Combination of Lenalidomide and Rituximab (R²) Targets Immune Dysfunction in FL By Activating T and NK Cells Ex Vivo and Is Differentiated from Chemotherapy

Introduction: Follicular lymphoma (FL) is an indolent disease characterized by a dysfunctional immune compartment. The standard of care in FL is chemotherapy-based treatment (R-CHOP/R-CVP or R-Bendamustine), with a high overall response rate of >90% but low complete response (CR) rates between 25% to 31% (Flinn 2014). The majority of patients still relapse and often experience immune suppression resulting from chemo-related cytotoxicity that may ultimately impact outcomes. Lenalidomide (Len) has both potent direct antineoplastic and immunomodulatory activity. Len in combination with rituximab (Rtx) has shown enhanced antibody-dependent cellular cytotoxicity (ADCC) and direct apoptosis in B-cell lymphoma in vitro and in animal models (Wu 2008; Hernandez-Ilizaliturri 2005). Multiple phase II studies showed that R² therapy achieves clinical synergy in treatment naive (Fowler 2014; Martin ICML 2017) and relapsed/refractory settings (Leonard 2015; Andorsky ASCO 2017). Here we provide a mechanistic rationale beyond ADCC for the activity observed in the clinical setting using patient samples. We show T and NK cell activation by Len in FL patients ex vivo and demonstrate R²activity in FL cell lines providing clear differentiation from immunochemotherapy.

Methods: Peripheral blood mononuclear cells (PBMCs) from FL patients were treated with Len (1nM-10µM), analyzed for cytokine production by ELISA, T and NK-cell activation on day 3 and proliferation of T and NK-cells on day 5 by flow. Len, idelalisib, bendamustine (1nM-10µM), and ibrutinib (0.1nM-1µM) were each used in combination with Rtx in an ADCC and cell autonomous assay to measure apoptosis by Annexin V/ToPro-3 flow cytometry against FL cell lines DoHH2 and RL. For cell autonomous assay, cells were treated with therapeutic agents for 7 days followed by 24hr Rtx treatment. Synergy of Rtx in combination with each therapeutic agent was calculated using fractional product method (Chou, 1984). For ADCC assays, healthy donor (HD) or FL PBMC were treated with therapeutic agent for 3 days followed by a 4-hr incubation with Rtx-treated CFSE labeled target tumor cells. PBMC viability was measured immediately prior to co-culture by Annexin V/To-Pro.

Results: In ex vivo FL PBMCs, Len significantly enhanced proliferating CD4⁺and CD8⁺ T cells by up to 3 and 7-fold respectively (p<0.001) and proliferating NK and NKT by up to 2 and 5-fold vs DMSO. In FL PBMCs, Len significantly increased the production of TNF-α, IFN-γ and GMCSF up to 4, 6 and 10-fold respectively (p≤0.01) vs DMSO. Len increased IL-2 and RANTES by up to 3-fold vs DMSO. Len activates NK cells by a 2-fold increase in expression of CD56, CD25, NKp30, OX40 and Granzyme B in FL PBMCs vs DMSO. Len significantly enhanced expression of CD154, HLA-DR, CD278 and CD134 on CD8+T cells by up to 2-fold (p≤0.05) vs DMSO. The R² combination was more potent than R-chemo in Rtx mediated ADCC. Combination of Len (1µM) and Rtx (1µg/mL) significantly increased ADCC to 44% (p≤0.01) in DOHH2 and RL cells vs Rtx alone (4% in DoHH2 and 17% in RL) using HD PBMCs. Combination of Len (1µM) and Rtx (10ng/mL) significantly increased ADCC by 41% in DOHH2 and 31% in RL (p≤0.05) vs Rtx alone (12% in DoHH2 and 21% in RL cells) using FL PBMCs. Combination of Rtx with idelalisib, ibrutinib and bendamustine demonstrated no effect or an antagonistic effect on Rtx mediated ADCC against FL cells. In direct apoptosis assay, combination of Len (1µM) and Rtx (10µg/mL) was significantly synergistic and increased apoptosis to 77% (p≤0.0001) vs Len (29%) or Rtx alone (40%). In RL cells, R² was significantly additive and increased apoptosis by 55% (p≤0.001) vs Len (37%) or Rtx alone (34%). No synergy was observed with Rtx in combination with the other chemo agents in ADCC assays. In PBMC viability assays, Len had no effect whilst idelalisib (2µM), ibrutinib (1µM) and bendamustine (10µM) significantly decreased viability by 25, 37 and 46 % respectively (p≤0.01-0.05).

Conclusion: R² is a novel and effective combination immunotherapy in FL. The mechanism of action of this combination results in T and NK cell activation, which leads to enhanced ADCC and addresses the known immune dysfunction in FL. This combination is clearly differentiated from chemo-immunotherapy. The R² combination immunotherapy is currently being assessed in the FL frontline phase 3 RELEVANCE trial (NCT01476787) and in the relapsed FL setting in the AUGMENT trial (NCT01938001).