BRIC: Packaging cell lines for inherently manufacturable viral vectors

Lead Research Organisation: King's College London
Department Name: Unlisted

Abstract

Viruses dominate overwhelmingly the types of vectors currently being tested in clinical gene therapy trials and of these retro- and lentiviruses are the most numerous. Until recently two technological problems have hampered progress in gene therapy; production of high titre clinical grade virus and efficient tissue specific targeting. Research at Cambridge and King's College London has addressed the former and led to the development of a novel lentiviral vector packaging cell line in which manufacturability is built into the genome of the packaging cell and co-expressed on the surface of the viruses produced thereafter. We initially used simple retroviral vectors, and latterly the more complex lentiviral vectors based on a core of HIV-1, and have developed strategies for increasing the titre by several orders of magnitude. This is an active area of research amongst which our preliminary work with novel chromatographic techniques and paramagnetic particles set the foundation for a practical and efficient alternative technique to cumbersome ultracentrifugal concentration. For lentiviral vectors we engineered a new producer cell type that provides a biotin tag amenable to various lentiviral vectors produced from these cells using either VSV-G or MLV amphotropic envelopes. We have shown that these bio-lentiviral vectors are produced in the normal manner and only require the presence of biotin in the culture medium to manifest their affinity for streptavidin. Vectors can thus be retained on streptavidin Paramagnetic Magnespheres for infection, or eluted from streptavidin adsorbents. This cell line allows the capture of multiple envelope pseudotypes of lentiviral or MLV derived vectors, enabling production and concentration to titres that are several orders of magnitude higher. Using this scalable protocol we have concentrated lentivirus in excess of 4500-fold in only 3 h and have provided titers for both VSV-G and MLV amphotropic envelope pseudotypes of 1010 IU/ml. However, these viruses could not be easily eluted from adsorbents and required the addition of biotin to the growth medium of the packaging cells. This proposal aims to express the alternative desthiobiotin ligand on the surface of lentiviruses in such a way that elution from adsorbents may be more readily preformed to give higher process yields and the addition of an affinity ligand binding precursor to growth medium is avoided.

Technical Summary

It addresses Improved Downstream Processing for nanoscale vectors, transferring in-built manufacturability from laboratory to GMP practice. It provides virus manufacturers with tools to engineer viral envelopes in a way that uncertainties in downstream processing are avoided whilst overcoming problems of low titre that have hampered the field. These tools will bring increased predictability for bioprocessing, including improved scale-up and reproducibility and so enable increased speed to clinic and market.

Publications


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Chen R (2010) Affinity recovery of lentivirus by diaminopelargonic acid mediated desthiobiotin labelling. in Journal of chromatography. B, Analytical technologies in the biomedical and life sciences






 
Description Development of a cell line (BL15) in which introduction of a series of bacterial genes has enabled the conversion of a precursor of biotin (DAPA), provided by addition to the culture media, into biotin which is then displayed on the surface of the cell, thus allowing the biotinylation of lentivirus vector produced from these cells. These cells are now used for the generation of virus that can be readily captured, purified and concentrated on streptavidin paramagnetic beads. This development has results in attraction of several million of industrial contracts from the pharmaceutical industry and a large number of subsequent grants form research councils, NIHR and the Leukaemia Lymphoma Research (now Blood Wise)
Exploitation Route Novel manufacturing activities for the production of viral vectors for cell and gene therapy applications are now being developed in collaboration with other academic centres, supported by research grants, and in collaboration with Industry (Cell Therapy Catapult, Cellectis, Autolus and Pfizer).
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description This project, in combination with BB/D014301/1, has allowed us to develop novel procedure for attachment of biomarkers to viral vectors, and to establish GLP and GMP compliant procedures for the conduct of immunological assays on patient samples and for the manufacture of vectors that are used in clinical trials. The IP, techniques and facilities developed have directly contributed to the attraction of substantial grant funding and3 major industrial collaborations: 1) Roche Pharmaceuticals for the development and assessment of clinical potential of glycoengineered antibodies - value of contract research to KCL £2,400,000 between 2009 and 2013. 2) Northwest Biotech (USA) for the production of dendritic cell vaccine (DCVax) used in a sponsored Phase-III clinical trial - Value £2,600,000 between 2012 and 2016. 3) Cellectis (France) for the development of lentivirus vectors for the manufacture of Chimeric Antigen Receptor T cells (CAR-T cells) - Value £10,100,000 between 2014 and 2019.
First Year Of Impact 2009
Sector Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description An immunisation strategy to prevent hepatocellular carcinoma
Amount $1,200,000 (SGD)
Organisation National Medical Research Council NMRC 
Sector Public
Country Singapore, Republic of
Start 08/2012 
End 07/2015
 
Description Core Suppport for GMP facility
Amount £150,000 (GBP)
Organisation Experimental Cancer Medicine Centre Network (ECMC) 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2012 
End 03/2017
 
