Team:Montpellier/Perspectives
PESPECTIVES
KARMA is a molecular strategy to program the specific degradation of protein targets in a complex biological environment. Our vision was to develop adaptable and modular molecular scissors that could be used to target any protein known to play an important role in the development of a pathology. Over the summer, our team successfully implemented a proof of concept to show that the KARMA strategy can effectively be used to selectively degrade a target of choice while leaving other non-target proteins unharmed. KARMA is comprised of two modules, one that codes for its intrinsic protease activity and one that modulates the effective activity by determining its specificity. These modules can be independently changed to meet the requirements of many applications. As antibodies can in principle be derived against any target protein and since a great diversity of proteases have been discovered and engineered, the KARMA strategy can theoretically be used to eliminate any extracellular protein. We describe below several potential applications we found most interesting.
1- KARMA to fight antibiotic resistance
2- KARMA in oncotherapy, neurodegenerative diseases, and beyond
3- Improving the properties of KARMA
1- KARMA to fight antibiotics resistance
Many examples of antibiotic resistance are not directly targetable by KARMA because they rely on intracellular mechanisms. Beta-lactamases, however, are a particularly widespread and effective family of enzymes causing resistance to beta-lactams, the class to which penicillins belong. As beta-lactams act at the cell-wall, beta-lactamases need to be secreted to be active. In recent years, the emergence of super resistance to beta-lactam antibiotics has been particularly worrying. For example, New Delhi Metallo-Beta-lactamase (NDM1) confers resistance to a very broad range of beta-lactams and is extremely resistant to available commercial inhibitors.
Due to time and biosafety concerns, we opted not to engineer strains producing NDM1 for the iGEM competition; however, other beta-lactam resistances are routinely used in laboratories. For example, researchers use the ampicillin resistance gene (bla) that codes for a conventional beta-lactamase as a selective marker. As such, we easily identified from the literature a VHH capable of recognizing beta-lactamase (1). We also identified a non-specific protease, glutamate carboxypeptidase II, that targets motifs present in the beta-lactamase but not in the identified VHH(2). As beta-lactamase is secreted, the KARMA strategy can be used to resensitize a strain that possesses this resistance. To do so, we wanted to add KARMA at different concentrations in fresh liquid cultures of ampicillin-resistant bacteria, then treat with ampicillin and then measure the Optical Density of the samples by comparing them with a control (bacteria not treated with KARMA). The same experiment can be repeated but on agar media by measuring inhibition haloes. The final goal would be to see if we could perceive a decrease in bacterial growth. Additionally, we could measure the MIC (Mean Inhibitory Concentration) with resistant bacteria treated by KARMA.
2- KARMA in oncotherapy, neurodegenerative diseases, and beyond
a- Oncotherapy
As scientists and especially for biologists, we are all very sensitive to cancer, which remains one of the most deadly diseases and poorly understood to this day. Modern science is constantly developing new therapies that are increasingly effective against this class of disease, but despite this, it remains extremely difficult to treat depending on the type of cancer. Surgical removal is often the most effective method for the most advanced stages, but this surgery remains extremely invasive.
Immunotechnology has been booming in the fight against cancer for the past 15 years. New biomedicines are increasingly being developed to act as immunomodulators. Often these therapeutic antibodies are developed to inhibit target proteins, known to be important in tumour development. Several of these antibodies have been commercialized against different targets
Ipilimumab (Yervoy™): anti-CTLA-4 monoclonal antibody
Pembrolizumab (Keytruda™): anti-PD-1 monoclonal antibody
Nivolumab (Opdivo™): anti-PD-L1 monoclonal antibody
With KARMA, we could target these already known targets with the added benefit of cleaving them via our protease activity. Additionally, we thought about TGF-beta (tumoral growth factor beta) as a target because it is known for its important role in tumour formation and the literature has many examples of compatible antibodies and proteases that we could integrate into KARMA. The power of KARMA is that it does not only inhibit its target, as do the current immunomodulators, but it irreversibly degrades it. Inhibition by an antibody can be reversible because immunoglobulin can always detach at any time.
b- Neurodegenerative diseases
While meeting with the other French iGEM teams, we were inspired by the iGEM Grenoble team's 2019 project to work on an early detection method for Parkinson's disease by measuring the level of alpha synuclein in tears. As this protein is known to be one of the major causes of this neurodegenerative disease, we thought it might be interesting to apply our molecular degradation tool to this target. By browsing the literature we have also found some anti-alpha synuclein antibodies and of course proteases capable of acting on the target to be degraded.(4)
c- Additional applications
Prions are misfolded proteins that can transmit their 3D structural conformations to other similar proteins, thus causing different fetal and neurodegenerative diseases. As we explained earlier, karma targets a protein sequence and the protease attached to it clives the target , thus eliminating these accidentally-appeared prions and help fighting prion-derived diseases like Sporadic CJD-desease , far more frequent than the Familiar CJD , which is a fatal neurodegenerative brain disorder which is caused by accumulation of misfolded proteins in the brain.
