Circumventing Pediatric Solid Tumor Microenvironment Resistance by Combinatorial CAR NK and Immunomodulating Therapy

Abstract

Background & Aim

Patients diagnosed with relapsed/refractory (R/R) Neuroblastoma (NB) and Sarcoma (SARC) have a dismal outcome. Lines of evidence have shown the importance of NK cell immunity against NB and SARC. However, NK cell number and function are low in NB and SARC patients, in large part due to the immunosuppressive tumor microenvironment (TME). Our aim is to improve NK therapy against R/R NB and SARC by strategic combinations that simultaneously enhance tumor specific targeting of NK cells via chimeric antigen receptor (CAR), increase NK migration and tumor infiltration by engineering CAR NK cells to express tumor attracting chemokine receptor CXCR2, improve NK cell ADCC by a GD2 antibody dinutuximab, enhance NK cell persistence and the immune synapse between cancer and NK cells via a tri-specific killer engager Cam1615B7H3, and increase expression of NK activating receptor ligands (MICA/B) on tumor cells by a HDAC inhibitor romidepsin.

Methodology

NK cells were expanded using K562-mbIL-21-41BBL feeder cells and IL-2. Expanded NK cells were engineered to co-express ROR1 CAR and CXCR2 using mRNA electroporation. NK cell cytotoxicity was evaluated by luciferase-based cytotoxicity assay. In vitro transwell assays were performed to determine the migratory ability of CAR NK and CAR CXCR2 NK cells. Flow cytometry was utilized to evaluate MICA/B expression on tumor cells treated with or without romidepsin.

Results

We found that compared to mock NK cells, ROR1 CAR NK cells had significantly enhanced cytotoxicity against NB (SKNFI) and SARC (A673 and U2OS) cells at various T:E ratios (p<0.05). While the in vitro cytotoxicity of CAR NK and CAR CXCR2 NK cells was very similar (Fig.1A), co-expression of CXCR2 on CAR NK cells significantly enhanced their migration towards the conditioned media of tumor cells (p<0.05 and p<0.01) (Fig.1B). Both dinutuximab and cam1615B7H3 significantly enhanced in vitro cytotoxicity of CAR CXCR2 NK cells against tumor cells (p<0.05 and p<0.01) (Fig.1C and 1D). MICA/B expression on U2OS but not A673 or SKNFI cells was markedly increased (33% vs 72%) after romidepsin treatment (Fig.1E). We observed significantly enhanced cytotoxicity of CAR NK cells when combined with romidepsin compared to CAR NK cells alone against U2OS cells (p<0.01) (Fig.1F).

Conclusion

Our results demonstrated the in vitro anti-tumor efficacy of ROR1 CAR CXCR2 NK cells in combination with dinutuximab, cam1615B7H3 and/or romidepsin against malignant NB and SARC. Supported by U54 CA232561 and ALSF reach grant.