Abstract P2-20-03: Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model
Authors: Pereira Marcelo , Trikha Prashant , Vasu Sumithira , Li Zihai , Stover Daniel , Lee Dean , Gatti-Mays Margaret 
Published: March 2023

Abstract
Background: Intrinsic resistance to immunotherapy observed in breast cancer is attributed to low neoantigen levels, defective antigen presentation, low mutational burden, reduced programmed death ligand (PD-L1) expression and the presence of immunosuppressive signals like transforming growth factor-beta (TGFβ) in the tumor microenvironment (TME). These collectively attenuate the effector functions of T cells and natural killer cells (NK cells). NK cells are an important component of innate immunity. While generally TGFβ dampens the NK cell response in the breast cancer TME, chronic stimulation in vivo with TGFβ during the expansion process of donor NK cells produces TGFβ imprinted NK cells which exhibit high cytotoxicity and resistance to suppression by TGFβ due to down regulation of SMAD3 and hypersecretion of interferon gamma and tumor necrosis factor alpha. NK cell activation also promotes antibody dependent cellular cytotoxicity (ADCC) and augments monoclonal antibody directed killing. GD2 is a disialoganglioside and a tumor-associated antigen with limited expression in healthy tissues and overexpression in a variety of cancers, including breast cancer. Up to 60% of high grade breast cancers have GD2 expression with higher expression linked to worse overall survival. Given GD2 expression in aggressive BC subtypes and the role of NK cells in ADCC and antigen presentation, there is a strong rationale for evaluating an anti-GD2 antibody along with NK cells in the immunosuppressive breast TME. Based upon established preclinical data, we hypothesized that combining (1) an IgG1 anti-GD2 monoclonal antibody (anti-GD2), (2) TGFβ imprinted, IL-21 expanded allogenic universal donor NK cells (TGFβiNK) and (3) the standard chemotherapy agent gemcitabine (gem) which has immunomodulatory effects including improved NK cell function, will improve tumor control and survival in a TNBC mouse model. Methods: First, we evaluated if anti-GD2 treatment would increase ADCC against MD-MBA-231 tumor cells. To do this, tumor cells with or without anti-GD2 were cocultured with TGFβiNK + anti-GD2 and evaluated by real time cell analysis (RTCA). Next we evaluated the effect of gem on both tumor and immune cells. Finally to evaluate the effect of our novel combination therapy, female NSG mice were injected with 1 × 10e6 MDA-MB-231 cells into the mammary fat on day 0 and tumor burden was followed for 83 days. On day 21, mice were divided into 4 groups: control, gem only (standard of care), TGFβiNK+anti-GD2 (NK-GD2), and gem+TGFβiNK+anti-GD2 (G-NK-GD2) and tumor growth was measured weekly. Mice were observed for toxicity and bioluminescence evaluation for NK cells were performed prior to each cycle. Results: RTCA data confirmed anti-GD2 treatment increased ADCC mediated cell lysis by TGFβiNK. Furthermore, at clinically relevant doses, gem did not alter GD2 expression by flow cytometry on MDA-MB-231 tumor cells but induced MICA expression which suggests potential improved NK cell activity. In the mouse model, monitoring of mouse weights showed greater weight stability in the G-NK-GD2 group, suggesting a tolerable regimen. Serial measurement of tumors showed a significant reduction in tumor growth in NK-GD2 and G-NK-GD2 arms when compared to Gem or control. Additionally, mice who received G-NK-GD2 or NK-GD2 had significantly improved survival. Histological evaluation and immune assessment of the mice are ongoing. Conclusion: Our data showed no significant toxicities in the murine model and improved efficacy of G-NK-GD2 over the standard of care gemcitabine. Our preclinical data supports moving forward with a phase 1b/2 Bench-To-Bedside clinical trial, that will evaluate this combination in patients with aggressive subtypes of metastatic breast cancer where there is a great clinical need for effective treatments.
Citation Format: Marcelo Pereira, Prashant Trikha, Sumithira Vasu, Zihai Li, Daniel Stover, Dean Lee, Margaret Gatti-Mays. Combination Therapy with an Anti-GD2 Antibody, Transforming Growth Factor Beta Imprinted Natural Killer Cells and Gemcitabine Improve Tumor Control and Survival in a Triple Negative Breast Cancer Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-03.