC lysates. In the clinical study M12-175, we had data from 26 patients at 6h timepoint and 27 patients at 24h timepoint. Data was limited to patients with sufficient amounts of total protein (&gt;7 ug) in their PBMC lysates which was the minimum required for this immunoassay. Since post- treatment samples were only taken after the first dose, this limited the evaluation of most patient samples to the starting dose of ABT-199, 20 mg in 3 patients, 50 mg in 23 patients and 200 mg in 1 patient evaluated, with most patients subsequently receiving intra-patient dose escalation to the target doses, the highest tolerable dose tested so far is 600 mg. Strongest decrease in Bcl-2:Bim complex was observed at 200 mg which was the highest dose tested with this assay. Variable decrease in the Bcl-2:Bim complex was observed at the 20 mg and 50 mg doses, range from 1-3 in fold change and 22 % of them with a 2 fold decrease at 24h. A concomitant increase in Mcl-1:Bim was observed in a subset of patients. 14% of them with a 2 fold increase at 24h.

Conclusions

ABT-199 treatment resulted in a decrease in Bcl-2:Bim complexes measured using our MSD immunoassay run on PBMC lysates from patients on the M12-175 monotherapy study in CLL/SLL patients. Measurable decreases were observed in the majority of patients (although to variable extents) even at the lowest doses of 20 mg and 50 mg, with stronger decreases in the patient at 200 mg.

Citation Format: Jessica Li, Jun Chen, Ward Kadel, Brenda Chyla, evelyn McKeegan, Elizabeth Punnoose, Walter Darbonne. An immunoassay detecting Bcl-2:Bim and Mcl-1:Bim complexes in the phase 1 monotherapy study of the Bcl-2 inhibitor, ABT-199/GDC-0199, in chronic lymphocytic leukemia patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-72. doi:10.1158/1538-7445.AM2014-LB-72