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Link to original content: http://pubmed.ncbi.nlm.nih.gov/33809981/
Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor - PubMed Skip to main page content
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. 2021 Mar 22;13(6):1443.
doi: 10.3390/cancers13061443.

Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor

Leonie D H Gossel  1 , Catrin Heim  1 , Lisa-Marie Pfeffermann  2 , Laura M Moser  1   3   4   5 , Halvard B Bönig  2   6 , Thomas E Klingebiel  3   4   5   7 , Peter Bader  1   3   4   5 , Winfried S Wels  3   4   5   8 , Michael Merker  1   3   4   5 , Eva Rettinger  1   3   4   5
Affiliations

Retargeting of NK-92 Cells against High-Risk Rhabdomyosarcomas by Means of an ERBB2 (HER2/Neu)-Specific Chimeric Antigen Receptor

Leonie D H Gossel et al. Cancers (Basel). .

Abstract

The dismal prognosis of pediatric and young adult patients with high-risk rhabdomyosarcoma (RMS) underscores the need for novel treatment options for this patient group. In previous studies, the tumor-associated surface antigen ERBB2 (HER2/neu) was identified as targetable in high-risk RMS. As a proof of concept, in this study, a novel treatment approach against RMS tumors using a genetically modified natural killer (NK)-92 cell line (NK-92/5.28.z) as an off-the-shelf ERBB2-chimeric antigen receptor (CAR)-engineered cell product was preclinically explored. In cytotoxicity assays, NK-92/5.28.z cells specifically recognized and efficiently eliminated RMS cell suspensions, tumor cell monolayers, and 3D tumor spheroids via the ERBB2-CAR even at effector-to-target ratios as low as 1:1. In contrast to unmodified parental NK-92 cells, which failed to lyse RMS cells, NK-92/5.28.z cells proliferated and became further activated through contact with ERBB2-positive tumor cells. Furthermore, high amounts of effector molecules, such as proinflammatory and antitumoral cytokines, were found in cocultures of NK-92/5.28.z cells with tumor cells. Taken together, our data suggest the enormous potential of this approach for improving the immunotherapy of treatment-resistant tumors, revealing the dual role of NK-92/5.28.z cells as CAR-targeted killers and modulators of endogenous adaptive immunity even in the inhibitory tumor microenvironment of high-risk RMS.

Keywords: CAR; ERBB2; HER2/neu; NK-92; RMS; cancer immunotherapy.

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Conflict of interest statement

W.S.W. is named an inventor on patents and patent applications in the field of cancer immunotherapy owned by Georg-Speyer-Haus. The authors declare no other potential conflicts of interest.

Figures

Figure 1
Figure 1
Chimeric antigen receptor (CAR) expressed by NK-92/5.28.z cells. The coding regions of the lentiviral CAR vector and the CAR molecule with its protein domains are schematically shown.
Figure 2
Figure 2
ERBB2 expression on alveolar rhabdomyosarcoma (aRMS) cells. RH30, RH41, and (A) primary tumor cell suspensions showed different variability in size (forward (FSC) vs. side scatter (SSC)) and displayed low but homogenous ERBB2 expression compared to isotype controls. The MDA-MB-453 and MDA-MB-468 breast cancer cell lines served as positive and negative controls, respectively (B,D). Compared to tumor cell suspensions stable ERBB2 expression was detectable on tumor spheroids during tumor growth (C,E).
Figure 3
Figure 3
Specific cytotoxicity of NK-92/5.28.z cells against aRMS cell lines growing in suspension or as tumor cell monolayers. NK-92/5.28.z cells were compared with unmodified parental NK-92 cells in a 3-h cytotoxicity assay against cell suspensions of (A) RH30 cells, (B) RH41 cells, (E) primary aRMS cells, (C) MDA-MB-453 cells (which served as a positive control), and (D) MDA-MB-468 cells (which were used as a negative control). The effector-to-target (E:T) ratios ranged from 40:1 to 5:1 or from 40:1 to 1:1 (in primary RMS cells). Tumor cell lysis was ERBB2-specific and significantly increased when NK-92/5.28.z cells were used, even at low E:T ratios. In a 16-h cytotoxicity assay, the killing capacity of NK-92/5.28.z cells against monolayers of (F) RH30, (G) RH41, (H) MDA-MB-453, and (I) MDA-MB-468 tumor cells was assessed in comparison to that of parental NK-92 cells by a Celigo cell cytometer. ARMS monolayers were lysed to a significantly greater extent by NK-92/5.28.z cells than by unmodified parental NK-92 cells. Differences were considered significant for p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), and p < 0.0001 (****), or not significant (ns).
Figure 4
Figure 4
Time-lapse microscopy analysis of NK-92/5.28.z cell activity over 18 h. Images of established RH30 cell monolayers were recorded during coincubation with NK-92/5.28.z (A) or parental NK-92 (B) cells (n = 1). NK-92/5.28.z cells proliferated and almost completely lysed RH30 cell monolayers, while NK-92 cells decreased in number and were not able to lyse RH30 cell monolayers or inhibit tumor growth. The observed effects are shown as exact cell counts (C). Here target and effector cells were discriminated by their different microscopic morphology as well as by their different culture behaviors (see Section 2.5).
Figure 5
Figure 5
Activity of NK-92/5.28.z cells against aRMS tumor spheroids. Tumor spheroids were established from RH30 (A) and RH41 cells (B,C). NK-92/5.28.z or NK-92 cells were added as effector cells at an E:T ratio below 1:1, and their growing and antitumoral activity were followed over 10 days. NK-92/5.28.z cells exhibited fast and efficient lysis of aRMS tumor spheroids within two (A,B) to five days (C, fewer effector cells), while parental NK-92 cells were not able to efficiently lyse the tumor cells in 3D culture. Growing behaviors of target and effector cells (D) alone as well as in cocultures are shown in parallel, which allows for discrimination between tumor and effector cells.
Figure 6
Figure 6
Secretion of immunoregulatory factors by activated NK-92/5.28.z cells. The supernatants of cocultures of RH30 (A) and RH41 (B) cells with NK-92/5.28.z and NK-92 cells at E:T ratios of 20:1 (absolute cell count per well: 1 × 105 effector cells and 5 × 103 target cells) were assessed for effector molecules and immunoregulatory factors. Mean concentrations are shown ± SD. The lytic molecules granzyme A and B, perforin and granulysin (orange bars), interleukin (IL)-17A (blue bars), interferon (IFN)-γ, and Fas ligand (FasL) (red bars) were detectable at increased concentrations in NK-92/5.28.z compared with NK-92 cocultures. Cocultures with NK-92 cells showed higher levels of FasL (red bars) and IL-10 (purple bars) than cocultures with NK-92/5.28.z cells. Tumor necrosis factor (TNF)-α, IL-2, IL-4, and IL-6 were not secreted at considerable levels. To assess their degranulation capacity, NK-92 (C,D) or NK-92/5.28.z cells (E,F) were coincubated with RH30 cells (D,F) or remained without target cells (C,E). Cells stained with CD107a and CD56 antibodies represented degranulating immune effector cells. Differences were considered significant for p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), or not significant (ns).
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