Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Abstract

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (»30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti-Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.

Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice

“…Camelid VHHs are a popular tool for constructing recombinant antibodies to detect and neutralize a range of targets [18,19,20,21,22,23,24]. In particular, it has been shown that HC-only antibodies or VHHs derived from HC-only antibodies, produced by camelids demonstrate strong anti-BoNT activities in animal models [6,43,44].…”

“…Clear advantages include the ability to recognize hidden antigenic sites that are inaccessible for conventional antibodies due to their structure; stability over a wide range of temperatures and pH; high solubility, as well as economical and facile expression and production in large quantities in microorganisms [15,17]. Several studies were conducted to test the potency of VHHs as inhibitors of viral infections [18] and different toxins, produced by such plants and microorganisms as Ricinus communis [19], Mycoplasma hominis [20], Clostridium tetani [21], Clostridium difficile [22], Crotalus durissus terrificus [23], and Shiga toxigenic Escherichia coli (STEC) [24]. It has been demonstrated that the antigen-binding region of VHHs produced by camelids showed strong anti-BoNT activities in animal models [25,26].…”

Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment

“…A solution to this health problem could be offered using recombinant single domain antibodies, referred to as nanobodies, which currently constitute a highly versatile and efficient alternative for the development of therapeutic agents [ 37 , 38 ]. Nanobodies are of interest due to their small size favoring rapid bioavailability and fast delivery to sites of toxin uptake [ 39 , 40 , 41 ]. In the last couple of years, several groups have generated nanobodies against Stx2 toxins.…”

Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains

“…All other assay conditions remained the same. An irrelevant dimer T5/TC9 [32] was used at 1 μM as a negative control while the TcdB-binding mAb bezlotoxumab (MDX-1388) was used at 250 nM as a positive control.…”