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Using an innovative design approach we have developed a new class of nanosized antibody- drug conjugate using camelid Variable Heavy chain (VHH) antibody fragments, combined with antimicrobial peptides ( AMP)  by a cleavable linker and have demonstrated the effectiveness of this strategy against drug resistant forms of three different medically important pathogens, a gram positive ( S.aureus) gram negative ( P.aeruginosa) and a yeast ( C. albicans and C. auris).

Called AbTids, these molecules are small, 17-20 kDa in size, economically and efficiently generated as a single chain fusion protein in microbial production systems. They are extremely specific, stable in plasma and activated in the presence of 10000 CFU mL^-1 of the pathogens and have an efficacy in the sub-micromolar range (6.25-12.5 µg mL^-1, 250 nM), inhibiting pathogen growth within 2 hrs of administration.  The VHHs had a Minimum Inhibitory Concentration 90 ( MIC) of 125 µg mL^-1 (2.5mM) in vitro.  Conjugation of the AMP resulted in 10 -20 times increase in efficacy due to a dual mode of action - the inhibitory action of the VHH on surface target (transporters or metabolic enzymes) and the bactericidal activity of the AMPs released from the conjugates by the pathogen surface proteases. AbTids demonstrated a dose dependent antimicrobial activity, was plasma stable and non-toxic to human cells. The extreme specificity will not disrupt the patient microbiota reducing morbidity, possibly resulting in faster recovery of patients in critical care settings. The Pseudomonas AbTid was found to destroy biofilms, resilient to resistance and cleared systemic infection of a carbapenem resistant P. aeruginosa from a neutropenic mouse with a significant reduction of bacterial counts at a dose of 5 mg kg^-1 within 24 hrs of administration, establishing the effectiveness of the design concept in vivo.

The challenge

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs. Although  small molecule antibiotics are being developed constantly, resistance will appear in a short while after deployment, not only making the treatment ineffective but also dissuading further research and investments, resulting in drying up of the antibiotic pipeline. Hence newer strategies using biologicals like antimicrobial peptides, phage therapy, vaccines, and antibodies are thus being developed to tackle the problem. Called non-traditional approach (1), these methods are very specific and are generally resilient to resistance.  Antibodies derived from camels are the latest class of therapeutic molecules being deployed to treat human diseases with five released into the market for immunological indications in recent years (2). We felt that if this class of molecules was used to combat the  superbugs, it could be a gamechanger, with the potential to radically redefine the drug discovery process.  We trained our guns on ESKAPE pathogens, that are a group of six bacteria that pose the maximum risk due to the ease with which they acquire resistance particularly in hospital settings. Critically ill patients with prior antimicrobial exposure or comorbidities are particularly vulnerable, with increased mortality with lengthy costly hospital stays. We wanted to develop a platform technology that could be used to rapidly generate molecules to control the emerging pathogen variants. The AbTids, strategy outlined in the paper if properly deployed with the existing ineffective antimicrobials would reduce the ten year development cycle and the billion dollars cost of newer molecules that might also result in rejuvenating the ineffective/shelved antimicrobials.

The approach

Anticipating the problem of superbugs years earlier, we started working on the camelid antibody platform and constructed 16 immunised camelid antibody libraries against the major bacterial and fungal pathogens containing a billion molecules each, from which antibodies can be isolated within weeks against drug targets and used as a backbone to develop  therapeutic products. To test the concept, we first isolated a neutralizing antibody fragment VHH hit (PsC23) against a component of the transmembrane C4-dicarboxylate transporter in P. aeruginosa that was effective in reversing carbapenem resistance and identified the mechanism of action to be the metabolic shift from glyoxylate shunt to the TCA cycle that re-potentiates the antibiotic by free radical generation and additionally blocking efflux mediated resistance (3).  We combined this antibody fragment with the AMP histatin and generated the first AbTid where we saw in vitro efficacy in the nanomolar range against multiple strains of drug-resistant Pseudomonas.

Using the same design philosophy, we developed the two other molecules against Candida and Staphylococcus, but chose a common surface target, the enzyme enolase with pathogen specific cleavage sites linked to histatin. This was done to study the effects of protease mediated cleavage in activation of the AbTids. The molecules were activated by cleavage and release of the AMP payload  in the presence of specific pathogens followed by their specific neutralization. This experiment proved the concept of AbTids and opened up the possibility of developing new molecules rapidly by shuffling the components like antibodies, cleavage sites or peptides using the basic design blueprint.

Why the results matter

This is a new approach to antimicrobials discovery based on a platform technology that can be extended to other bacteria or emerging pathogens. AbTids  have three very important properties that sets them apart from small molecule antibiotics.

1. Resilience to resistance due to multiple modes of action: As the antibody component of AbTids interfere with the working of the surface targets (enzymes or transporters), the VHH is bactericidal at slightly higher doses and reverses the resistance to antibiotics by interfering with the metabolic pathways. Additionally, the antimicrobial peptide exerts its bactericidal action and combined with its multiple points of attachment to the target results in a complex biological that cannot be neutralized by the pathogen easily by simple point mutations. Since AbTids attach to the surface of the bacteria and is not internalized, efflux or permeability barrier based resistance mechanisms become ineffective as well.  
2. Specificity of action: Since the molecule is structured around an antibody fragment that binds specifically to its target, other bacteria that might be beneficial for the patients are not targeted. The microbiome of the hospitalized patient is not disrupted that might result in lesser morbidity, resulting in faster patient recovery thereby reducing the burden on the healthcare systems.  
3. Evolvable: AbTids are modular molecules with three components that can be rapidly shuffled, in case resistance develops. The antibody or the cleavage site can be replaced rapidly, nullifying the emerging resistance phenotype. These molecules can be designed synthetically in a matter of days and expressed in microbial production systems in a single step reducing the time for generating the first lead molecule, making evolved variations  versions a practical option.

