2B8T2M Enhances exPBNK Expression, in Vitro Cytotoxicity in Rituximab-Sensitive/Resistant Burkitt Lymphoma

Yaya Chu, PhD

2B8T2M, a fusion of the IL-15 superantagonist ALT-803, and 4 single-chains of rituximab (Rituxan) significantly boosted the cytotoxicity of expanded peripheral blood natural killer cells (exPBNK) against Burkitt lymphoma cells in in vitro testing, according to preclinical results presented at the 2018 ASPHO Conference.

Lead study author Yaya Chu, PhD, assistant professor of pediatrics, New York Medical College, said that 2B8T2M significantly enhanced exPBNK cytotoxicity against both rituximab-sensitive Raji Burkitt lymphoma cells and rituximab-resistant Raji-2R and Raji-4RH Burkitt lymphoma cells.

“2B8T2M significantly enhanced exPBNK activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and [rituximab]-resistant Burkitt lymphoma cells,” investigators concluded. “The in vivo functions of 2B8T2M with exPBNK against rituximab-sensitive and [rituximab]-resistant Burkitt lymphoma cells using humanized NSG models are under investigation.”

Rituximab is a common first-line treatment for patients with Burkitt lymphoma, but Chu explained that there is a desperate need for novel therapies to treat patients with relapsed/refractory disease. Among the strategies under investigation is engineering immune cells with chimeric antigen receptors (CAR), as well as second-generation CD20-targeted antibodies.

While natural killer (NK) cells, which help drive tumor rejection, have potential for treating non-Hodgkin lymphomas, including Burkitt lymphoma, there are challenges to using NK therapy. The population of active NK cells is small in unmodified peripheral blood, and tumor cells have multiple mechanisms for escaping immunosurveillance.

However, Chu et al worked to develop and grow the population of exPBNK. Results from earlier studies in mice and human tumor models showed that ALT-803 “significantly enhanced” expression of exPBNK activating receptors and the in vitro cytotoxicity of anti-CD20 CAR against CD20-positive rituximab-resistant and rituximab-sensitive pediatric Burkitt lymphoma.

Furthermore, the combination of ALT-803 and rituximab demonstrated enhanced cytotoxicity in primary NK cells, which resulted in improved survival in Raji-xenografted NSG mice.

Chu et al hypothesized that, compared with ALT-803/rituximab, 2B8T2M would augment the antitumor activity of exPBNK against Burkitt lymphoma cells that were resistant and sensitive to rituximab, both in vitro and in vivo.

2B8T2M has a longer circulation half-life in mice than ALT-803 (184 vs 18 hours) and promotes effector cell proliferation and cytotoxicity. Through its recognition of the CD20 molecule on tumor cells, 2B8T2M has demonstrated tri-specific binding activity, activated NK cells to boost antibody-dependent cell-mediated cytotoxicity, and induced apoptosis in B-lymphoma cells.

If validated in clinical trials, 2B8T2M could be a replacement for the combination of ALT-803 and rituximab.

Investigators expanded peripheral blood mononuclear cell (PBMNC) with lethally-irradiated K562-mbIL21-41BBL, producing CAR mRNA that was later processed into exPBNK. They then compared the effect of 2B8T2M in NSG mice against equal doses of rituximab, ALT-803, ALT-803/rituximab, and obinutuzumab (Gazyva), a humanized CD20-targeted monoclonal antibody. Immunoglobulin G (IgG) was used as a control. Investigators targeted rituximab-sensitive Raji and rituximab-resistant Raji-2R and Raji-4RH Burkitt lymphoma cells.

Compared with IgG, rituximab, ALT-803, ALT-803/rituximab, and obinutuzumab, 2B8T2M significantly augmented exPBNK cytotoxicity against rituximab-sensitive Raji cells and rituximab-resistant Raji-2R and Raji-4RH cells (P <.001).

Chu et al found that 2B8T2M boosted expression of the exPBNK activating receptors CD16, NKp30, and NKG2D and enhanced levels of Ki67 and P-Stat-5 compared with ALT-803/rituximab. Compared with obinutuzumab plus ALT-803, 2B8T2M also enhanced the in vivo cytotoxicity of exPBNK against CD20-reduced, rituximab-resistant BL.

Investigators evaluated cytotoxicity by measuring interferon-gamma and granzyme B production. 2B8T2M significantly increased interferon-gamma and granzyme B production from exPBNK compared with rituximab (P <.001), ALT-803 (P <.001), ALT-803/rituximab (P <.001), obinutuzumab (P <.001), and IgG (P <.001), against Raji, Raji-2R, and Raji-4RH cells.

Investigators also determined that the combination of 2B8T2M and exPBNK demonstrated equivalent in vitro cytotoxicity to anti-CD20 CAR exPBNK cells against rituximab-sensitive Raji cells and rituximab-resistant Raji-2R and Raji-4RH cells (P <.05). Furthermore, compared with the combination of ALT-803/rituximab/exPBNK, 2B8T2M/exPBNK reduced tumor burden and extended survival in Raji-2R xenografted NSG mice.

Chu said investigators plan to build on these findings by exploring the antitumor effect of combining 2B8T2M with expanded NK cells using human Burkitt lymphoma xenographs, mechanisms of tumor relapse after treatment, and the combination of 2B8T2M with anti-CD19 or anti-CD22 CAR T-cell therapy and NK.

Chu Y, Su NK, Alter S, et al. Therapeutic effects of a novel fusion of ALT-803, an IL-15 superagonist, with 4 single-chains of anti-CD20 antibody (2B8T2M) in combination with expanded natural killer cells against rituximab sensitive and resistant Burkitt lymphoma (BL). In: Proceedings from the 2018 ASPHO Conference; May 2-5, 2018; Pittsburgh, Pennsylvania. Abstract 1025.

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