DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study

Abstract

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.

Keywords: NK cells; activating receptor; adoptive transfer of NK and CAR-NK cells; chimeric receptor; immunomodulation; immunotherapy combined therapy.

Figure 1

Schematic diagram of DNAM-1-based chimeric receptors. The amino acids (aa) sequences of DNAM-1 (red), CD3ζ (yellow) and 2B4 (blue) are shown below each diagram of DNAM-1-based chimeric receptors. EC, extracellular; TM, transmembrane; CYP, cytoplasm.

Figure 2

Enhanced surface expression of DNAM-1 receptor on DNAM-1-engineered NK cells. (A) Flow cytometry analysis of DNAM-1 surface expression in NK cells transfected with the indicated DNAM-1-based vectors (red) compared to that of NK cells transfected with empty vector (gray). Isotype-matched negative control antibody is displayed as gray and red dashed lines for NK cells transfected with empty vector and each indicated DNAM-1-based vector, respectively. A representative experiment of eight performed in NK cells isolated from independent healthy donors and transfected with DNAM-1-constructs is shown. (B) Summary of flow cytometry analyses performed in NK cells isolated by eight independent healthy donors and transfected with DNAM-1-based constructs. MFI, mean fluorescence intensity. Mean ± SD; *p<0.05; p value (two-tailed non-parametric Mann-Whitney test). (C) Flow cytometry analysis of NKG2D, NKp46, NKp30, CD96, TIGIT, PD-1 and CD57 surface expression in NK cells transfected with the indicated DNAM-1-based vectors. Isotype-matched negative control antibody is displayed as dashed lines for NK cells transfected with empty vector and each indicated DNAM-1-based vector. A representative experiment of seven performed in NK cells isolated from independent healthy donors and transfected with DNAM-1-based vectors is shown.

Figure 3

Enhanced degranulation and cytokine production of DNAM-1-engineered NK cells against K562. DNAM-1-engineered NK cells were tested for degranulation and cytokine production assays in response to K562. (A) Degranulation of NK cells engineered for the empty vector as control or for the four DNAM-1-based constructs as indicated, in the absence (medium) or presence of neutralizing anti-NKG2D or anti-DNAM-1 antibodies, measured as CD107a expression on co-culture with or without K562. The percentages of CD107a in NK cell subset are indicated in each plot. A representative experiment of four performed in NK cells isolated from independent healthy donors and transfected with DNAM-1-constructs is shown. (B) Summary of degranulation of NK cells isolated from four independent healthy donors, transfected with DNAM-1-constructs, in the absence or presence of neutralizing antibodies. Dots correspond to the percentage of CD107a⁺ NK cells from each healthy donor transfected with the indicated DNAM-1-based constructs; horizontal bars indicate the mean; *p<0.05; p value (two-tailed nonparametric Mann-Whitney test). (C) DNAM-1-engineered NK cells production of IFNγ and TNFα in co-culture with or without K562. The percentages of IFNγ+ and TNFα+ in NK cell subset are indicated in each plot. A representative experiment of four performed in NK cells isolated from independent healthy donors and transfected with DNAM-1-constructs is shown. (D) Summary of cytokine production of NK cells isolated from four independent healthy donors and transfected with DNAM-1-based constructs. Mean ± SD; *p<0.05; p value (two-tailed non-parametric Mann-Whitney test).

Figure 4

Nutlin-3a boosted the susceptibility of NB LA-N-5 and SMS-KCNR cells to DNAM-1-engineered NK cells. NB LA-N-5 and SMS-KCNR cell lines were left untreated (DMSO) or treated with Nutlin-3a at 2 μmol/L for 48 hours and used as targets for degranulation assay of DNAM-1-engineered NK cells. (A) Degranulation of NK cells, measured as CD107a expression upon stimulation with LA-N-5 or SMS-KCNR cells treated with DMSO or Nutlin-3a. The percentages of CD107a⁺ DNAM-1-engineered NK cells are indicated in each plot. A representative experiment of four performed in NK cells isolated from independent healthy donors and transfected with DNAM-1-constructs is shown. (B) Summary of degranulation of NK cells isolated from four independent healthy donors and transfected with DNAM-1-constructs, in response to LA-N-5 or SMS-KCNR cells treated with DMSO (D) or Nutlin-3a (Nut-3a). Dots correspond to the percentage of CD107a⁺ NK cells from each healthy donor transfected with each indicated DNAM-1-based constructs; horizontal bars indicate the mean; *p<0.05; p value (two-tailed non-parametric Mann-Whitney test).