We have additionally brainstormed other applications for KARMA, such as the degradation of naked viruses and bacterial toxins as well as treatment of atherosclerosis (Table 1).
Using KARMA as a modular protein scissor can offer diverse and varied applications in many fields, and we are convinced that this type of tool can generate new innovative health technology, ranging from diagnosis in biosensor systems to active therapeutic molecules directed against targets.
Table 1) Antibodies and Proteases that can be used for more diseases
3- Improving the properties of KARMA
-Injection
In addition to the applications, we also thought about how KARMA could be pharmacologically delivered. As maintaining the 3D structure of our peptide is critical, it would be very complicated to carry out an oral formulation, so we think the best way to administer it is via intravenous delivery. One of the major problems we had considered at the pharmacological level is its life-time. This parameter is extremely important as it is known that therapeutic antibodies have low half-lives, but fortunately many strategies exist to increase it.
-Increasing the half-life :
First we could use a complete antibody, having a constant fraction, so KARMA would be able to be recycled by using the neonatal receptors (4). We could also use a carrier protein by coupling it to an albumin binding domain.
One of the other innovative strategies that can be considered to prevent the catabolism of the tool by the patient's endogenous protease is the use of unnatural amino acids. Conventional proteases have great difficulty recognizing modified amino acids with altered chemistry. The use of unnatural amino acids can also be a solution to avoid degradation of the antibody by the non-specific protease of our tool.
To conclude :
Karma has as many applications as you could think of ! Prions, Naked (or not) viruses, cancer, neurodegenerative diseases, antibiotic resistance, one problematic protein, one KARMA.
1- Conrath, K. E. et al. 2001. «B-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae ». Antimicrobial Agents and Chemotherapy 45(10): 2807‑12.
2- B-Lactamase Inhibitors Derived from Single-Domain Antibody Fragments Elicited in the Camelidae Antimicrob Agents Chemother. 2001 Oct;45(10):2807-12.
3- De Genst, Erwin J. et al. 2010. « Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody ». Journal of Molecular Biology 402(2): 326‑43.
4- Coutinho, Ester, et Angela Vincent. 2016. « Autoimmunity in Neuropsychiatric Disorders ». In Handbook of Clinical Neurology, Elsevier, 269‑82. https:// linkinghub.elsevier.com/retrieve/pii/B9780444634320000153 (9 octobre 2019).
5- De Genst et al., Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody, Journal of Molecular Biology, Volume 402, Issue 2, 2010, Pages 326-343, ISSN 0022-2836, https://doi.org/10.1016/j.jmb.2010.07.001.
6- Mejías, Maria P. « Development of Camelid Single Chain Antibodies against Shiga Toxin Type 2 (Stx2) with Therapeutic Potential against Hemolytic Uremic Syndrome (HUS) ». Scientific Reports: 11. https://doi.org/10.1038/srep24913
7- van Dyck, Christopher H. 2019. « Anti-Amyloid-β Monoclonal Antibodies for Alzheimer’s Disease: Pitfalls and Promise »
8- Tzong-Yueh Chen et al. “A Nontoxic Pseudomonas Exotoxin A Induces Active Immunity and Passive Protective Antibody against Pseudomonas Exotoxin A Intoxication”, J Biomed Sci 1999;6:357-363
9- Hiroyuki et al. « A Monoclonal Antibody against Oxidized Lipoprotein Recognizes Foam Cells in Atherosclerotic Lesions.” Vol. 269, No. 21, Issue of May 27, pp. 15274-15279,1994
10- Ratti, G., R. Rappuoli, et G. Giannini. 1983. « The Complete Nucleotide Sequence of the Gene Coding for Diphtheria Toxin in the Corynephage Omega (Tox + ) Genome ». Nucleic Acids Research 11(19): 6589‑95.
11- Atsushi Iwata et al. Alpha-synuclein degradation by serine protease neurosin: implication for pathogenesis of synucleinopathies Human Molecular Genetics, Volume 12, Issue 20, 15 October 2003, Pages 2625–2635, https://doi.org/10.1093/hmg/ddg283
12- https://www.ebi.ac.uk/merops/cgi-bin/pepsum?id=A26.001
13- Saido, Takaomi, et Malcolm A Leissring. « Proteolytic Degradation of Amyloid B-Protein »?
14- Wang, Jing et al. 2015. « Cathepsin G Activity Lowers Plasma LDL and Reduces Atherosclerosis ».
15- https://www.ebi.ac.uk/merops/cgi-bin/pepsum?id=M28.010;type=P (it doesn’t cleave the VHHs) it cleaves after EE or DE