Overcoming HLA-E-mediated inhibition via KLRC1 knockout to improve NK cell antitumor activity in solid tumors
Posted: April 15, 2024
Written by: Bigitha Bennychen
Edited by: Elizabeth Balint
In their work published in Frontiers in Immunology, PhD student Alice Mac Donald and post-doctoral fellow Dr. Delphine Guipouy from Dr. Elie Haddad's lab showed that knocking down the KLRC1 gene, which encodes the inhibitory receptor NKG2A, improves NK cell anti-tumour activity against HLA-E+ tumours.
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Adoptive cell therapies have shown remarkable success in treating haematological malignancies. The use of NK cells in cancer immunotherapy is promising due to their potential for allogeneic use. However, due to the immunosuppressive nature of the tumour microenvironment (TME), NK cells are limited in their effectiveness against solid tumours. One way solid tumours evade NK-cell anti-tumour activity is through the interaction of the NK inhibitory receptor NKG2A with a non-classical MHC Class I molecule, HLA-E, which is frequently overexpressed on tumour cells. To abrogate HLA-E mediated inhibition of NK cell cytotoxicity, Alice Mac Donald, Dr. Delphine Guipouy, and colleagues established KLRC1- knockout human NK cells (KLRC1KO NK) using a CRISPR-based system.
To generate KLRC1KO NK cells, human NK Cells were freshly isolated from peripheral blood and expanded using the irradiated K562-mb-IL21 feeder cell co-culture system. Next, they used the Feldan Shuttle technology, a peptide-based delivery system that allows safe and efficient delivery of proteins and other therapeutic cargo into cells. Using this technology, they delivered a ribonucleoprotein complex targeting exon 2 of the KLRC1 locus into the expanded NK cells. This led to a significant reduction of NKG2A-expressing NK cells. Cell sorting was performed to further enrich NKG2A-depleted NK cells, and this depleted state was stably maintained over several weeks of expansion.
Next, they investigated the ability of KLRC1KO NK cells to overcome HLA-E-mediated inhibition of NK cell-mediated cytotoxicity by overexpressing HLA-E on solid tumour cell lines that normally exhibit low levels of HLA-E such as breast cancer cell lines, MDA-MB-231 and SK-BR3, lung carcinoma A549, and colorectal adenocarcinoma HT-29. They discovered that KLRC1KO NK cells could overcome the HLA-E-mediated inhibition compared to WT NK cells and demonstrated higher levels of tumour cell lysis. Furthermore, using the Gompertz curve model, they determined that a knockdown of KLRC1 that results in at least 50% of NKG2A deficient NK cells would be enough to restore the NK cell's anti-tumour activity against high HLA-E -expressing tumours.
Given that the activating receptor NKG2C can promote NK cell activity upon recognising HLA-E, they next investigated the effect of NKG2A depletion on NKG2C expression in NK cells. They found that the expression of NKG2C was significantly higher in KLRC1KO NK cells compared to WT NK cells. Additionally, blocking NKG2C using an anti-NKG2C antibody significantly reduced the cytotoxic activity of KLRC1KO NK cells. This suggests that the NKG2C/HLA-E interaction may play an important role in the improved cytotoxicity of KLRC1KO NK cells against HLA-E tumours.
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Finally, the in vivo capacity of KLRC1KO NK cells was tested using human IL-15 transgenic NSG mice that were injected with HLA-E expressing MDA-MB-231 tumor cells. They observed that not only was the tumour growth delayed, but that tumour burden was significantly lower in mice treated with KLRC1KO NK cells compared to WT NK cell-treated mice. Median survival of mice was significantly higher in KLRC1KO treated (59 days) vs WT-treated (48 days).
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Altogether, the findings of this study present a potential solution for effectively using NK cell-based immunotherapy against solid tumours through CRISPR-mediated knockdown of the inhibitory receptor NKG2A. The cytotoxic activity of KLRC1KO NK cells can be further enhanced with the addition of chimeric antigen receptors (CARs) to allow recognition of tumour-specific antigens on the surface of the high HLA-E expressing tumour cells.