Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine
Inventors
Nemunaitis, John J. • Senzer, Neil • Maples, Phillip B. • Rao, Donald
Assignees
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Publication Number
US-10253331-B2
Publication Date
2019-04-09
Expiration Date
Abstract
Compositions and methods for cancer treatment are disclosed herein. More specifically, the present invention describes an autologous cancer vaccine genetically modified for Furin knockdown and GM-CSF expression. The vaccine described herein attenuates the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs to knock down the expression of furin in cancer cells, and to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.
Core Innovation
The invention relates to a Furin-knockdown and GM-CSF-augmented (FANG) autologous cancer vaccine. It addresses immunosuppression associated with TGF-b2 activity amplification by using furin knockdown to inhibit TGF-b2 effects. The vaccine includes tumor cells transfected with an expression vector plasmid that encodes human GM-CSF cDNA together with a bi-functional short hairpin RNA (bi-shRNA) that hybridizes to a furin mRNA transcript.
The bi-shRNA is described as bi-functional and capable of hybridizing to furin mRNA to inhibit furin mRNA and associated furin activity, thereby reducing TGF-b2 activity. The described rationale includes that furin activation of multiple TGF-b2 isoforms and a TGF-b2 amplification loop contribute to immunosuppression, and that furin knockdown reduces TGF-b21, TGF-b22, and/or related TGF-b23 effects while augmenting GM-CSF.
The document further describes expression vector architecture for the GM-CSF and bi-shRNA components, including a CMV promoter with regulatory elements, and optional use of a picornaviral 2A ribosomal skip peptide linkage. It describes targeting the coding region and/or 3UTR of furin transcripts. Supporting evidence described in the partial content includes observed cytokine changes after plasmid transfection, including reductions in TGF-b21 and TGF-b22 with elevated GM-CSF, with manufacturing/clinical context using GMP-produced patient vaccines and evaluation concepts such as RT-qPCR and ELISA.
Claims Coverage
Independent claim coverage includes manufacturing a bi-shRNA furin/GMCSF cancer vaccine using a two-insert expression vector plasmid and a manufacturing workflow that ends with harvesting and freezing of transfected tumor cells. The inventive features across the independent claim are focused on 3 inventive features.
Tumor cell suspension formation and transfection workflow
Forming a suspension of tumor cells; transfecting the tumor cells with a bi-shRNA furin/GMCSF expression vector plasmid; harvesting the transfected tumor cells; and freezing the transfected tumor cells.
Bi-shRNA furin/GMCSF expression vector with dual inserts
A bi-shRNA furin/GMCSF expression vector plasmid comprising a first insert encoding a human Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA and a second insert encoding a bi-functional short hairpin RNA (bi-shRNA) capable of hybridizing to a furin mRNA transcript.
The independent claim centers on a manufacturing method in which tumor cells are suspended, transfected with a plasmid carrying human GM-CSF cDNA and a bi-functional furin bi-shRNA that hybridizes to furin mRNA, followed by harvesting and freezing of the transfected tumor cells.
Stated Advantages
Reduces immunosuppression associated with TGF-b2 activity by inhibiting furin activity via furin mRNA knockdown.
Reduces TGF-b21 and TGF-b22 while elevating GM-CSF, as reported after plasmid transfection.
Documented Applications
Autologous cancer vaccination using GMP-produced patient vaccines, with described clinical outcome statements including overall survival comparison and a PET-CT response example, including contexts such as NSCLC and melanoma.
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