Abstract A47: Superior expansion of central memory CD8+ T cells using NKG2D-targeted delivery of IL-2: Implications for adoptive T cell immunotherapy
Authors: Li Kang , Shi Lei , Wang Qing , Onyema Oscar , Guo Yizhan , Krupnick Alexander Sasha 
Published: February 2019

Infusion of activated autologous lymphocytes or CAR-T cells is a promising clinical treatment for both solid and liquid tumors. Traditional methods for ex vivo T cell expansion have relied on transient stimulation of the T cell receptor in the presence of the common γ-chain cytokines. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL2) to support T cell expansion, this cytokine can result in expansion of regulatory T cells (Tregs) and activation induced death of effector cells. Furthermore, prolonged exposure to IL2 may result in differentiation of CD8+ T cells toward an effector phenotype, a terminally differentiated state that provides only short-lived and highly limited protection from malignancy. Alternate gamma chain cytokines, such as IL7 and IL15, do not expand Tregs and may offer an advantage for the expansion of central memory CD8+ T cells, that are superior in mediating tumor regression. Nevertheless, optimal protocols for T cell expansion have yet to be defined. We have recently demonstrated that IL2 targeted to NKG2D-expressing cells using the viral decoy ligand known as orthopox major histocompatibility complex class I-like protein (or OMCP for short), offers a superior method for NK cell expansion and NK mediated immunotherapy (Nat Commun 2016 Sep 21;7. doi: 10.1038/ncomms12878). The possibility of using this redirected cytokine (called OMCP-IL2 for short) for ex vivo CD8+ T cell expansion has not been explored.

To directly compare several common gamma cytokines bulk T cells from C57BL/6 mice were cultured with transient CD3/CD28 stimulation in the presence of wild-type IL2, OMCP-IL2 or combination IL15/IL7 at 100IU/ml. While both IL2 and IL15/IL7 resulted in substantial T cell expansion by day 12 of culture (5.8±0.7 and 3.4±0.16-fold expansion respectively) OMCP-IL2 treated cultures resulted in 10±0.8 fold expansion by the same time point. Furthermore, IL2 and IL15/IL7-treated cultures did not expand further after two weeks of cytokine treatment while OMCP-IL2 treated cultures expanded for 40 days (reaching a 27±0.7-fold expansion). Flow cytometric analysis revealed that the higher CD8+ T cell numbers in OMCP-IL2 treated cultures were due to both increased proliferation (%KI67 positivity in 49±9%, 70±2% and 86±3% of CD8+ T cells for IL2, IL15/IL7 and OMCP-IL2 treated cultures respectively) and improved viability (27±2%, 20±2% and 79±1.8% viable cells for IL2, IL15/IL7 and OMCP-IL2-treated cultures respectively). While wild-type IL2 expanded both CD8+ and CD4+ T cells OMCP-IL2 preferentially expanded CD8+ T cells. Phenotypic analysis revealed that the majority of CD8+ T cells expanded in OMCP-IL2 were CD44hiCD62Lhi central memory T cells while those expanded in IL2 were primarily CD44hiCD62Llow effector cells. Consistent with this a starting population of 1 million T cells resulted in 60,756±21,813; 368,756±30,217; and 7,605,870±872,870 CD8+ central memory T cells after three weeks of culture in IL2, IL15/7 and OMCP-IL2 respectively. Furthermore, both IL2 and IL15/7 expanded cells expressed high levels of surface PD-1 (67.8% and 40.9% respectively) while minimum PD-1 was expressed on OMCP-IL2 expanded cells (18.4%)

In summary, we now demonstrate that the use of an NKG2D-targeted form of IL2 provides a significant quantitative and qualitative advantage for CD8+ T cell expansion in vitro. Such an advantage might be the result of precise delivery of IL2 to cytotoxic T cells or novel signaling pathways induced by engagement of both the NKG2D and IL2 receptor. Since the ability to obtain a significant number of CAR T cells limits the clinical application of this technology the possibility of quickly and efficiently expanding T cells will be key to improving clinical therapy.

Citation Format: Kang Li, Lei Shi, Qing Wang, Oscar Onyema, Yizhan Guo, Alexander Sasha Krupnick. Superior expansion of central memory CD8+ T cells using NKG2D-targeted delivery of IL-2: Implications for adoptive T cell immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A47.