Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xix, 255 pages) : color illustrations.

Dissertation/Thesis Chair

Nicholas J Mantis

Committee Members

Rajendra K Agrawal, Gabriele Fuchs, Yi-Pin Lin, Anne Messer, Charles B Shoemaker

Keywords

Antibody, Intrabody, Nanobody, Ribosome, Ricin, Toxin, Ribosomes, Apoptosis, Immunoglobulins, Monoclonal antibodies, Antigenic determinants

Subject Categories

Biology | Biomedical Engineering and Bioengineering | Immunology of Infectious Disease

Abstract

Ricin is a highly lethal protein toxin derived from the seeds of the castor plant, Ricinus communis. It is a Type II ribosome inactivating protein (RIP), meaning it is a heterodimer with one subunit, ricin toxin B (RTB), that mediates cell surface attachment and intracellular trafficking and a second subunit, ricin toxin A (RTA), that irreversibly shuts down protein synthesis in the cytosol. During trafficking, RTA and RTB necessarily separate in the endoplasmic reticulum, wherein RTA unfolds and translocates into the cytosol where it refolds into an enzymatically active conformation. RTA is remarkably fast acting and efficient, with few molecules required for cell death to occur, but the exact determinants of cytotoxicity and critical steps involved in ribosomal inactivation remain unclear. The current understanding of RTA’s enzymatic activity is a two-step model, where it first binds the acidic P-stalk proteins of the ribosome (“recruitment”) and is then guided to the sarcin-ricin loop (SRL) where it cleaves a specific residue required for protein synthesis (“depurination”). The goal of my dissertation was to determine whether RTA follows the two-step model in the context of cellular intoxication and to specifically evaluate whether both steps contribute equally to ribosome inactivation and cytotoxicity using single domain antibodies to block either step. Single domain antibodies (VHHs) are incredibly powerful and versatile biological tools with high therapeutic potential. While conventional IgG antibodies are large 150 kDa protein complexes characterized in part by their strong and highly specific binding interactions with target antigens, VHHs are small antibody fragments (12-15 kDa) that retain these high affinities and specificities. These proteins are highly stable and easily expressed in prokaryotic and eukaryotic systems, facilitating library generation and screening. Our lab has isolated and characterized a large library of alpaca-derived VHHs against ricin toxin. Through extensive phage display and epitope mapping, we have identified VHHs that bind to nearly all surfaces on the ricin holotoxin and many that specifically bind sites that are inaccessible in the holotoxin structure (i.e., bind RTA or RTB, but not ricin). Many of these VHHs strongly neutralize ricin, though have little in vivo efficacy due to short half-lives in circulation. Regardless, they have been useful tools for understanding the intoxication pathway as well as targetable neutralizing hotspots on the toxin. Until now the only tools to probe cytosolic events and neutralize the toxin by targeting RTA’s function have been low affinity small molecules with little success. Recent work with antibody fragments, including VHHs, has exploited their size and ease of expression to target intracellular antigens. These antibodies can be delivered inside the cells, encoded on DNA or RNA vectors or as proteins. Intracellular antibodies, or intrabodies, are a promising tool with numerous applications ranging from neutralization of host- or foreign antigens to labeling and tracking intracellular processes without disrupting normal cell or antigenic functions. Here I have identified several VHHs that bind RTA at its ribosomal interfaces and developed a model to test their ability to neutralize ricin cytotoxicity when prophylactically transfected into Vero cells. RTA’s ribosomal recruitment site (RRS) has been determined by structural analysis, but these analyses have been complicated by the P-stalk proteins’ inherent flexibility. Thus many studies focus solely on the more stable C-terminal amino acids that form a short helical peptide which binds RTA. Mutating residues at or near this site or using small molecule inhibitors renders the enzyme unable to depurinate the 28S rRNA in the context of the ribosomal macromolecule by preventing this initial binding event, but does not specifically prevent enzymatic activity. Cell-based assays have yielded similar results using RTA-expression vectors that harbor mutations at the RRS, but these lack critical physiological context to conclude this initial binding event is necessary within the cell. In Chapter 3, I have evaluated a panel of VHHs that bind at or near the RRS with high affinity (< 1 nM) in cell-free assays and the intrabody cytotoxicity model. I have shown that VHHs whose epitopes overlap the RRS (e.g., V9E1, V9B2, & V9F9) inhibit RTA activity strongly, strongly driven by affinity and two VHHs slightly offset from the RRS (V9F6 & V9D5) do not. When expressed as an intrabody, V9E1 strongly neutralizes ricin’s cytotoxicity, while the “offset” intrabodies do not. These results demonstrate that ribosomal recruitment is an important step for ricin cytotoxicity in a model that allows for trafficking and RTA conformational changes to occur. Furthermore, by discovering non-neutralizing VHHs we have refined the critical residues on RTA for recruitment in the context of the intact ribosome and not only the stalk peptide. The role of RTA’s active site (AS) has been well established down to the specific mechanism of depurination. It has been the obvious target for intracellular small molecule inhibitors with limited success for decades. Because of this and the obvious importance of this site in ribosomal inactivation, in Chapter 4 I sought to evaluate VHHs that target the AS for their ability neutralize ricin as intrabodies with the ultimate goal of comparing these results to RRS targeting. Using a panel of 7 AS VHHs, I found that three (V2A11, V2G10, & V8E6) strongly inhibit RTA due to their high affinities. Likewise, each of these three neutralize the toxin as intrabodies despite being ineffective when used in a conventional extracellular cytotoxicity assay. These results reaffirm the importance of affinity on neutralizing the highly active enzyme, RTA, intracellularly. Importantly, comparative analysis between V9E1 (Chapter 3) and V2A11 (Chapter 4), indicates that occupying the RRS is as effective toward toxin neutralization as the AS. While I have shown both the RRS and AS to be important for cytotoxicity by blocking either site intracellularly, defining their relative importance has required a combinatorial approach utilized in the Chapter 5. I hypothesized that, in accordance with the two-step model, neutralization via VHHs targeting the RRS or AS would be redundant with both sites being equally important for cytotoxicity. In a cell-free assay with low VHH concentrations, a combination of equal parts V9E1 (RRS) and V2A11 (AS) inhibits RTA activity similarly to either VHH alone, indicating there is no in vitro advantage to binding multiple sites on the toxin. Not only do both sites contribute equally to enzymatic activity, but these results reinforce that they are correspond to steps along the same interaction pathway. To determine whether there is an intracellular advantage to targeting both sites, I transfected Vero cells with an intrabody cocktail and rationally designed heterodimer (V2A11-(G4S)4-V9E1). I hypothesized that within the cytosolic environment, there may be an unanticipated disadvantage to combining neutralizing VHHs or conversely an additive or even synergistic effect on ricin protection. Both approaches result in equally robust increases in toxin neutralization. Finally, I have stably transfected cells with three intrabodies (V9E1, V2A11-(G4S)4-V9E1, and V9F6) to maximize expression efficiency and shown near complete resistance to ricin in cells expressing the RRS/AS heterodimer. I hypothesize that this increase compared to V9E1 alone is due to redundant neutralization mechanisms and an increased avidity accomplished by targeting non-competing epitopes. Based on these combinatorial experiments, I have concluded that recruitment is as important to cytotoxicity as depurination, itself, thus demonstrating that the two-step model for ribosomal inactivation occurs intracellularly. The results of this dissertation present the first strong neutralization of ricin toxin by targeting the enzymatic subunit, RTA, intracellularly. Antibody fragments expressed intracellularly are uniquely qualified to this task due to their ease of delivery and solubility in addition to their high affinities, typically several orders of magnitude greater than small molecule alternatives. By blocking both ribosomal interfaces separately and in combination intracellularly, I have demonstrated that the two-step model for ribosomal inactivation applies in the cytosolic context and that disrupting either step can significantly reduce or potentially eliminate cytotoxicity. I have also shown that despite sub-nanomolar affinities, intracellular inhibitors in the form of intrabodies or other constructs can be improved by enhancing affinity, likely due to RTA’s extraordinary catalytic efficiency. Importantly, these results may also be applicable to other ribosome inactivating proteins, such as Shiga toxins (Stx), in terms of neutralizing targets and toxin mechanisms. While ricin is a promiscuous toxin in terms of tissue and cell type tropism, in vivo therapeutic applications of intrabodies for other toxins with more specific targets, like Stx, may be an invaluable alternative or addition to current approaches.

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