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Developing a Cell Line

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Cobra Biologics have recently developed a cell line for Alligator Bioscience, expressing monoclonal antibodies for a Phase 1 clinical trial which commenced in April 2015. The work they carried required the use of novel technologies  in order to  overcome the many challenges confronted throughout the development process. 
To learn more about the work carried out and, the partnership between the two companies in general we spoke to Philip Ridley-Smith, Sales and Marketing Director, at Cobra Biologics.

JR: 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?

PR: 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 markedly 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 Ambr™ robotic system bring?

PR: 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?

PR: 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 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, pharmacodynamics, antitumor activity, as well as 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.

Philip Ridley-Smith was speaking to Jack Rudd, Editor for Technology Networks.