Immunotherapy with LAK Cells and Anti-CD20 Monoclonal Antibodies for Follicular Lymphoma: Enhanced Antibody-Dependent Cell Cytotoxicity of LAK Cells in Association with GA101 Rather Than Rituximab
Authors: Panizo Carlos , Inoges Susana , de Cerio Ascensión López Díaz , Muñoz Ricardo García , Pastor Fernando , García Carlos Grande , Olave Mayte , Palomera Luis , Rifon Jose J. , Barea Sara , Alfonso Ana , Bendandi Maurizio 
Published: October 2019

Abstract
Abstract 4881

Background
The current gold standard treatment for follicular lymphoma (FL) consists in the association of rituximab, that is an anti-CD20 monoclonal antibody (mAb), and one among many possible chemotherapy regimens, followed by maintenance with the same mAb. However, LF is still considered incurable. Hence, it is necessary to keep investigating new strategies aiming at improving the overall clinical results.
As an anti-tumor mechanism, antibody-dependent cellular cytotoxicity (ADCC) depends on both the immune system effector arm and the antibody structure. The culture of peripheral blood lymphocytes with IL-2 activates killer cell subpopulations (LAK cells), mainly consisting of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), both with significant cytotoxic capacity. The combination of LAK cells with a mAb could achieve a synergistic effect by enhancing ADCC and potentially achieving clinical efficacy. As structural modifications in mABs may alter their biological activity, new anti-CD20 with structural changes in the Fc portion of the immunoglobulin have been synthesized in order to obtain a greater therapeutic effect. GA101 is a new anti-CD20 mAB that binds with higher affinity to the CD20 type II epitope, increasing the ADCC effect as compared to to rituximab. Currently, no data support the notion of a possible synergistic effect of GA101 and LAK cells.

Aim
The objective of this work was to assess the cytotoxic capacity of LAK cells generated from the peripheral blood of patients with FL. In addition, we aimed at ascertaining whether the combination of rituximab and LAK could improve the functional activity of LAK alone. Finally, we conducted a comparative study of two anti-CD20 antibodies structurally different, namely rituximab and GA101, used in combination with LAK cells.

Material and methods
LAK cells were expanded in vitro from 39 peripheral blood samples of patients with FL. Cytotoxicity studies were performed using chromium release assays in which LAK cells targeted K562 (as NK target), Daudi (as LAK cells target) and CRL-1596 (CD20 positive) cells. Overall, mononuclear cells were isolated and basal cytotoxicity was measured in 39 peripheral blood samples from patients with FL. The ability of two anti-CD20 antibody (rituximab and GA101) to enhance LAK cell activity was evaluated against the CRL-1596 cell line. As a control, an irrelevant antibody (cetuximab) was used. To perform these studies, target cells were incubated with effector cells and antibody at 10 mg/ml concentration.

Results
Results are expressed as mean percentages. Basal cytotoxicity against K562, Daudi and CRL-1596 cells was 19.4%, 11.9% and 1.5% respectively. After culture with IL-2, the cytotoxicity activity against the same cell lines was reassessed and in all cases a statistically significant increase was observed (p<0,05). In particular, cytotoxicity against K562, Daudi and CRL-1596 was 39.5%, 39.6% and 21.1%, respectively.
Furthermore, we studied the rituximab effect on the cytotoxic capacity against CRL-1596. The observed cytotoxicity of LAK cells with rituximab was 39.9% vs 23.6% for LAK cells alone (p<0,001). In parallel, the GA101 effect on the cytotoxic capacity against CRL-1596 was also evaluated. The cytotoxicity of LAK cells with GA101 was 52.3% vs 23.6% for LAK cells alone (p<0,001). Vice versa, no difference was observed in the cytotoxic activity of LAK cells against CRL1596 cell line when the effector cells were incubated with the irrelevant antibody cetuximab (26.0% vs 23.6%).
Finally, when comparing the ability of both anti-CD20 antibodies to enhance ADCC, we observed a significant difference in favor of GA101 (GA101: 52.3%, rituximab: 39.9%; p<0,001).

Conclusions
LAK cells generated from peripheral blood lymphocytes by culture with IL-2 in patients with LF show a higher cytotoxic activity than naive lymphocytes. The observed cytotoxic capacity of these LAK cells against a CD20 positive cell line (CRL-1596) is enhanced by the addition of anti-CD20 mAbs. Interestingly, GA101 was consistently more effective than rituximab in enhancing the cytotoxic capacity of LAK cells.

Disclosures:
No relevant conflicts of interest to declare.