What is next

We have identified a new target, in Pseudomonas that is involved with the operation of the Glyoxylate shunt with a role in drug resistance. It was later discovered that the target is present in all the bacterial pathogens, but is absent in the human host. A new “broad spectrum” molecule targeting all the ESKAPE pathogens can be developed using a different antibody binding to the same target. A proper clinical trial protocol has to be designed around AbTids for standalone or adjunct therapy application. We are currently proposing an IV dosing for lower respiratory tract or urinary tract infection but different formulations like topical or even oral (VHHs are known to survive gastric passage) can be developed for other applications as well.  As AMR is a global problem, this technology will be relevant in all parts of the world but must overcome regional regulatory bottlenecks and find acceptance among major pharma players who can formulate it with the small molecules in their portfolio to tackle drug resistant superbugs.

We are trying to established formal working relationships with pharma companies who might be willing to invest in the technology platform, but require more validation and confidence that can come with support from larger specialized research groups and funding agencies. We are also working with an international group specializing in superbug solutions and they can be included as a development partner. So, a combination of a small company (us), a major pharma company and a specialized academic institute supported by competitive grants might be the perfect recipe for developing the novel technology that might contribute a small drop in the vast ocean of overcoming the challenges of controlling superbugs.

Behind the scenes

This project took us around eight years to execute as this type of strategy was without precedence. We were not sure of the eventual path of drug discovery and were absolutely clueless of the following aspects:

Would we be able to generate neutralizing antibodies against pathogen surface targets?

Multiple groups worked on the project. We started with the Pseudomonas target as we felt that since it colonized multiple organs of the body like lungs and urinary tract as well as caused sepsis, multiple applications could be thought of during clinical trials. We first isolated a neutralizing antibody by panning the library against intact bacteria, but the real surprise came when we discovered that it bound to a new uncharacterized target. Then came a mad scramble to characterize the target, the results of which was published as a separate paper. We identified a new mechanism of drug resistance by metabolic shift and found that the target was present in all the ESKAPE pathogens but not the human host, opening up possibilities for developing newer molecules.

Could we produce an antimicrobial peptide in a microbe?

We chose a common linear AMP histatin, for the three molecules and fused it to the antibody by a cleavable linker. We were worried that the histatin would kill the E. coli producing it and that was exactly what happened in the initial experiments when the growth slowed down as soon as we induced the culture. We worked on the induction conditions constantly and were able to do a quick induction so that the AbTids went into inclusion bodies and toxicity was reduced considerably as the antibody wrapped around the peptide tightly and prevented its bactericidal action. Still the yields were comparatively low, but they increased when the Pichia system was used probably as the mode of action of the peptide in yeasts was different (not lytic).

Will the specific cleavage mechanisms work, what if cleavage happened in the human plasma?

The choice of the cleavage sites was very challenging as we had to ensure that it was specific to the pathogens and stable in human plasma. For that purpose, we chose proteases that are pathogenicity factors and are responsible for establishment of the infection and subsequent tissue invasion. The cleavage sites were studied closely and engineered to be specific and stable. By multiple trial and errors, we were able to design these sites so that there was no cross reactivity and specific activation was achieved, that went a long way in ensuring the non- traditional character of the molecule. 

What pathogens should we choose to demonstrate the proof of concept?

To convincingly demonstrate the concept, we chose three bacteria with different surface properties, Gram positive S. aureus, Gram negative P. aeruginosa and a fungus Candida sp, all of which are a serious healthcare challenge particularly in hospitalized patients. We expected the AbTids to work in gram positive bacteria due to its relatively simple cell wall, but the AbTids worked for the fungus and gram negatives equally well due to the small size of the antibodies with excellent permeability properties.

On a personal note

When I started my new lab, I was unsure if the AbTids concept would work at all  as it was totally untested and camelid antibodies was not a popular format for researchers, particularly for treating bacterial infections. The enthusiasm of the people working with me removed these doubts from my mind and made the project a success. Multiple teams worked on the idea intensely and the lab was full of life and energy. We had a heartbreak as one of our cofounders Mr. Anil Nahar passed away after being infected with carbapenem resistant P. aeruginosa, against which we had successfully developed an AbTid, but at the end of it all there will be a happy outcome as two of the co-authors Agasthya Suresh and Rija Nada are getting married next week - the first AbGenics couple.

References

1. Umarje, S. C., & Banerjee, S. K. (2023). Non-traditional approaches for control of antibiotic resistance. Expert opinion on biological therapy, 23(11), 1113–1135. https://doi.org/10.1080/14712598.2023.2279644

  2. Evers, A., Guarnera, E., Pekar, L., & Zielonka, S. (2025). From discovery to the clinic: structural         insights, engineering options, clinical, and ‘next wave’ applications of camelid-derived               single-domain antibodies. mAbs, 17(1). https://doi.org/10.1080/19420862.2025.2583210

    3. Nagraj AK, Shukla M, Kulkarni M, Patil P, Borgave M, Banerjee SK. Reversal of carbapenem       resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter. J Antibiotics (Tokyo). 2024 Sep;77(9):612-626. https://doi.org/10.1038/s41429-024-00748-w