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<p>Development of effective, long-lasting antibiotics is a challenge due to the rapid rate at which the microbes acquire resistance, prompting the major pharma players to omit them from their portfolio. We present a simple non-traditional biologics approach for developing highly effective anti-infectives that are resilient to adaptive resistance. Antibody fragment-drug conjugates were developed against three different pathogens <i>Pseudomonas aeruginosa</i> (ABG 14), <i>Staphylococcus aureus</i> (ABG 16) and <i>Candida albicans</i> (ABG 07) that neutralised them specifically irrespective of their drug resistance profiles. Neutralising heavy chain camelid antibody fragments (nanobodies, VHH) isolated from immunised camel libraries of the three pathogens by phage display demonstrated a Minimum Inhibitory Concentration (MIC) 90 value of 125&#xa0;µg mL<sup>− 1</sup> (2.5mM) <i>in vitro.</i> An Antimicrobial Peptide (AMP) was then conjugated to them by pathogen-specific cleavable linkers resulting 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. Called AbTids, these molecules are small, 17–20&#xa0;kDa in size and 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 10<sup>4</sup> to 10<sup>5</sup> CFU mL<sup>− 1</sup> of the pathogens and have an efficacy in the sub-micromolar range (6.25–12.5&#xa0;µg mL<sup>− 1</sup>, 250 nM), inhibiting pathogen growth within 2&#xa0;h of administration. One of the AbTids, ABG 14 was characterised further and found to be <i>P. aeruginosa</i> specific, destroyed biofilms, non-toxic to the host, had low resistance frequency and cleared a systemic infection in a mouse with a carbapenem resistant strain of the bacteria at a dose of 5&#xa0;mg kg<sup>− 1</sup>. This strategy can be used to generate new antimicrobial biologicals against medically important pathogens in a matter of weeks by using different VHHs, linkers and AMP, bypassing years of expensive drug development efforts and can potentially rejuvenate drug discovery efforts against emerging superbugs.</p>

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A Rapid Nanobody Based Approach for Developing Antimicrobials Against Drug-resistant Bacteria and Yeasts

  • 期刊论文

  • 发表日期：

    2025年12月18日

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  • Sanjiban K. Banerjee,
  • Pratik Patil,
  • Agasthya Suresh Babu,
  • Rija Nada,
  • Gayatri Naik,
  • Manjiri Shukla

摘要

Development of effective, long-lasting antibiotics is a challenge due to the rapid rate at which the microbes acquire resistance, prompting the major pharma players to omit them from their portfolio. We present a simple non-traditional biologics approach for developing highly effective anti-infectives that are resilient to adaptive resistance. Antibody fragment-drug conjugates were developed against three different pathogens Pseudomonas aeruginosa (ABG 14), Staphylococcus aureus (ABG 16) and Candida albicans (ABG 07) that neutralised them specifically irrespective of their drug resistance profiles. Neutralising heavy chain camelid antibody fragments (nanobodies, VHH) isolated from immunised camel libraries of the three pathogens by phage display demonstrated a Minimum Inhibitory Concentration (MIC) 90 value of 125 µg mL− 1 (2.5mM) in vitro. An Antimicrobial Peptide (AMP) was then conjugated to them by pathogen-specific cleavable linkers resulting 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. Called AbTids, these molecules are small, 17–20 kDa in size and 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 104 to 105 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 h of administration. One of the AbTids, ABG 14 was characterised further and found to be P. aeruginosa specific, destroyed biofilms, non-toxic to the host, had low resistance frequency and cleared a systemic infection in a mouse with a carbapenem resistant strain of the bacteria at a dose of 5 mg kg− 1. This strategy can be used to generate new antimicrobial biologicals against medically important pathogens in a matter of weeks by using different VHHs, linkers and AMP, bypassing years of expensive drug development efforts and can potentially rejuvenate drug discovery efforts against emerging superbugs.