AbDEN

Along every step of our ideation process, we did extensive research and spoke to several experts to make sure that the product we developed would be applicable in the real world.

We considered the downstream steps of production and purification, including possible alternative chassis and vectors.

We compiled a battery of assays that would be performed on the path to approving the drug for public use, including binding assays, virus neutralization assays, effector function assays and animal trials to obtain information on effectiveness and toxicity, as well as pharmacokinetic parameters like half-life. Finally, we also tried to address the questions of ground usage of our therapeutic in terms of stability of the drug - in storage and within the human body. We also included considerations of formulation in our plans.

There are many steps between theory and implementation for any therapeutic, even before those of live animal and human trials.

We were successful in demonstrating that the antibodies we produced were correctly folded, bound to their target, and could successfully neutralize the virus.

General assays for the drug on its journey to approval include the following components.

Antigen-binding Assays

These are assays performed to test the binding of the antibody to its target. It is not a measure of its therapeutic efficacy, merely binding strength - but is still a very helpful initial indication. Other than ELISA, these may also be performed using Surface Plasmon Resonance (SPR) assays, which do not require any molecular labels to obtain a readout. SPR can also allow continuous monitoring of antigen-antibody reactions, which is challenging to achieve with ELISA[1]. In addition, as the efficacy of our antibody is dependent on competitive binding with naturally produced antibodies, it would be important to assess their on-rate and off-rate in comparison to their competitors. This is also possible with SPR[2], as well as isothermal calorimetry.

Virus Neutralization Assays

These are conducted to measure the ability of the protein to inhibit the virus' infectious power. As our drug is neutralizing in nature, and works by binding to the E-dimer to inhibit virus entry into cells. The most common quantification of this value is Half Maximal Inhibitory Concentration or IC50, which is a measurement of the drug concentration required to inhibit a process (viral infection in this case) to half the uninhibited value. There are other measured values like VN50 that are more specific to antiviral drugs, but similar in spirit to IC50.

The standard test to find the potency of an anti-DENV neutralizing antibody is the PRNT (Plaque Reduction Neutralization Assay), where an incubated mixture of the virus and the antibody is poured over a monolayer of host cells, and the number of plaques are counted after a few days. PRNT50 is defined as the amount of antibody required to reduce the number of plaques by half compared to infection with un-neutralised viral particles[3][4].

We conducted a VLP Fusion assay to study the effect of our fragment on the specific infectious step where the dengue virus fuses with the lysosomes wall to avoid hydrolysis.

Effector Function Assays

Since one of the goals of this therapy is to neutralize the infection without involving the immune system, it must be demonstrated that the protein does not bind to the various Fc and complement receptors.

This can be accomplished in a similar manner to the antigen-binding assays using cell-surface ELISA or SPR.

As pH-dependent binding is an important factor for the antibody's interaction with the FcRn receptor, it must also be calculated. The performance of the protein in the recycling pathway can be studied through a cellular recycling transcytosis assay[5].

Toxicity Assays

Monoclonal antibody therapy has been known to have off-target detrimental effects, especially in large concentrations - hepatotoxicity and dermal toxicity being the most common. While in-vitro toxicity assays can be performed with various cell lines to test for these, they tend to have limitations - several antibodies have been discontinued and withdrawn for adverse impact, and several approved monoclonals have been associated with detrimental health effects[6].

In-vitro assays like the WST-1 cell proliferation assay[7] are vitally important in the preclinical stage to catch early warning signs, but useless if they are not followed up with tests on clinically relevant model systems - like monkeys.

However, the specificity of monoclonal therapy to humans does mean that antibodies can have effects in clinical trials that weren’t predicted in any prior tests, both in the positive and negative sense[6].

Half-life Evaluations

The half-life of our proposed full-length antibodies should be approximately the same as native IgG, as glycosylation does not affect binding to the FcRn receptor.

As for our proposed scFvs, the FcRn-binding peptide will increase the half-life considerably from a matter of hours to days. The effect of the peptide can be studied in-vitro through cell-based assays, such as the recycling assay described in this paper[5].

The exact value must, of course, be calculated through live animal and human trials. As ordinary mice may prove insufficient due to the differences in properties of human and mouse FcRn receptors, it may be worth using transgenic mice engineered to have human FcRn receptors[8].

FcRn binding Assay

Dosage Implementation

There are computer models for predicting such values, and several preliminary in-vitro tests, but the only reliable method of finding the appropriate dose are live animal and human trials, where the subjects are treated with varying amounts of the drug to evaluate the value where the drug is maximally effective and minimally toxic for the majority of the population.

In-vivo trials typically begin with mice, measuring various pharmacokinetic parameters - how the drug is absorbed, where the drug goes within the body, how and where it is metabolized and how it is removed from the system.

Dosage evaluation is typically done through an extensive set of tests on non-humans, whose responses are extrapolated to human trials. Every country has a different set of recommendations, but most are derived from the guidelines of the WHO, which prescribes “adequately powered, randomized and controlled clinical trials”[9]. They also recommend immunogenicity testing, and pharmacovigilance post-market release.

Drug Transport and Usage

As we are trying to address a problem found almost exclusively in developing tropical countries, we would be remiss if we ignored the issues associated with cold chain processing. The drug we are proposing is a protein - that must be stored at low temperatures if it is to remain active. This is difficult to achieve in places with poor transport and energy infrastructure - it would be ideal to avoid it.

In addition, the protein must also be functional within the human body for several days, making the question of its stability a vital one.

A therapeutic for a disease like dengue must be stable in storage for long periods at fairly high temperatures(28-32°C is the average for India).

