Activation of T- and NK- Cells through CD19xCD3 and CD19xCD16A Bispecific TandAb Antibodies in Patients with B-Cell Non-Hodgkin's Lymphoma.

Bispecific, tetravalent.antibodies (TandAbs) directed against the B-cell-surface marker CD19 and activating receptors on T or NK cells (CD19xCD3 or CD19xCD16b) have shown promising effects in vitro and in preclinical studies. The present study examines their effects on the cytotoxic responses of viable T and NK cells isolated from patients with B-cell Non Hodgkin Lymphoma (NHL).

Peripheral Blood MNCs were obtained from 30 patients with B-NHL (High grade=6, low grade=24) after successful prior therapy and from 17 healthy donors (HD) and were enriched for NK cells by immunomagnetic separation with CD56-microbeads, the other one was enriched for T cells. After overnight stimulation (RPMI-1640 with 10% FCS, 1% Pen/Strep and human recombinant IL-2 (Novartis) (NK: 1000U/ml; T: 100U/ml) cells were stained with CD107a, a surface marker indicating cytotoxicity. Cells were exposed to K562 alone, to Raji cells alone and finally to the TandAbs CD19xCD3 (T cells) or CD19xCD16A (NK cells) (conc. 1μg/ml). Using FACS analysis we determined the percent fraction of activated CD3 or CD56 cells being positive for CD107a.

To look for functional NK cells and T cells of HD and NHL patients first we exposed the cells to the K562 cell line. Both groups, HD and NHL patients expressed CD107a similarly (NK: 27.7±12.0% vs. 28.5±14.6%; T: 9.75±7.1% vs. 8.1±5.7%), indicating functional NK and T cells. To observe if NK or T cells would upregulate CD107a expression when exposed to lymphoma cells alone we performed experiments with Raji cells and measured the CD107a response. NK cells of both, patients and HD responded with similar levels of CD107a expression. T-cells of patients showed more cytotoxic activity towards Raji cells than observed in HD (3.1±8.4% vs. 0.1±0.2%). Next we added the CD19xCD3 or the CD19xCD16 TandAb to T/NK cells in the presence of Raji cells. In the patients′ NK-cell-population the fraction of activated cells increased from 10.7% (Raji cells alone) to 30.7±19.2% when TandAb was added. The mean reaction in the group of HD was increased from 9.2% up to 27.7%. CD107a expression of T cells from NHL patients increased from 3.1% when exposed to Raji alone up to 21.3±14.3% when TandAb was added which was comparable to the results of HD (19.6%±11.4%).

Patients with NHL have functional intact effector cells which show cytotoxic activity in response to TandAbs in presence of lymphoma cells similar to that of HD. These TandAb antibodies are therefore suitable for patients with B-cell NHL as potential maintenance therapy.

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