Bispecific antibody-activated T cells enhance NK cell-mediated antibody-dependent cellular cytotoxicity

Abstract

Resistance to anti-cancer monoclonal antibody (mAb) therapy remains a clinical challenge. Previous work in our laboratory has shown that T cell help in the form of interleukin-2 maintains long-term NK cell viability and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Lack of such T cell help may be a potential mechanism for resistance to mAb therapy. Here, we evaluate whether concomitant treatment with anti-CD3 × anti-cancer bispecific antibodies (bsAbs) can overcome this resistance by enhancing T cell help, and thereby maintaining long-term NK cell-mediated ADCC. Normal donor peripheral blood mononuclear cells were depleted of T cells, replenished with defined numbers of autologous T cells (from 0.75 to 50%) and co-cultured with mono-/bispecific antibody-treated target tumor cells for up to 7 days. At low T cell concentrations, bsAb-activated T cells (mainly CD4⁺ T cells) were more effective than resting T cells at maintaining NK cell viability and ADCC. Brief (4 h to 2 day) bsAb exposure was sufficient to enhance long-term ADCC by NK cells. These findings raise the hypothesis that local T cell activation mediated by systemic treatment with anti-CD3 X anti-cancer bsAb may enhance the anti-tumor efficacy of monospecific mAbs that mediate their primary therapeutic effect via NK-mediated ADCC.

Keywords: ADCC; Anti-CD20; Bispecific antibody; Blinatumomab; NK cell.

Fig. 1

Blinatumomab enhances RTX-mediated NK cell response. PBMCs depleted of CD3⁺ T cells were cocultured with Raji cells and RTX or trastuzumab (TRA) as the control for 7 days. Serial dilutions (from 0.75 to 50% of PBMCs) of autologous CD3⁺ T cells were added back as was blinatumomab to select samples. NK cell response was measured on day 7. A, B RTX-mediated NK cell elimination of CD19⁺ target cells and NK cell viability increase in a T cell dose-dependent manner. These changes are enhanced by blinatumomab (1 ng/mL or 10 ng/mL) at low T cell percent from 0.75 to 6%. n = 6. C, D Lower concentrations of blinatumomab at 0.1 ng/mL or 0.01 ng/mL minimally impact on RTX-mediated NK cell ADCC or viability. n = 5. Student’s t test was used to calculate statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001 indicate RTX versus RTX + blina 10 ng/mL. blina blinatumomab

Fig. 2

Short-term blinatumomab exposure enhances NK cell ADCC and viability. PBMCs depleted of T cells were cocultured with Raji cells and RTX for 7 days. Serial dilutions (from 0.75 to 50% of PBMCs) of autologous T cells were added back. A Blinatumomab (1 ng/mL) was supplemented for the first 4 h (4 h), 2 days (d1-2), 7 days (d1-7) or not added (0 h). After the indicated time, blinatumomab was washed out and the coculture was refreshed with RTX-containing medium. B, C 2-day blinatumomab enhances RTX-mediated NK cell killing of CD19⁺ target cells and viability. 4-h blinatumomab enhances NK cell ADCC at 12% T cells but fails to increase NK cell viability. n = 6. Student’s t test was used to calculate statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001 are the comparisons between RTX + blina d1-7 versus RTX + blina 0 h; #p < 0.05, ##p < 0.01 indicate RTX + blina d1-2 versus RTX + blina 0 h; × p < 0.05 indicates RTX + blina 4 h versus RTX + blina 0 h. D PBMCs depleted of T cells were cocultured with Raji cells and RTX for 7 days. Recombinant IL-2 (20 ng/mL) was supplemented for 4 h (4 h), 2 days (d1-2), 7 days (d1-7) or not added (0 h). IL-2 was washed out and replaced by RTX-containing medium after the indicated time. E, F 2-day or 4-h IL-2 exposure enhances NK cell ADCC of CD19⁺ target cells. Short-term IL-2 treatment also increases the number of viable NK cells, although not statistically significant. n = 5. One-way ANOVA was used to calculate statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001