The protein could be lyophilised, if it can be demonstrated that it does not lose functionality upon reconstitution. A percentage moisture of 1-8% has been shown to be the most favorable to preserve function[10]. This can be improved with the presence of carbohydrates (like sucrose and trehalose) in the solid-state to fulfill the hydrogen bonding requirements of the protein. A second option could be ensilication, where the protein is protected by silica cages. It has been shown to preserve function better than freeze-drying.

The above would make transport easier, but it is also important to make the protein stable in its native, aqueous form - so that it can remain and work as intended in the body after administration. Unstable proteins also tend to aggregate, causing an immune response against them.

We can try to make the protein itself more stable by mutating the sequence to change the Gibbs free energy of unfolding, either by stabilizing the folded form of the protein or by destabilizing the unfolded form[11].

The broader question in both the above points is that of formulation, which is important to consider early in the development of the therapeutic, for the same purpose of maximizing stability in transport and usage.

Research regarding favorable excipients in the formulation of antibody fragments is scant. They have a known tendency to aggregate in solution, which makes it important to include polysorbates or similar elements to prevent it from doing so.

Other considerations will be dependent on the chosen method of drug delivery - intravenous, intramuscular or subcutaneous. The primary concerns for answering the above is the safety and efficacy of each method of delivery, the secondary concern being ease of administration and patient comfort.

Summary

We envision a final product that is effective and easy to transport and use. Outside of the efficacy of the drug, it is also important to address the details of production, to make the final product as cheap and accessible as it can be.

  • SHuffle is not suitable for large-scale fermentors due to its sensitivity to oxidising conditions due to the build-up of H2O2. One solution is to use another strain with the ability to form disulfide bonds in the cytoplasm, so as to still allow over-expression, but is less sensitive. SHuffle is a trxB-gor knockout, knocking out only one of the two genes has been shown to make an improvement.
    Another solution is to make SHuffle less sensitive though the use of a chaperone like Gpx7-PDI, where Gpx7 shuttles the oxidizing power of H2O2 to PDI, allowing the formation of more soluble product[12].
  • While bacterial production of therapeutic proteins has its advantages, gram negative bacteria contaminate the product with endotoxins that must be purified in later steps. The solutions could be to use alternative gram-positive strains like B.subtilis which has proven to be effective at protein production.
  • In addition, our choice of vector backbone could also be improved. Antibiotic selection markers are excellent for the easy elimination of most contaminants, but they lead to trace amounts of antibiotics and resistance-conferring genetic material in the product, which is unfavorable from a biomedical standpoint[13]. Many cutting-edge alternative expression vectors have been described, reliant on phenomena like post-segregational killing and essential gene complementation.

In addition to these, a couple of other points to keep in mind are:

  • We made use of a His-tag to purify our protein through Ni-NTA affinity chromatography. For therapeutic usage, it is recommended that all affinity and solubility tags be made removable[14] - which can be possible through the usage of specific proteases, like TEV protease.
  • For those peptide sequences that are essential for the physiological behaviour of the protein, but are foreign to the human body - the protein can be subjected to de-immunisation. This is an in-silico process that identifies and modifies T-cell epitopes from a query, thereby decreasing the immune response towards it[15].

References

  1. Surface plasmon resonance
  2. Hunter, S. A., & Cochran, J. R. (2016). Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions: Technologies and Considerations. Methods in enzymology, 580, 21. DOI
  3. Plaque reduction neutralization test
  4. Thomas SJ, Nisalak A, Anderson KB, Libraty DH, Kalayanarooj S, Vaughn DW, Putnak R, Gibbons RV, Jarman R, Endy TP. Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infections: How alterations in assay conditions impact performance. Am J Trop Med Hyg. 2009 Nov; 81(5):825-33. DOI
    PMID: 19861618; PMCID: PMC2835862.
  5. Vince W. Kelly and Shannon J. Sirk. ACS Chemical Biology 2022 17 (2), 404-413 DOI
  6. Kizhedath A, Wilkinson S, Glassey J. Applicability of predictive toxicology methods for monoclonal antibody therapeutics: status Quo and scope. Arch Toxicol. 2017 Apr;91(4):1595-1612. DOI
    Epub 2016 Oct 20. PMID: 27766364; PMCID: PMC5364268.
  7. Kizhedath A, Wilkinson S, Glassey J. Applicability of Traditional In Vitro Toxicity Tests for Assessing Adverse Effects of Monoclonal Antibodies: A Case Study of Rituximab and Trastuzumab. Antibodies (Basel). 2018 Aug 17;7(3):30. DOI
    PMID: 31544882; PMCID: PMC6640679.
  8. Proetzel, G., & Roopenian, D. C. (2014). Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies. Methods (San Diego, Calif.), 65(1), 148–153. DOI
  9. The World Health Organization's guidelines on clinical trials
  10. Breen, E.D., Curley, J.G., Overcashier, D.E. et al. Effect of Moisture on the Stability of a Lyophilized Humanized Monoclonal Antibody Formulation. Pharm Res 18, 1345–1353 (2001). DOI
  11. Romas Kazlauskas. Engineering more stable proteins. Chem. Soc. Rev., 2018,47, 9026-9045. DOI
  12. Lénon, M., Ke, N., Szady, C. et al. Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions. Appl Microbiol Biotechnol 104, 9693–9706 (2020). DOI
  13. Peubez, I., Chaudet, N., Mignon, C. et al. Antibiotic-free selection in E. coli: new considerations for optimal design and improved production. Microb Cell Fact 9, 65 (2010). DOI
  14. Waugh, D. S. (2011). An overview of enzymatic reagents for the removal of affinity tags. Protein Expression and Purification, 80(2), 283-293. DOI
  15. De Groot AS, Knopp PM, Martin W. De-immunization of therapeutic proteins by T-cell epitope modification. Dev Biol (Basel). 2005;122:171-94. PMID: 16375261.