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Developing a Cell Line
22nd June 2015 | General
Cobra have recently developed a cell line for Alligator Bioscience. Jack Rudd, Editor for
Technology Networks talks to Cobra's Philip Ridley-Smith to learn more about the work
carried out.
JRudd: Can you tell us more about the maxXpress service and Ubiquitous Chromatin
Opening Element technology which were used to develop the cell line for Alligator
Bioscience?
PRidley-Smith: Eukaryotic genes are organised into chromatin domains, which exist in
either a 'closed' state in which the genes are tightly condensed with proteins and are
transcriptionally silent, or in an 'open' de-condensed state: a prerequisite for efficient gene
expression.
When transfected genes integrate into mammalian chromosomes the structure of the
chromatin at the site of integration has a profound effect on expression of the transgene.
Consequently, only relatively rare transfected clones in which the expression vector has
integrated into open chromatin show efficient expression of the transgene.
Ubiquitous Chromatin Opening Elements (UCOEs) have been isolated which ensure efficient
expression in CHO-S cells. UCOE containing vectors have been shown to enhance the
expression of a wide variety of intracellular, secreted and membrane-bound
proteins. Inclusion of a UCOE (4-8 kb in size) in a eukaryotic expression vector permits
efficient expression in the vast majority of stable clones, whereas with conventional vectors
only a minor proportion of transfectants demonstrate high-level expression.
There is therefore no need for amplification and expression has been demonstrated to be
stable over 130 generations. The combination of UCOE and CHO-S allows the generation of
high yields of recombinant proteins.
JR: What benefits does the AmbrTM robotic system bring?
PRS: The 24 mini bioreactors allow a number of parameters to be assessed in a single
experiment during cell line development. Cobra has also been able to show that the
microscale bioreactors mimic very closely large scale bioreactors from 5-250L. Finally,
there are significant reductions in development costs and time for customers compared to
the traditional 5L bioreactors.
JR: Did you face any challenges when developing the cell line?
PRS: Transfection was performed using commercial lipid based transfection system. After
two rounds of limiting dilution to ensure monoclonality there were several promising
candidates for further evaluation in bioreactors. The maxXpress platform is proven well
suited for development of antibody expressing cell lines.
JR: How did the Cobra/Alligator partnership come about?
PR: Cobra and Alligator had been in contact for more than two years prior to Alligator's
need for a CDMO to develop their antibody. The selection of Cobra was the outcome of a
thorough vendor selection process including a request for information step, and a request
for proposal step, bid-defence and negotiations. The focus was on quality followed by cost
and speed. Cobra won the contract due to our previous experience in protein development
programs, timelines and maxXpress offering.
JR: Can you tell us more about the ADC-1013 antibody being used in the clinical trial?
PR: Alligator develops antibody-based immune-oncology therapeutics. ADC-1013 is an
agonistic fully human monoclonal antibody targeting CD40, an immune-stimulatory receptor
found on antigen-presenting cells such as dendritic cells. Stimulation of CD40 on dendritic
cells initiates a process leading to a dramatic increase in T effector cells attacking the
tumour. In addition, a tumour-specific memory is established leading to long term immunity
to the cancer. The phase 1 trial is a first-in-human, multicentre, open-label, multiple
ascending dose study in patients with advanced solid tumours to determine the safety,
pharmacokinetics and pharmacodynamics of intra-tumourally administered ADC-1013. The
study includes a dose escalation part followed by an expansion at the optimal biological
dose level. The primary endpoints are to identify the maximum tolerated dose and to study
the safety and tolerability of ADC-1013. In addition, the trial will evaluate
pharmacokinetics, immunogenicity, pharamacodynamics, antitumour activity, as well as
the mechanism of action of ADC-1013. The study will enrol up to 40 patients during the
dose escalation and expansion phases at five centres in the UK, Denmark and Sweden.