ABSTRACT
Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.
ABSTRACT
Background: Ide-cel, a BCMA directed CAR T-cell therapy, was FDA approved 3/26/2021 for the treatment of RRMM after 4 prior lines of therapy. We evaluated the real-world outcomes of patients treated with standard of care ide-cel under the commercial FDA label. Methods: Ten US academic centers contributed data to this effort independent of the manufacturer. As of 1/10/2022, 138 patients were leukapheresed with overall manufacturing failure in 6 (4%). 108 patients were infused ≥ 30 days prior to data-cut off and constitute the study population for this retrospective analysis. Results: Table describes the study population compared to the pivotal KarMMa-1 trial (Munshi et al, NEJM 2021). Patients in our study were less likely to have ECOG PS of 0/1 (77%) and more likely to be penta-refractory (41%). 67% of patients would not have met eligibility criteria for KarMMa. Common reasons for ineligibility (> 1 reason in 22% patients) were co-morbidities (28%), cytopenias (22%), prior therapy with alloSCT/ CAR-T/other BCMA therapy (19%), CNS myeloma/non-measurable disease/plasma cell leukemia (13%), and fitness (12%). 81% of patients received bridging therapy. Toxicity was comparable to that seen in KarMMa-1. Cytokine release syndrome (CRS) was seen in 82% (> grade 3: 4%) and immune effector cell-associated neurotoxicity syndrome (ICANS) in 15% (> grade 3: 5%) of patients, respectively. Tocilizumab and steroids were used in 72% and 25% of patients, respectively. Infections were seen in 34% of patients. Day 30 response was evaluable in 104 patients. Response rates were: ≥ partial response, 83%;≥ very good partial response, 64%;and ≥ complete response (CR), 34%. 11% of patients have died by data cut-off, 7 due to disease progression and 5 due to other causes (1 grade 5 CRS, 1 hemophagocytic lymphohistiocytosis, 1 progressive neurological weakness, 2 COVID-19). Conclusions: This multicenter retrospective study delineates the real-world outcomes of ide-cel CAR T-cell therapy for RRMM. Despite more patients being penta-refractory and less fit compared to the pivotal KarMMa trial, safety and 30-day responses in the real-world setting (overall response rate: 83%, CR: 34%) are comparable to the clinical trial population. Follow-up is ongoing and updated data will be presented.
ABSTRACT
Background: Galectin-9 (Gal-9) is a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis. Multiple recent reports demonstrate that plasma levels of Gal-9 are elevated in the setting of severe COVID-19 disease. However, a causal role of Gal-9 in SARS-CoV-2 pathology remains to be elucidated. Here, we determined the impact of Gal-9 on SARS-CoV-2 replication and pro-inflammatory signaling in immortalized and primary human airway epithelial cells (AECs). Methods: Dose-dependent cytotoxicity of recombinant human Gal-9 in the Calu-3 AEC line was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 lineage P.1 (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the nucleocapsid (N) gene, immunofluorescence assay (IFA) of N protein, and titration of supernatant (TCID50). Viral entry was measured using luciferase activity of VSV-SARS-CoV-2 S-ΔG-Luciferase reporter pseudovirus. ACE2 and TMPRSS2 cell-surface expression were measured by flow cytometry. Pro-inflammatory factors (IL-6, IL-8, and TNFα) were detected by RT-PCR. Total RNA-seq was used to evaluate Gal-9 effects on the host transcriptome. Groups were compared by Student's t-test, and differential expression analyses were performed using DESeq2. Results: Gal-9 reached 50% cytotoxicity in Calu-3 cells at 597 nM. Gal-9 significantly increased SARS-CoV-2 expression (8.1 to 25.5 fold;p<0.0001) and infectious virus release (1.9 to 17.8 fold;p<0.038) in a dose-dependent manner in Calu-3 cells. Pseudovirus entry into Calu-3 cells was enhanced by Gal-9 (2.4 to 5.6 fold;p<0.0016), and the enhanced entry was inhibited by anti-ACE2 antibody (p<0.0027). Cell surface ACE2 and TMPRSS2 expression were unaffected by Gal-9. Gal-9 treatment accelerated virus-induced expression of IL-6, IL-8, and TNFα (p<0.018) in Calu-3 cells. Gal-9 increased SARS-CoV-2 production (p=0.03) and pro-inflammatory factor expression (p<0.05) in primary AECs (N=5 donors). RNA-seq data revealed that Gal-9 significantly induced IL-17, EIF2, IL-8 and IL-6 signaling pathways in the setting of SARS-CoV-2 infection. Conclusion: Gal-9 facilitates SARS-CoV-2 entry, replication, and virus-induced pro-inflammatory signaling in AECs ex vivo. Our data suggest that pharmacologic manipulation of Gal-9 should be explored as a SARS-CoV-2 therapeutic strategy.
