June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Maintenance of CNTF secreting vector in genetically engineered ARPE-19 cells over clinical cell scale up
Author Affiliations & Notes
  • Sid Plakkot
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Matthew Fera
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Arne Nystuen
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Crystal Cortellessa
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Sandy Sherman
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Konrad Kauper
    Neurotech Pharmaceuticals, Cumberland, Rhode Island, United States
  • Footnotes
    Commercial Relationships   Sid Plakkot Neurotech Pharmaceuticals, Code E (Employment); Matthew Fera Neurotech Pharmaceuticals, Code E (Employment); Arne Nystuen Neurotech Pharmaceuticals, Code E (Employment); Crystal Cortellessa Neurotech Pharmaceuticals, Code E (Employment); Sandy Sherman Neurotech Pharmaceuticals, Code E (Employment); Konrad Kauper Neurotech Pharmaceuticals, Code E (Employment)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3662. doi:
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      Sid Plakkot, Matthew Fera, Arne Nystuen, Crystal Cortellessa, Sandy Sherman, Konrad Kauper; Maintenance of CNTF secreting vector in genetically engineered ARPE-19 cells over clinical cell scale up. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3662.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Neurotech’s proprietary drug product utilizes a genetically engineered cell line called NTC-201.6A designed to deliver ciliary neurotrophic factor (CNTF) to the retina to slow the progression of photoreceptor degradation in Macular Telangiectasia (MacTel) patients. NTC-201.6A was created by transfecting retinal pigment epithelial parental (ARPE-19) cells with a unique CNTF expression vector. The expression of human CNTF undergoes glycosylation and is secreted out of the cell via the Golgi apparatus (GA). Despite having characteristics of a clone in terms of stability, NTC-201.6A was never experimentally cloned which may allow for non-transfected cells in the cell line. Therefore, an evaluation of the maintenance of the expression of the CNTF secreting vector by determining percent of transfected cells over the clinical cell line scale is necessary.

Methods : To determine the percent transfection of the NTC-201.6A cell line, an immunofluorescence method was developed that leverages differences in subcellular localization of CNTF with Golgin97 between exogenous CNTF in transfected cells and endogenous CNTF in non-transfected cells. Golgin97 is a peripheral membrane protein unique to the GA. Colocalization of CNTF to the GA will only occur in a transfected cell. A double labelled immunofluorescence method using antibodies against CNTF and Golgin97 combined with a Neurotech developed machine learning cell classification algorithm was developed to objectively distinguish between colocalized, transfected NTC-201.6A cells and non-colocalized, non-transfected parental cells.

Results : The Master (MCB), Working (WCB), and End of Production (EOP) cell banks were evaluated for the NTC-201.6A cell line. On average, the percent of transfected cells in the MCB, WCB, and EOP was 85.25%, 88.54%, and 85.84% respectively and were statistically equivalent (ANOVA, p = 0.6752, ns). The parental cell line was evaluated for percent of transfected cells and had 0% percent transfection. The cell classification algorithm could reliably evaluate varying levels of transfected cells.

Conclusions : Using a novel machine learning based cell counting and classification algorithm, the percentage of transfected cells in Neurotech’s engineered cell line over cell scale up was evaluated and remained steady. These methods can be used as a platform to monitor the maintenance of the vector expression in future cell lines.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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