Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection

Abstract

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.

Keywords: Banana Lectin; CAR-NK cells; COVID-19; SARS-CoV-2; glycobiology; immunotherapy.

Figure 1

H84T-BanLec.4-1BB.ζ CAR expression in human NK cells. (A) Schema defining CAR components. (B) Quantification of retroviral vector copy number (VCN) in transduced NK cells (CAR). Untransduced/unmodified (UTD) NK cells served as negative controls. n = 4 NK cell donors. (C) Western blot detection of protein. UTD: untransduced NK cell lysate, CAR: H84T-BanLec CAR-NK cell lysate. Blue arrows: CAR, red arrows: endogenous zeta chain. GAPDH was used as a loading control. (D) Representative histogram showing detection of CAR-expression with flow cytometry. Gray: UTD, Blue: CAR-NK. (E) CAR detection on primary NK cell surface on days 4 and 14 post-transduction. Each dot representative of single transduction. n = 6 total replicates from 4 independent NK cell donors. *p < 0.05.

Figure 2

Recombinant SARS-CoV-2 proteins bind hACE2.293T. (A) Schema of 293T engineered with hACE2. (B) Flow cytometric analysis of 293T expressing hACE2. (C) Detection of recombinant SARS-CoV-2 spike (S)-proteins bound to hACE2-expressing 293T cells. 293T without hACE2 expression used as negative control.

Figure 3

H84T-BanLec CAR-NK cells decrease S-pseudotyped viral infection. (A) Schema of SARS-CoV-2 pseudovirus infection of hACE2.293T. Pseudoviral particles contain plasmids encoding ffLuc. Following viral entry, cells emit bioluminescence (BL) after D-Luciferin metabolism. (B) Measurement of target cell BL emission following transduction with S-protein pseudotyped virus carrying ffLuc reporter gene. Assay performed in triplicate. (C) Schematic representation of the BanLec-CAR NK cells blocking hACE2.293T infection. (D) NK cells plated with target cells (hACE2.293T) and pseudovirus at indicated effector to target (E:T) ratios. Percent bioluminescence (BL) reduction from maximal measured on day 2. The condition with 293T.ACE2 and pseudovirus alone (100%) represents the positive control (E:T = 0.4, p = 0.034; E:T = 1, p = 0.054, n = 6, 2 separate experiments using 3 independent NK cell donors, each experiment performed in triplicate). (E) Quantification (pg/mL) of IFNγ and TNFα present in culture media of NK cells at baseline and in co-culture with S-pseudotyped virus infected hACE2.293T (black: no target, red: co-culture; n=3 donors; Mean value+/- SEM; baseline vs. co-culture *p < 0.05, **p < 0.01, ***p < 0.001, not significant if not indicated).

Figure 4

H84T-Banana Lectin (BanLec) CAR-NK cells are activated by binding high mannose glycosites that decorate the SARS-CoV-2 envelope. This binding can diminish S-protein pseudotyped viral infectivity.