ABSTRACT
Intravenous (IV) ascorbic acid (AA) improves organ function and reduces inflammation in sepsis, an inflammatory state similar to the post-hematopoietic cell transplant (HCT) milieu. This salutary effect is mediated by antioxidant activity as well transcriptional modulation by AA. HCT recipients are deficient in AA, therefore we evaluated the safety and efficacy of patients receiving parenteral AA after myeloablative conditioning for allogeneic HCT compared to similarly treated historical controls who did not receive AA. Methods: Patients with hematologic malignancies, AML (48% of patients), ALL (28%), and CML+MDS (25%) were enrolled in an IRB approved prospective phase 2 clinical trial (NCT03613727). IV AA 50 mg/kg/d divided in 3 doses was given on days 1-14 after HCT, followed by oral AA 500 mg bid from day 15 until 6 months post HCT (FDA-IND 138924). Conditioning regimens utilized included;fludarabine & melphalan (45%), cyclophosphamide with either busulfan (30%) or total body irradiation (25%). GVHD prophylaxis included calcineurin inhibitors and methotrexate or cellcept along with anti-thymocyte globulin (ATG). Primary endpoint was reduction in TRM at 1 year. Propensity score matching was used for matching study patients with similarly treated historical controls, matching for diagnosis, conditioning regimen, and CIBMTR disease risk category for comparison of clinical outcomes. Cox-proportional hazard models were used to estimate adjusted hazard ratios (AHR) between the time-to-event outcomes and study group, adjusted for patient age, donor type, stem cell source, diagnosis, conditioning regimen, and CIBMTR disease risk. Results of an interim analysis following a period of COVID 19 mandated suspension of study accrual are reported. Results: As of March 2020, 40 patients have received IV AA: these include HLA-matched related donor (MRD;n=11), and either 10/10 or 9/10 HLA- matched unrelated donor (MUD;n=22 & 7 respectively) recipients. Graft source was either peripheral blood (n=38) or bone marrow (n=2);88% patients had CIBMTR high risk disease. Median age was 55 years;males (19). All patients enrolled were deficient in AA at day 0, median AA level 0.3 mg/dL (range: 0.1-0.5);post AA infusion level was normal at 1.6 (1.2-5.7) on day 14. Median neutrophil and platelet recovery was by 12 days (range: 9-15 & 8-21 days respectively) with sustained donor engraftment. Median absolute CD3+ cell count at day 30 was 330 cells/microL. With a median follow up of 220 days in AA recipients, no statistically significant difference was observed in transplant related mortality between propensity matched historical controls and study patients (AHR 0.6, 95% CI: 0.2-1.5;p-value = 0.27);univariate survival analysis is depicted in Figure 1. Relapse was also similar (AHR 1.2, 95% CI: 0.3-4.5;p-value = 0.82), and despite a larger number of HLA mismatched unrelated donor recipients, acute GVHD (Grade II-IV) rates were similar in the two groups for both grade II-IV (AHR 0.8, 95% CI: 0.7-1.7;p-value = 0.65) and grade III-IV disease (AHR 0.6, 95% CI: 0.2-1.6;p-value = 0.32). Chronic GVHD rates were also similar (AHR 0.4, 95% CI: 0.1-2.7;p-value = 0.74). There are no attributable grade 3 - 4 toxicities from AA;CMV and EBV reactivation rates were not different in the two groups. Conclusions: In patients undergoing myeloablative allogeneic HCT the administration of IV ascorbic acid is safe and does not negatively impact myeloid engraftment or immune reconstitution. In this interim analysis, transplant related mortality, relapse and GVHD are not increased in IV AA recipients compared to historical controls. Thus, given its safety and tolerability, and possible salutary impact on survival and relapse in these high-risk patients, we posit the feasibility of a randomized phase 3 trial with IV AA in the post-transplant setting to determine its effect on relapse and TRM. [Formula presented] Disclosures: No relevant conflicts of interest to declare.
ABSTRACT
Background/Case Studies: The efficacy of COVID-19 convalescent plasma (CCP) to treat COVID-19 is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS-CoV-2. While high capacity, automated serologic assays to detect binding antibodies (bAb) have been developed, complex nAb assays are not easily adaptable to high-throughput testing. We sought to determine the effectiveness of using surrogate bAb signalto- cutoff ratio (S/CO) in predicting nAb titers using a pseudovirus reporter viral neutralization (RVPN) assay. Study Design/Methods: CCP donor serum collected by 3 large US blood collectors was tested with a bAb assay (Ortho Diagnostic VITROS® Anti-SARS-CoV-2 Total, CoV2T) and a nAb RVPN assay. Although EUA approved as a qualitative assay, CoV2T reports a semi-quantitative S/CO. The RVPN assay uses a pseudovirus construct with native S-protein and target cell lines overexpressing ACE2 receptor and TMPRSS2 protease. Serially diluted serum is mixed with SARS-CoV-2 pseudovirus to assess inhibition of viral entry in culture and reported as titers resulting in 50% neutralization of virus infectivity (NT50) by nonlinear regression analysis. CoV2T prediction effectiveness at several S/CO thresholds was evaluated for various RVPN nAb NT50 titers using receiver operating characteristic analysis. Results/Findings: 753 CCP donations were tested with median CoV2T S/CO of 71.2 (range 0.1-919) and median NT50 of 527.5 (range <40 to >10,240). The prevalence of CCP donors with NT50 over various target n-Ab titers were 86% >80, 76% >160, and 45% >640. Increasing CoV2T reactivity threshold reduces sensitivity to predict the target NT50 titer while specificity to identify those below nAb threshold increases for all targeted NT50s (Table 1). As the targeted NT50 is increased from >80 to >640, the positive predictive value falls dramatically while the negative predictive value increases, thus S/CO thresholds are less able to predict donors who have the target NT50 titer but more able to predict those donors who do not meet it. Conclusions: The selection of targeted nAb titer for clinical use will significantly impact availability of CCP for transfusion. Product release with CoV2T assay S/CO thresholds must balance the risk of releasing products below minimum target nAb titer and the cost of false negatives (CCP units below the threshold with adequate nAb titers). A two-step testing scheme may be optimal, with nAb testing performed on CoV2T reactive samples with S/CO values below the release threshold.