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Background: Patients with relapsed/refractory follicular lymphoma (R/R FL) often experience multiple relapses and require various lines of therapy. The ELARA and ZUMA-5 trials demonstrated high response rates along with acceptable safety profiles. We perform a phase 1b/2 single-center clinical trial of autologous point-of-care (POC) academic anti-CD19 chimeric antigen receptor (CAR) T-cells for patients with R/R FL treated with at least 2 lines of systemic therapy (NCT02772198). Aims: To report outcomes of POC CAR T-cell therapy in patients with R/R FL. Methods: Adults with R/R FL underwent a single leukapheresis procedure. Fresh peripheral blood mononuclear cells were isolated, activated, and transduced with a gammaretrovirus encoding for a CD19 CAR (based on an FMC63-derived ScFv, a CD28 costimulatory domain, and a CD3-ζ signaling domain). Lymphodepletion included fludarabine 25 mg/m2 over 3 days (days-4 to-2) and cyclophosphamide 900 mg/m2 once (day-2), followed by infusion of 1×106/kg CAR T-cells in the inpatient setting. Primary endpoints were response (by PET-CT, per Lugano criteria) at day 28, best response, and safety. Secondary endpoints included overall survival, progression-free survival (PFS), and production feasibility. Last follow-up was as of 02/2022. Results: All 19 patients enrolled received CAR T-cell infusion in a median of 11 days (IQR 10-11) after leukapheresis. The median age was 61 years (IQR 52-66). Five (26%) patients had Karnofsky performance status < 90%. Disease stage at enrollment was III-IV in 16 (84%) patients. Two (11%) patients had bulky disease;8 (42%) had LDH > upper limit of normal;and 16 (84%) had Follicular Lymphoma International Prognostic Index ≥ 3. Disease status at enrollment was progressive disease (n=14, 74%), stable disease (n=3, 16%), or partial response (PR;n=2, 11%). Twelve patients (64%) were refractory to last treatment. Disease grade at most recent lymph node biopsy was 1 (n=3, 16%), 2 (n=11, 58%), or 3a (n=5, 26%). The median time from FL diagnosis was 3.9 years (IQR 2.5-4.6). Sixteen (84%) patients had progression of disease within 24 months of initial therapy. The number of prior therapies was ≥ 4 in 6 (32%) patients;and 5 (26%) patients underwent prior autologous transplantation. Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 1 (5%) and 4 (21%) patients, respectively. One patient was infected with COVID-19 on the 5th day following cell infusion and was admitted to the intensive care unit. One patient had grade 3 atrial fibrillation. Severe neutropenia (absolute neutrophil count <500/μL), thrombocytopenia (platelets <50K/μL) and anemia (hemoglobin <10g/dl) occurred in 15 (79%), 5 (26%), and 7 (37%) patients, respectively. No bleeding events or death were recorded following cell infusion. Response was evaluated in all patients. Overall response rate on day 28 was 84% (79% complete response [CR]). One patient with PR on day 28 achieved a CR after a year of follow-up. Three patients (16%) continued to progress following CAR infusion. All patients were alive at the last follow-up (median follow-up, 11.5 months [IQR 4-21]). One-year PFS was 74% (95% CI, 53-100). The median duration of response (DOR) was not reached (95% CI, 12.5-not reached). Estimated DOR at 1-year was 89% (95% CI, 71-100). Image: Summary/Conclusion: Point-of-Care anti-CD19 CAR T-cell therapy, performed following a very short production time, induced high CR rate with an acceptable safety profile in a cohort of patients with high-risk R/R FL.
ABSTRACT
This second edition contains 24 new and updated chapters on aetiology, epidemiology, prevalence, pathogenesis, clinical signs, treatment, prevention and control of infectious diseases in cats, dogs and exotic small companion mammals in animal shelters. These include an introduction to infectious disease management in animal shelters, wellness, data surveillance, diagnostic testing, necropsy techniques, outbreak management, pharmacology, sanitation, canine and feline vaccinations and immunology, canine infectious respiratory disease, canine distemper virus, canine influenza, feline infectious respiratory disease, canine parvovirus and other canine enteropathogens, feline panleukopenia, feline coronavirus and feline infectious peritonitis, internal parasites, heartworm disease, external parasites, dermatophytoses, zoonoses, rabies, feline leukaemia and feline immunodeficiency viruses and conditions in exotic companion mammals (ferrets, rabbits, guineapigs and rodents). It is intended for shelter veterinarians, managers and workers.
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.