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821 Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Lymphoma: Pre-Clinical – Chemotherapy and Biologic Agents
Program: Oral and Poster Abstracts
Type: Oral
Session: 625. Lymphoma: Pre-Clinical – Chemotherapy and Biologic Agents: Immune Modulation and Microenvironment in Lymphoma
Monday, December 7, 2015: 5:30 PM
Hall E2, Level 2 (Orange County Convention Center)

Patrick Hagner, PhD1*, Hsiling Chiu1*, Maria Ortiz-Estevez2*, Tsvetan Biyukov3, Carrie Brachman4*, Marek Trneny5, Luca Arcaini6,7*, Franck Morschhauser, MD8*, Antoine Thyss9, Stephan Stilgenbauer10, Noel Milpied11, Reinhard Marks12, Pellegrino Musto13, Giovanni Martinelli, MD, PhD14, Carla Heise4, Thomas Daniel15*, Rajesh Chopra1, James Carmichael15*, Matthew Trotter2*, Anita K. Gandhi, PhD1* and Anjan Thakurta1*

1Celgene Corporation, Summit, NJ
2Celgene Corporation, Sevilla, Spain
3Celgene Corporation, Boudry, Switzerland
4Celgene Corporation, San Francisco, CA
5Department of Hematology, Charles University Hospital, Prague, Czech Republic
6Department of Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
7Department of Molecular Medicine, University of Pavia, Pavia, Italy
8Hematology Department, Centre Hospitalier Régional Universitaire de Lille, Lille, France
9Department of Medical Oncology, Centre Antoine Lacassagne, Nice, France
10Department of Internal Medicine III, Ulm University, Ulm, Germany
11Service d’Hematologie Clinique et Therapie Cellulaire, CHU Haut-Lévêque, Bordeaux, France
12Department of Hematology and Oncology, University Medical Center Freiburg, Freiburg, Germany
13Scientific Direction, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture (Pz), Italy
14Bologna University School of Medicine, Bologna, Italy
15Celgene Corporation, San Diego, CA

Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170).  The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models.

Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay.

Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm.  Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit.

The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours.  Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells.  Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014).

Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro‑apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B.

Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL.

Disclosures: Hagner: Celgene: Employment , Equity Ownership . Chiu: Celgene: Employment , Equity Ownership . Ortiz-Estevez: Celgene: Employment , Equity Ownership . Biyukov: Celgene: Employment , Equity Ownership . Brachman: Celgene: Employment , Equity Ownership . Trneny: Celgene: Consultancy , Honoraria , Other: Travel, accommodations, expenses , Research Funding . Morschhauser: Genentech Inc./Roche: Other: Advisory boards . Stilgenbauer: AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy , Honoraria , Research Funding . Milpied: Celgene: Honoraria , Research Funding . Musto: Sandoz: Consultancy ; Celgene: Honoraria ; Roche: Honoraria ; Sanofi: Consultancy ; Genzyme: Consultancy ; Novartis: Honoraria ; Janssen: Honoraria ; Mundipharma: Honoraria . Martinelli: AMGEN: Consultancy ; Ariad: Consultancy ; Pfizer: Consultancy ; ROCHE: Consultancy ; BMS: Consultancy , Speakers Bureau ; Novartis: Consultancy , Speakers Bureau ; MSD: Consultancy . Heise: Celgene: Employment , Equity Ownership . Daniel: Celgene: Employment , Equity Ownership . Chopra: Celgene: Employment , Equity Ownership . Carmichael: Celgene: Employment , Equity Ownership . Trotter: Celgene Corporation: Employment . Gandhi: Celgene: Employment , Equity Ownership . Thakurta: Celgene Corporation: Employment , Equity Ownership .

*signifies non-member of ASH