Abstract 1716P
Background
SARS-CoV-2 genome reveals a unique furin cleavage site change at S1/S2 junction and furin-like S2’ cleavage site which promotes membrane fusogenic pathway entry and exit to human host cells. Clinical testing of VP, used in an autologous tumor vaccine (Vigil), demonstrates >90% knockdown of TGFβ, a downstream furin protease product, elevation of GMCSF and safety and benefit in solid tumor cancer patients.
Methods
Lyophilized (Lyo) VP blended with GFP was stabilized with w/v Trehalose to ≤5 micron particle size for deep lung entry. Significant GFP expression was demonstrated (Nexcelom Cellometer) and restriction enzyme mapping confirmed molecular structure. GMCSF and TGFβ expression (Protein Simple ELLA cytokine production) in CCL247 and RDES cell lines validated for performance of FDA defined cancer clinical product release assays was done. Function was determined for both electroporation (BioRad) and lipid-based reagent [Lipofectamine (Lipo) 3000].
Results
Transfection efficiency and cytokine expression of non Lyo and Lyo VP with and without electroporation (Zap) are shown below: Table: 1716P
| Cell Line | CCL247 | RDES | ||||
| Time Point | Day 2 | Day 3 | ||||
| Treatment Condition | Zap + Frozen DNA | Lipo + Frozen DNA | Lipo + Lyo DNA (5% Tre) | Zap + Frozen DNA | Lipo + Frozen DNA | Lipo + Lyo DNA (5% Tre) |
| GFP (Transfection efficiency) | 65% | 50% | 70% | 69% | 50% | 52% |
| GMCSF (pg/mL) | 1.85 e6 | 3.01 e6 | 3.82 e6 | 3.95 e6 | 4.84 e6 | 5.08 e6 |
| TGFβ1 (% Knockdown) | 54% | 23% | 35% | 59% | 30% | 41% |
Conclusions
Lyophilization of VP is feasible for pulmonary aerosol delivery. Inhaler delivery provides direct access of VP to the primary infectious site of SARS-CoV-2, establishes a novel opportunity for lung active uptake by alveolar cells as well as a home use therapy. Favorable results pending virus plaque assay and in vivo studies will inform initiation of large animal safety concurrent with phase I testing (FDA Tech Watch Committee March 31, 2020).
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Gradalis, Inc.
Funding
Has not received any funding.
Disclosure
C. Jay, F. Kerneis, L. Manning, G. Wallraven, E. Bognar: Shareholder/Stockholder/Stock options: Gradalis, Inc. J. Nemunaitis, D. Shanahan: Leadership role, Shareholder/Stockholder/Stock options, Officer/Board of Directors: Gradalis, Inc. Y. Shen, N. Senzer: Advisory/Consultancy: Gradalis, Inc. All other authors have declared no conflicts of interest.