ABSTRACT
Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6;Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6;Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3;Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3;Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK;65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05;78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03;n=6;Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. [Formula presented] Disclosures: Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding;BMS: Research Funding;Kiadis Pharma: Rese rch Funding.
ABSTRACT
Background: For patients of reproductive age with cancer, counseling regarding reproductive risks associated with systemic therapy and facilitating access to fertility services are essential to quality care. We conducted a quality improvement study to improve rates of fertility preservation counseling at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center (SKCCC) in Baltimore, MD. Methods: We formed a multidisciplinary team as part of the ASCO Quality Training Program. We aimed to increase the rate of fertility preservation counseling for patients of reproductive age (18-40 years) with newly diagnosed cancer who were initiating systemic therapy from a baseline (June 2019-January 2020) of 36.7% (25/68) to 70.0% by February, 2021. Data sources included the electronic medical record and direct verification with patients by phone. We surveyed patients, oncologists, and experts in reproductive endocrinology and urology to identify barriers to optimal care. After considering a prioritization matrix, we implemented Plan-DoStudy-Act (PDSA) cycles. Results: We identified the following improvement opportunities: (1) oncologist under confidence about counseling, (2) oncologist lack of knowledge about local fertility preservation options/processes, and (3) lack of a standardized referral mechanism to reproductive endocrinology/urology. The first PDSA cycle was disrupted due to COVID-19;from February 2020-August 2020, we introduced the initiative at oncology disease site meetings (e.g., leukemia). In September 2020, we implemented a second PDSA cycle. Our interventions included (1) presenting the baseline data and fellow-led initiative at Oncology Grand Rounds (attended by 150 staff members), (2) creating and distributing paper and electronic informative pamphlets to oncologists and patients, and (3) implementing an electronic medical record order set. This order set included instructions for providers and patients, necessary laboratory studies, and a referral to reproductive endocrinology or orders for cryopreservation of sperm. It also added the following to a patient's after visit summary: Contact information for a dedicated fertility coordinator, estimated costs of services, and financial assistance programs. Postimplementation (September 2020-February 2021), the percentage of patients who reported receiving fertility preservation counseling increased from 36.7% to 80.9% (38/47). A sustained shift in the process was apparent on a control chart. Conclusions: Despite disruptions caused by the COVID-19 pandemic, we demonstrate how a trainee-led, patientcentered initiative improved fertility care services for people with cancer. Ongoing work focuses on ensuring sustainability of change, assessing the quality of counseling, and evaluating the impact on utilization of fertility care services.