CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation.

BACKGROUND: The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. METHODS: Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. RESULTS: Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell-cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. CONCLUSIONS: Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer.

Natural killer (NK) cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies using chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety. In order to further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, preclinical screening assays using patient-derived tumor samples are needed. Zebrafish (Danio rerio) embryos and larvae represent an attractive xenograft model to study growth and dissemination of patient-derived tumor cells because of their superb live cell imaging properties. Injection into the organism's circulation allows investigation of metastasis, cancer cell-to-immune cell-interactions and studies of the tumor cell response to anti-cancer drugs. Here, we established a zebrafish larval xenograft model to test the efficacy of CAR-NK cells against metastatic breast cancer in vivo by injecting metastatic breast cancer cells followed by CAR-NK cell injection into the Duct of Cuvier (DoC). We validated the functionality of the system with two different CAR-NK cell lines specific for PD-L1 and ErbB2 (PD-L1.CAR NK-92 and ErbB2.CAR NK-92 cells) against the PD-L1-expressing MDA-MB-231 and ErbB2-expressing MDA-MB-453 breast cancer cell lines. Injected cancer cells were viable and populated peripheral regions of the larvae, including the caudal hematopoietic tissue (CHT), simulating homing of cancer cells to blood forming sites. CAR-NK cells injected 2.5 hours later migrated to the CHT and rapidly eliminated individual cancer cells throughout the organism. Unmodified NK-92 also demonstrated minor in vivo cytotoxicity. Confocal live-cell imaging demonstrated intravascular migration and real-time interaction of CAR-NK cells with MDA-MB-231 cells, explaining the rapid and effective in vivo cytotoxicity. Thus, our data suggest that zebrafish larvae can be used for rapid and cost-effective in vivo assessment of CAR-NK cell potency and to predict patient response to therapy.

Genome sequences of two clinical Escherichia coli isolates harboring the novel colistin-resistance gene variants mcr-1.26 and mcr-1.27.

BACKGROUND: Colistin is still a widely used antibiotic in veterinary medicine although it is a last-line treatment option for hospitalized patients with infections caused by multidrug-resistant Gram-negative bacteria. Colistin resistance has gained additional importance since the recent emergence of mobile colistin resistance (mcr) genes. In the scope of a study on colistin resistance in clinical Escherichia coli isolates from human patients in Germany we characterized the mcr-1 gene variants. RESULTS: Our PCR-based screening for mcr-carrying E. coli from German patients revealed the presence of mcr-1-like genes in 60 isolates. Subsequent whole-genome sequence-based analyses detected one non-synonymous mutation in the mcr-1 gene for two isolates. The mutations were verified by Sanger sequencing and resulted in amino acid changes Met1Thr (isolate 803-18) and Tyr9Cys (isolate 844-18). Genotyping revealed no relationship between the isolates. The two clinical isolates were assigned to sequence types ST155 (isolate 803-18) and ST69 (isolate 844-18). Both mcr-1 variants were found to be located on IncX4 plasmids of 33 kb size; these plasmids were successfully conjugated into sodium azide resistant E. coli J53 Azir in a broth mating experiment. CONCLUSIONS: Here we present the draft sequences of E. coli isolate 803-18 carrying the novel variant mcr-1.26 and isolate 844-14 carrying the novel variant mcr-1.27. The results highlight the increasing issue of transferable colistin resistance.