ABT-199 (venetoclax) Synergizes Cytotoxicity of Cytotoxic T-Cell (CTL) to B-Cell Lymphomas

[Introduction] Therapeutic success of immune checkpoint inhibitors, nivolumab and pembrolizumab for several types of B-cell malignancies have shown the significance of tumor immunity for the treatment. Accordingly, the enhancement of tumor immunity has been one of the directions of therapeutic development. Bcl-2 family proteins regulate apoptosis by controlling cytochrome c release from mitochondria. Apoptosis induced in tumor cells by CTL is known to be caused through mitochondrial pathway. It is considered that ABT-199, BH3 mimetic bcl-2 inhibitor synergizes cytotoxicity of cytotoxic T-cell (CTL) to B-cell lymphomas with overexpression of bcl-2 by genetical alterations such as 14:18 translocation. Therefore, we have evaluated thsynergistic effect of ABT-199 to the cytotoxicity of CTL to B-cell lymphoma cell lines.

[Materials & Methods] We investigated in this study synergistic effect of ABT-199 to CTL using cytomegalovirus (CMV) pp65 antigen-specific cytotoxic T-cells (CMV-CTLs) and pp65 antigen-expressing B-cell lymphoma cell lines, VAL, FARAGE, REC1, SUDHL-10, P3HR1 and etc. as target cells. The main reasons why we used CMV-CTLs instead of tumor specific-CTLs is that it is very difficult to prepare enough tumor specific-CTLs for study and in general, the molecular mechanisms involved in killing target cells by the virus-CTLs, including CMVpp65-CTLs, are the same as those used by tumor specific-CTLs. CMV-CTLs were prepared by mixed lymphocyte peptide cultures followed by the CD137-guided CTL isolation method from peripheral blood mononuclear cells (PBMCs) of healthy donors. Pp65 antigen-expressing B-cell lymphoma cell lines (BCL-pp65) were prepared by the infection with Lentiviral vector, pLVSIN-CMV Neo_pp65. Target cells, BCL-pp65 were co-cultured with HLA-type matched CMV-CTL in the presence of serial concentrations of ABT-199 for 4 hours. The co-cultured cells were stained with Annexin V-FITC, anti-CD8-BV511 and anti-CD20-APC, and early apoptotic cells in both the target cells and CTLs were analyzed by flowcytometry.

[Results] Synergistic effect of ABT-199 to CTLs was observed when ABT-199 sensitive BCL-pp65 lines (5μM<IC50) were used as target cells. Representative results using VAP-pp65 as target cell was shown in the Figure. Annexin V positivity of the VAL-pp65 in the CD20 positive gate was increased in the ABT-199 in a dose-dependent manner (31.25 - 500 nM). The annexin V positivity in the each ABT-199 concentration points were 43.8%, 60.4%, 77.1%, 87.6% and 91.2% respectively, while in the presence of CMV-CTLs at the E/T ratio of 0.2, those were increased to 70.4%, 80.2%, 90.3%, 94.4% and 95.5% respectively. In contrast, when VAL-MOCK was used as target cells, the annexin V positivity in each ABT-199 concentration points was not different between in the presence and absence of CMV-CTL. These observations suggest that ABT-199 synergize the antigen specific cytotoxicity of CTL to B-cell lymphomas. However, when B-cell lines in which the IC50 exceed 5 μM were used as the target cells, the synergistic effect was decreased according to IC50 value because ABT-199 induces apoptosis to CTL at the concentration above 5 μM.

[Conclusions] We have directly investigated the synergistic effect of the bcl-2 inhibitor, ABT-199 to cytotoxicity of CTL to B-cell lymphomas in vitro. What our results suggest are as follows.

1) ABT-199 therapeutic effects are elicited not only by the direct cytotoxic effect to B-cell lymphomas but also the enhancement of sensitivity of B-cell lymphomas to CTLs.

2) Developments of the combination therapies of ABT-199 with immunotherapies such as immune-checkpoint inhibitors, antigen vaccinations, adoptive T-cell transfer and etc. are expected.

3) Maximum tolerated concentration of ABT-199 in vivo in the combination therapy with immunotherapies is considered to be 5 μM because ABT-199 induces apoptosis to CTL at the concentration above 5μM.

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