Description Next generation CAR19 studies - Collaboration with Martin Pule et al at UCL
Amount £900,000 (GBP)
Funding ID II-C3-0714-20005 
Organisation National Institute for Health Research (NIHR) 
Department NIHR i4i Invention for Innovation (i4i) Programme
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 05/2016 
End 04/2019
 
Description Pre-emptive immune therapy to prevent relapse of myeloid malignancies
Amount £1,093,000 (GBP)
Funding ID LLR 13007 
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 07/2013 
End 02/2017
 
Description Translational Research Program: Activation of multiple adjuvant pathways to improve AML vaccine efficacy
Amount $540,000 (USD)
Organisation The Leukemia & Lymphoma Society 
Sector Charity/Non Profit
Country United States of America
Start 07/2013 
End 06/2016
 
Description Cellectis: Production of viral vectors (primarily lentivirus), and gene modified cells, for clinical applications of cell and gene therapy 
Organisation Cellectis
Country France, French Republic 
Sector Private 
PI Contribution Development and production of multiple lentivirus and retrovirus vectors for a range of clinical studies in collaboration with both academic and industry partners, the largest of which is the collaboration with Cellectis culminating in over £1.6 million of funding todate, plus a new contract for £10.1 million over the next 3 years.
Collaborator Contribution Provision of funding and know-how in specific areas (e.g. site directed endonuclease mediated inhibition of endogenous T cell receptors, in order to allow the generation of allogeneic (off-the-shelf) Chimeric Antigen Receptor (CAR) T cells for the treatment of malignant disease. This project is directly supported by BB/N003853/1 and assisted by the outputs from our previous BBSRC grants: BB/E005896/1, BB/D014301/1 and BB/K013785/1.
Impact The development of allogeneic CAR-T cells (referred to as UniCAR-T) for the treatment of malignant disease. There has also been substantial inward investment (over £11,000,000 between 2016 and 2019 from Cellectis alone) underpining further developments that we expect to culminate in substantially larger collaborations with other pharmaceuticaal companies (active discussions in progress with Cell Therapy Catapult, Pfizer and Servier). This collaboration has also resulted in a separate collaboration with a UK based start-up company - Autolus (reported as a separate collaboration).
Start Year 2015
 
Description Vector Industrialisation Project 
Organisation Cell Therapy Catapult
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution The aim of this recently initiated project is the development of GMP compatible procedures for the industrialisation of gene therapy products. The main focus of this specific project is the development of cell line/s with characteristics needed for large scale manufacture of a retrovirus vector encoding a specific T cell receptor (TCR).
Collaborator Contribution Funding of the initial studies, providing the vector manufacturing cell line and expertise in project management, accurate calculation of costs of goods. Cell and Gene Therapy Catapult is also providing expertise in the development of strategies aimed at reducing the cost of goods, risk-reduction for manufacturing campaigns and strategies for efficient large scale manufacture of clinical grade vectors. This project is underpinned by two previous BBSRC grants and directly affected by our current BBSRC supported project.
Impact This project is contributing to the development of new therapies and therapeutic strategies, particularly with respect of the industrial scale manufacture of cell and gene therapy vectors, thus contributing both to better health care and to creation of wealth, including inward investment from outside the UK.
Start Year 2016
 
Title Development of GMP compliant manufacturing strategies for the production of clinical grade viral vectors 
Description The production of viral vectors, in particular lentivirus and gamma-retrovirus in sufficient quantities and able to meet the regulatory standards of quality is particularly challenging. Using the technologies that were developed as part of our BBSRC supported projects, we have established a range of manufacturing, purification and concentration strategies that have enabled us to manufacture the largest number (academia or industry) of retroviral and lentivirus vectors for regulatory approved clinical trials in Europe. This extensive research and development programme has now culminated in over £15 million pound of income (2012 to 2019) for King's College London from overseas based companies. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed Yes
Impact The background manufacturing IP and know-how is licensed (non-exclusive) to Cellectis and to Cell Therapy Catapult. Discussions are in progress with other organisations in taking similar non-exclusive licenses.
 
Title The processes developed in the course of this study have directly contributed to the success of subsequent contracts with the Industry, including Autolus and Cellectis (biotech and pharmaceutical companies. 
Description We have developed procedures for the fast manufacture of retrovirus and lentivirus vectors in compliance with the regulatory requirements for clinical use (GMP compliant procedures). These highly optimised procedures have enabled the production of high titre vectors (about 50,000 million infectious units of vector) from relatively small scale cultures (circa 10 litres), with greater than 50% recovery (frequently in excess of 70%) and minimal quantities of contaminating proteins and nucleic acids. This knowhow has recently been licensed on non-exclusive deals to the industry (Cellectis) in contracts producing in excess of £15 million pounds of income over the next 3 years. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed Yes
Impact We have produced, for regulatory approved clinical trials, the largest number of lenti- and retroviral vectors in Europe. Each of the 4 BBSRC supported projects have contributed to this outcome. We are now extending this expertise with a view to similarly innovative manufacture of Adeno Associated Virus (AAV) manufacture for clinical use.