Intracellular Neutralization of Ricin Toxin by Single Domain Antibodies Targeting the Active Site Pocket

Abstract

25The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which 26 inactivates mammalian ribosomes with near perfect efficiency. Here we characterized, at the 27 functional and structural levels, seven alpaca single-domain antibodies (VHHs) previously 28 reported to recognize epitopes in proximity to RTA's active site. Three of the VHHs, V2A11, 29 V8E6 and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin 30 when expressed as intracellular antibodies ("intrabodies"). Crystal structure analysis revealed 31 that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate 32 RTA's active site and interact with key catalytic residues. V2G10, in contrast, sits atop the 33 enzymatic pocket and occludes substrate accessibility. The other four VHHs also 34 penetrated/occluded RTA's active site, but lacked sufficient binding affinities to outcompete 35 RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may 36 offer a new avenue in the development of countermeasures against ricin toxin. 37 38 3 39 65 Glu177 stabilizes the actual cleavage of the N-glycosidic bond. The role of Trp211 in catalysis 66 remains unknown. These catalytic residues, as well as the chemistry of the SRL depurination 67 reaction is conserved among other members of the ribosome-inactivating protein (RIP) 68 superfamily of toxins, including Shiga toxins 1 (Stx1) and 2 (Stx2) from foodborne Escherichia 69 coli (9). 70 With the capacity to inactivate >1500 ribosomes per minute (10), RTA's active site is an 71 obvious target to consider when designing therapeutics to arrest the effects of ricin toxin 72 exposure. In fact, early efforts successfully identified substrate analogues (e.g., pteroic acid, 73 131 performed Spearman correlation analysis to examine the relationship between in vitro RTA 132 inhibition and VHH binding affinity at both high and low antibody:RTA molar ratios (Figure 133 1E,F). There was indeed a correlation between RTA inhibitory activity and VHH dissociation 134 constants (KD), although a stronger relationship with RTA inhibitory activity and dissociation 135 rates (Koff). These results indicate that the stability of the antibody-antigen complex is critical for 136 potent inhibition of ricin in cell based killing assays and RTA in in vitro assays. 137 138

A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication

“…From the standpoint of MCM development, RT is also challenging as it exerts its catalytic activities within the protective confines of the cell cytoplasm where it is off limits to conventional antibody neutralization mechanisms. However, we have shown in previous reports that RTA is susceptible to intracellular neutralization by antibodies targeting the active site (V2A11) or the RRS (V9E1) Rudolph et al, 2020). In the current study, we have advanced those observations and now demonstrate that the combination of V2A11 and V9E1 as a biparatopic intrabody virtually eliminates RTA's RIP activity and renders Vero cells impervious to the effects of RT in vitro.…”

“…In fact, at this point, RT-specific VHHs have been identified that are capable of interfering with virtually every step in RT's cytotoxic pathway, including cell attachment (Rudolph et al, 2021;, retrograde trafficking (Rudolph et al, 2021), ribosome recruitment , and SRL depurination (Rudolph et al, 2020). Moreover, a subset of VHHs were identified to be capable of neutralizing RT intracellularly when expressed as intrabodies Rudolph et al, 2020). Of particular interest are two structurally wellcharacterized VHHs, V2A11 and V9E1, each of which play a role in shielding the ribosome from RTA.…”

“…V2A11 targets RTA's active site, a solvent-exposed cleft on one face of the molecule that accommodates the protruding adenine (A) within the conserved GAGA motif of the mammalian SRL (Mlsna et al, 1993;Rutenber et al, 1991). Structural analysis revealed that V2A11's complementarity-determining region 3 (CDR3) penetrates the active site pocket and interacts with catalytic residues Tyr80 and Tyr123, thereby obstructing any possible interaction with the SRL (Rudolph et al, 2020). By contrast, V9E1 engages with RTA's ribosomal recruitment site (RRS), a hydrophobic pocket near the C-terminus of RTA that is normally masked by RTB (Chiou et al, 2008;Li et al, 2009;May et al, 2012).…”

“…Structural analysis of V9E1 reveals CDR3 occupancy of the RRS and antibody mimicry of the P-stalk protein interactions . When transiently expressed as intrabodies in the cell cytoplasm of Vero cells, V9E1 and V2A11 each conferred a high degree of resistance to RT-induced cytotoxicity Rudolph et al, 2020). In this report, we sought to investigate whether a biparatopic antibody targeting of RTA's active site (AS) with V2A11 and RTA's P-stalk ribosome recruitment site (RRS) with V9E1 would confer a degree of protection against RT not achievable with either of the antibodies independently.…”

“…Over the course of the past ten years, dozens of RT-specific alpaca-and llama-derived VHHs have been described in the literature (Anderson et al, 2008;Herrera et al, 2016;Poon et al, 2017;Rudolph et al, 2020;Vance et al, 2020;Vance et al, 2013). In fact, at this point, RT-specific VHHs have been identified that are capable of interfering with virtually every step in RT's cytotoxic pathway, including cell attachment (Rudolph et al, 2021;, retrograde trafficking (Rudolph et al, 2021), ribosome recruitment , and SRL depurination (Rudolph et al, 2020). Moreover, a subset of VHHs were identified to be capable of neutralizing RT intracellularly when expressed as intrabodies Rudolph et al, 2020).…”