Lenalidomide and rituximab combination activates anti-tumor immunity and tumor cell death in ex vivo and in vitro FL samples

On 14 February 2019, Hsiling Chiu from Celgene Corporation, Summit, New Jersey, USA, and colleagues, published in the British Journal of Haematology ex vivo data from primary cells of follicular lymphoma (FL) patients and in vitro FL cell lines, following lenalidomide and/or rituximab treatment.

In this study, the authors assessed the proliferating and activation potential of T-cells and Natural Killer (NK) cells from FL patients, healthy donors, and FL cell lines, following lenalidomide and rituximab combination treatment. Furthermore, they compared these results to primary cells of FL patients receiving rituximab-based chemotherapy in the RELEVANCE phase III trial (NCT01650701).

Study design

• Peripheral blood samples were collected from FL patients enrolled in the RELEVANCE phase III trial (n = 193), at screening and at the end of induction therapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP; n = 92) or lenalidomide plus rituximab (R²; n = 101)
• The FL cell lines DOHH2 and RL were also used in this study
• Immune synapse bioassays were performed on cryopreserved lymph node (single-cell suspension samples) from n = 6 treatment-naïve FL patients (Grade 1−3A)
• CD4⁺, CD8⁺, and malignant B cells were isolated from patient samples by magnetic separation
• CD56⁺ NK cells were isolated by magnetic separation from peripheral blood mononuclear cells (PBMC)

Key findings

• When primary CD3-stimualted PBMCs from FL patients were treated with lenalidomide, a significant 1.6−5-fold increase in the following surface markers, occurred: CD56, OX40, IL2Ra, and NKp30 on NK cells, as compared to control (dimethyl sulfoxide [DMSO]-treated cells; P < 0.05; n = 3−4)
• Lenalidomide increased the levels of intracellular granzyme B in FL NK cells by 1.6-fold, as compared to DMSO-treated cells
• Lenalidomide increased the expression of CD56 and OX40 by 1.7−0-fold in healthy donor NK cells, as compared to DMSO (P < 0.005; n = 3−4)
• Lenalidomide treatment led to a 2.3−6.6-fold increase in the absolute number of proliferating CD4⁺ T cells, CD8⁺ T cells and NK cells in PBMC from FL patients (P < 0.05)
• Lenalidomide treatment led to a 2.1−6.1-fold increase in the absolute number of proliferating CD4⁺ T cells, CD8⁺ T cells and NK cells in PBMC from healthy donors, as compared to DMSO-treated cells (P < 0.05)
• However, total CD4⁺ T cell count and CD8⁺ T cell count was unaffected by lenalidomide treatment in both FL and healthy donor samples
• In the FL patient samples from the RELEVANCE trial, flow cytometry revealed that:
  • In patients receiving R-CHOP (n = 92):
    • Median CD4⁺ T cells declined from baseline to week 24
    • Median CD8⁺ T cell counts remained unaltered
    • NK cell counts decreased significantly following therapy (P < 0.05)
  • In patients receiving R² (n = 101):
    • Median CD4⁺ T cells significantly increased from baseline to week 24 (P < 0.05)
    • Median CD8⁺ T cell counts were unaltered (no statistically significant difference)
    • NK cell counts remained unaltered
  • Rituximab, lenalidomide or combination treatment led to a significant increase in F-actin polymerization and increased granzyme B expression at NK cell:tumor cell immune synapses
    • The combination of rituximab and lenalidomide led to the highest increase in NK immunological synapse formation with FL B cells
  • Rituximab and lenalidomide combination led to a superior NK lytic killing activity and increased tumor cell death, when compared to either regimen alone
  • In drug-resistant FL cell lines:
    • Lenalidomide seemed to enhance the effect of rituximab on NK-mediated antibody-dependent cellular toxicity (ADCC) via a cereblon-dependent manner
  • PBMCs from healthy donors were treated with bendamustine, ibrutinib or lenalidomide and then co-cultured with rituximab-treated DOHH2 and RL cell lines:
    • PBMC treatment with bendamustine or ibrutinib led to a significant decrease in cell viability, as compared to DMSO-treated cells (46% and 41%, respectively)
    • PBMC treatment with lenalidomide had no impact on cell viability
    • Following co-culture, lenalidomide and rituximab led to significant increase in ADCC of DOHH2 and RL cells (44% and 45%, respectively), as compared to DMSO-treated cells
    • Following co-culture, rituximab and ibrutinib or bendamustine did not increase ADCC
  • Lenalidomide induced a reversible, non-cytotoxic block in neutrophil maturation, upon drug wash out (in contrast to bendamustine which was cytotoxic to neutrophil progenitor cells)

Conclusions

• Lenalidomide re-activated dysfunctional T and NK cells from FL patient samples ex vivo, by increasing their proliferative capacity
• Lenalidomide and rituximab combination led to an increased ADCC in both sensitive and drug-resistant FL cell lines, via a cereblon-dependent pathway
• Lenalidomide and rituximab combination was superior to single-agent treatment for either of the two drugs, in increasing the formation of lytic NK cell:FL tumor cell immunological synapses
• Immunophenotyping of FL patient samples from the RELEVANCE trial revealed that R2 treatment increased circulating T- and NK-cell numbers, while R-CHOP treatment was associated with a decrease in these cell numbers
• Through in vitro modelling, lenalidomide led to a non-cytotoxic block in neutrophil maturation, which was reversible by lenalidomide wash out