Abstract 2319: Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma
Authors: Lam Bao Quoc , Sugase Takahito , Terai Mizue , Danielson Meggie , Anikeyeva Nadezhda , Wilson Melissa A. , Sykulev Yuri , Sato Takami 
Published: August 2019

Introduction: Metastatic uveal melanoma (MUM) is resistant to currently available treatments and overall prognosis is poor. MUM is an “immune cold” tumor and immune checkpoint blockades have only a marginal effect on this tumor. To bring immune cells to the tumor site and change the tumor microenvironment, we developed Chimeric Antigen Receptor (CAR)-NK cells targeting high molecular weight melanoma associated antigen (HMW-MAA/CPSG4).

Methods: NK92 cell line derived from NK cell lymphoma was obtained from the American Type Culture Collection (ATCC). NK92 cells were transfected with retroviral pBMN-I vector co-expressing Green Fluorescent Protein (GFP) and anti-HMW-MAA-CD28-CD3z CAR construct. The efficacy of CAR construct transfection was confirmed by expression of His-Tag and F(ab)2 by using BD FACS Celesta flow cytometry. MUM cell line, TJU-UM001, was established from a hepatic metastasis of a uveal melanoma patient in our institution. TJU-UM001 cells have approximately 37,000 HMW-MAA molecules on their surface and are sensitive to NK92 cell killing. Tdtomato-transfected TJU-UM001 cells (106 cells) were intra-hepatically implanted into NOD-scid IL2Rgammanull (NSG) mice. Once establishment of hepatic tumor was confirmed by IVIS 200 imaging system (PerkinElmer) at Red Fluorescent Protein (RFP) excitation 535nm, 3 x 106 CAR-NK92 cells or NK92 cells without CAR constructs were injected into the spleen. Accumulations of GFP+ NK92 cells in the TJU-UM001 tumors in the liver were analyzed under fluorescence microscopy. In a separate experiment, 107 TJU-UM001 cells were subcutaneously implanted into NSG mice. The size of subcutaneous tumor was measured by Caliper. Once tumor became palpable (7-10 mm), 3 x 106 CAR- or non-CAR NK92 cells were directly injected to the MUM tumors. Forty thousand units of human IL-2 were intra-peritoneally injected into mouse once daily for 4 consecutive days after NK92 cell injections.

Results: There were expressions of both His-tag and F(ab)2 on CAR-GFP NK92 cells. GFP and CD54 (ICAM-1) were expressed on 95% and 90% of CAR-GFP NK92 cells, respectively. There was no cross-expression of GFP or human CD54 on mouse cells. Therefore, GFP and CD54 were confirmed to be reliable markers for tracking CAR-GFP NK92 cells in mice. Both CAR- and non-CAR NK92 cells exhibited suppression of TJU-UM001 cell proliferation in in vitro assay. Direct injections of both CAR- and non-CAR NK92 cells showed anti-tumor growth effects on subcutaneously implanted TJU-UM001 tumors. More importantly, via splenic injection, CAR-NK92 cells accumulated selectively in the MUM tumors in the liver, while non-CAR NK92 cells were randomly distributed to normal liver tissue and the MUM tumors. These results support the rationale for hepatic arterial infusion of CAR-NK cells as treatment for uveal melanoma patients with hepatic metastasis.

Citation Format: Bao Quoc Lam, Takahito Sugase, Mizue Terai, Meggie Danielson, Nadezhda Anikeyeva, Melissa A. Wilson, Yuri Sykulev, Takami Sato. Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2319.