SPONTANEOUSLY BIOTINYLATED LENTIVIRAL VECTORS FOR ENVELOPE INDEPENDENT TARGETING OF INFECTION

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

Abstract

As a result of a BBSRC funded project we have generated a new 293T based cell line specifically for the production of metabolically biotin labelled lentiviral vectors. These bio-lentiviral vectors have been demonstrated to efficiently complex with streptavidin paramagnetic particles. This combination has resulted in the most efficient purification and concentration method yet described for lentiviral vectors. We wish to use the platform of these new biotin-lentiviral packaging cells to exemplify their value to the gene therapy community. By virtue of their biotinylated surface these viruses can be attached to specific cell binding proteins so that they can be targeted to specific sites of disease. Their unique properties also allow us to increase our knowledge of the cell derived accessory proteins that are also resident on the lentiviral surface. Information on the influence of these proteins on the process of lentiviral infection can then be applied to more efficient targeting of gene therapy vectors to specific sites of infection.

Technical Summary

For lentiviral vectors we have succeeded in engineering an entirely new producer cell type in which the extracellular domain of LNGFR is fused to a biotin acceptor peptide (BAPref). Coordinate expression with the bacterial Bir A gene in human 293T cells results in metabolic biotinylation of a specific lysine residue in the BAP region. LNGFR-BAP-BIOTIN appears on the surface of lentiviral vector making them susceptible to Streptavidin capture. We now propose to use the platform bio-lentivector technology to increase the efficiency of targeting and to test the hypothesis that 'infection attenuated, and non-infectious lentiviral vectors can be manipulated to generate an exclusively ligand dependent, tissues specific infection of target cells'. To do this we will pursue two strategies: 1) Incorporation of cell/tissue specific ligands onto lentivirus particles, thus enabling them to regain infectivity only for the targeted cells. 2) Ligand dependent targeting of non-infectious vectors to specific cells, followed by a second stage targeting delivery of infection promoting factors, thus restricting infection to cells that have been successfully targeted by both components (the non-infectious virus, and the infection agent).
 
Description The main output of this project, in combination with BB/E005896/1 has been the development of novel procedures for biomarking of retrovirus and lentivirus based vectors. This technology, combined with the development of GMP facilities and development of GMP compliant procedures for the production of clinically suitable batches of these viral vectors have resulted in substantial further grant funding, two clinical trials for lentivirus based modification of autologous acute myeloid leukaemia (AML) cells to enable the expression of CD80 and IL-2, in order to generate an AML cell vaccine. These Phase-I clinical trials are currently in progress at King's College Hospital. In addition, the technologies developed and the facilities generated with the aid of these grants (BB/D014301/1 and BB/E005896/1) have enabled further collaborations with the pharmaceutical industry, including £2.4M worth projects with Roche Pharmaceuticals, £2.6M worth of contracts with Northwest Biotehrapeutics and £10.1M with Cellectis.
Exploitation Route The viral vector manufacturing technologies and GMP facilities that are developed with the aid of these BBSRC supported project grants, in conjunction with further support from NIHR and Experimental Cancer Medicine initiative are now providing the basis for further collaborations with the pharmaceutical industry. We are in active discussions with Autolus, Pfizer and MedImmune. Autolus has already contracted projects worth over £500,000 for 2016/2017).
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description This project has culminated in the development of novel manufacturing procedures for the generation of lentivirus and retrovirus based vectors for clinical use. This IP, and facilities generated has resulted in substantial collaborations with the biotech and pharmaceutical industries. Please see the Key Findings above for further details.
First Year Of Impact 2012
Sector Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description An immunisation strategy to prevent hepatocellular carcinoma
Amount $1,200,000 (SGD)
Organisation National Medical Research Council NMRC 
Sector Public
Country Singapore
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
Start 04/2012 
End 03/2017
 
Description Core Suppport for GMP facility
Amount £450,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Academic/University
Country United Kingdom
Start 04/2012 
End 03/2017
 
Description Efficacy/safety of CD80-IL-2 vs novel CD80-IL-15-IL-15R? vector for leukemia vaccine
Amount $275,000 (USD)
Funding ID NIH R21: AI099829-01A1 
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 10/2013 
End 09/2015
 
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 
Department NIHR i4i Invention for Innovation (i4i) Programme
Sector Public
Country United Kingdom
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
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
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 
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 Collaboration with the University fo Lausanne for the development and production of lentivirus lectors 
Organisation University of Lausanne
Country Switzerland 
Sector Academic/University 
PI Contribution We have set up a new collaboration with the University of Lausanne for the development of lentivirus vectors and their GMP manufacture over the next 3 years (2018 to 2020). University of Lausanne has provided a contract of £2.6M of which the first instalment of 20% has already been paid.
Collaborator Contribution The development of vectors that they have produced for a number of gene therapy based clinical studies, and the use of the vectors made under GMP at King's College London, in these clinical trials.
Impact Contracts of Collaboration Signed
Start Year 2018
 
Description Vector Industrialisation Project 
Organisation Cell Therapy Catapult
Country United Kingdom 
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.