The timeline

Delivered

Currently what we have done is selected particular mutant variants, based on literature, cloned them into a display capable, commercially available plasmid and inserted the mutations using site directed mutagenesis through E. coli. These were transfected into HEK 293 cells to look at expression, co-localization, followed by a panel of antibody based validation studies. In silico, modelling and immunogenicity studies were attempted in parallel. We chose these steps since we wanted to make sure we have a method for developing proteins that evade autoantibodies and later can be produced in E. coli. The in silico studies helped us understand what happens thermodynamically and structurally, as well as predicting immune response.

Scaling

In order to implement our therapeutic protein for combatting pernicious anaemia we would have to scale up what we have done so far. This would firstly need more engineering cycles, until the results are significant and exceed expectations! Furthermore, we would need to check our results with additional validation methods with more controls, more variants, more antibodies and so on. To scale up, we would take the pipeline we have developed and parallelize the procedures and speed them up to whatever extent possible without compromising on the quality of the work. This would be done for continued development of BiG-IF, and possibly other protein therapeutics using the same pipeline.

Approval

In order for our idea to reach and benefit the consumer we would have to design the product as a therapeutic drug. It can’t be sold as a supplement since it is intended to treat a disease [1]. This means that it has to be approved by the FDA and EMA in order to reach consumers.

Pharmacology

Pharmacological studies will give us answers around catabolism - involving important considerations like pharmacodynamics and pharmacokinetics, binding capacity, patient factors such as genetics and overall health, solubility, and uptake [2]. They are an important part of the preclinical studies. It is also important to consider potential side effects and possible interactions with other drugs. For the preclinical work the toxicity has to be assessed in vivo and in vitro, in cell and animal model organisms. Depending on these results, the therapeutic could move on to clinical studies [3].

Clinical Trials

For the phase I clinical studies the dosage and safety is assessed on a small cohort of normal and healthy humans. This will green light phase II - for determining potential side effects, as well as efficacy. In the second phase, the trial lasts longer and involves more participants with the disease. The third and fourth phase trials have the same purpose as the second phase trial but go on for longer or involve more participants. Finally, there is a drug review where the data is analyzed and assessed, weighing harms and benefits for the therapeutic [4].

Cell Factory

To produce our modified intrinsic factor we plan to use E. coli and engineer it into a cell factory. The cells would be grown in a bioreactor under optimal conditions. Then, the therapeutic would be induced, harvested, separated, purified, suspended in a suitable medium and delivered to the drug production facility. The proteins would then be checked to make sure the structure is intact and then packed into pills or another suitable method for delivery.

Challenges:

Engineering

Are the mutations successful in producing an human gastric intrinsic factor that can serve the intended purpose?
How reliably can the protein be expressed on the cell surface?
How reliable are the antibody based validation assays for confirming the evasion of auto-antibodies?
Will the engineered protein be able to treat all patients with different genotypes and corresponding phenotypes?

Production

How reliable is the E. coli as a cell factory to produce our proteins for therapeutic use?
Optimizing large scale production.

Efficacy and Economics

Does it have better efficacy with improved clinical outcomes for patients compared with traditional remedies?
How cost effective is this approach than other treatments?

Consumers

Now we have reached our consumers - people with pernicious anaemia! Ideally they would be able to take a pill which releases our modified intrinsic factor in the stomach where B12 would be taken up at a higher rate than previously. The upscaling and an optimized production would make the treatment reasonably cost effective and accessible to people all around the globe making pernicious anaemia a more manageable condition to live with!

  1. Editorial Staff, American Addiction Centers
    Drugs vs. Supplements: What's the Difference?
    American Addiction Centers, 2022
    Read it

  2. Roche
    What is a clinical trial and how does a trial work?
    Roche
    Read it

  3. U.S. Food & Drug Administration
    Step 1: Discovery and Development
    U.S. Food & Drug Administration, 2018
    Read it

  4. U.S. Food & Drug Administration
    Step 4: FDA Drug Review
    U.S. Food & Drug Administration, 2018
    Read it

Proof of concept

Safety