*4.1. Ricin-Based Immunotoxins*

In the 19th century, Paul Ehrlich proposed the "magic bullet concept", which states that drugs can directly enter target cells and hit only abnormal cells of the human body [264]. Since then, the idea of a selective action of drugs that are able to affect only specific types of cells has been dynamically developed. However, the specificity of this process is challenging. The concept is utilized particularly in the field of cancer therapy with the application of immunotoxins (ITs) [265–268]. ITs are chimeric proteins composed of a toxin or a part of the toxin conjugated with a monoclonal antibody (mAb) or its fragment. When toxins are coupled to other carriers: growth factors, hormones or lectins that preferentially bind to some cell types, they are more commonly referred to as "chimeric toxins" or "conjugates" [269]. Ricin is the most commonly used plant toxin in the construction of ITs [30,269]. The first ricin-based ITs were prepared by binding holotoxin to a specific mAb [270]. Despite high efficiency, a large non-specific toxic effect of these immunotoxins was observed and made them impossible to use in a clinical setting. In a different experimental approach, only the A-chain of ricin was used, but also such ITs exhibited non-specific toxicity [271], due to the fact that receptors present on many cell types can recognize mannose residues present on the RTA [272]. To solve this problem, new ITs were prepared with deglycosylated RTA (dgRTA) [269,273,274].

Ricin-based immunotoxins are promising in the treatment of many types of diseases. Autoimmunity, immunodeficiency and neoplasia are examples of diseases connected with deregulation of the immune system. These dysfunctions are characterized by changes in the normal amount or function of Th (helper) cells. ITs composed of RTA and cell-reactive antibodies can specifically target neoplastic cells. It was shown that treatment of Th cells with Fab' fragments of anti-L3T4 antibody bound with RTA (Fab'anti-L3T4-A) inhibit keyhole limpet hemocyanin (KLH)-specific Th cells from proliferation and differentiation of the antigen-specific B cells (trinitrophenyl-(TNP)-specific B cells) [275]. These results indicate that Fab'anti-L3T4-A is able to specially inhibit Th cells that activate B cells. Another immunotoxin RTA-4D5-KDEL was constructed by connecting the anti-HER2 single chain variable fragment 4D5 scFv and KDEL, the ER-targeting peptides, with the C-terminal part of the RTA. Experiments showed that RTA-4D5-KDEL had a strong inhibitory effect on the ovarian cancer cells, SKOV-3, which were HER-2 overexpressing, and caused little damage to H460 lung cancer cells and to kidney HEK 293 cells. The KDEL of the RTA-4D5-KDEL immunotoxin was able to direct the recombinant protein to the ER. In light of this information, it can be assumed that this immunotoxin has a strong inhibitory effect on ovarian cancer cells with overexpression of HER2, and that it will exhibit little toxicity in normal cells [44]. Bladder cancer is one of the most frequent tumors. This disease is treated with transurethral resections and additionally with local immunotherapy or chemotherapy with good results; however, there is no ideal therapy to heal invasive carcinoma. A new antibody-based immunotoxin BCMab1-Ra was generated by linking of BCMab1-, a novel mouse

monoclonal antibody, specific for aberrantly glycosylated Integrin a3b1 in this type of cancer with the ricin A-chain (Ra) [276]. The e ffect of the BCMab1-Ra on bladder cancer was investigated on a 57-year-old patient that refused radical surgery and chemotherapy. It has been demonstrated that the use of BCMab1-Ra first reduced the tumor, and that after 30 weeks of treatment there was no tumor observed by cystoscope examination. Moreover, human anti-mouse antibody (HAMA) that would indicate a strong immunologic response was not detectable in the blood circulation of this patient [276].

Various therapies are being utilized in the treatment of cancer. Traditional procedures such as radiation therapy, chemotherapy and surgery have some limitations and give serious side e ffects. Immunotoxins represent another technique with the possibility to increase the selectivity of action, but further development in this field is required.

## *4.2. Ricin Conjugated with Nanoparticles*

During recent years, nanoparticles (NPs) have been studied intensively both as carriers used for delivery of therapeutic drugs (conventional drugs, recombinant proteins, vaccines and nucleotides) to certain cells and as therapeutic agents that may act per se or modulate activity of other compounds [277–280]. One carrier that can deliver (NPs) to cells is the ricin B-chain. The internalization mechanism as well as intracellular transport of ricinB:Quantum dot (QD) nanoparticle conjugates have been studied in di fferent cells [41,281,282]. It was concluded that Qdots may have severe consequences on cell physiology [281,282]. Moreover, the internalization of ricinB:QDs in HeLa cells is dependent on dynamin and based on a macropinocytosis-like mechanism [41].

A complex of carbon dots (CDs) with RTB has been evaluated for enhanced immunomodulatory activity of RTB [283]. It was demonstrated that CDs-RTB can facilitate macrophage proliferation and increase the generation of nitric oxide (NO), IL-6 and TNFα in RAW 264.7 cells, indicating enhanced immunomodulatory activity of CDs-RTB in comparison to RTB acting alone [283].

Another interesting example is a recombinant version of a ricin nanoparticle (T22-mRTA-H6) containing the T22 peptide, an e fficient ligand of the cell surface marker CXCR4 (a cytokine receptor selectively overexpressed in metastatic cells of many cancer types) at the amino terminus followed by a mutated version of the ricin A chain and a hexahistidine tail at the carboxy terminus [284]. In this construct, mutation N132A was introduced to suppress the vascular leak syndrome (see below); a furin cleavage site was incorporated to allow the release of the N-terminal region in the endosome as well as a KDEL motif was added to mediate retrograde transport. Interestingly, this construct was engineered in order to allow for ricin A-chain aggregation and to become a targeting agen<sup>t</sup> for the precise tumor delivery of protein-only nanoparticles. The recombinant T22-mRTA-H6 was produced in *E. coli* and purified. The spontaneous formation of self-assembled nanoparticles was possibly due to the combination of the cationic peptides at the amino terminus and polyhistidines at the carboxy terminus. T22-mRTA-H6 nanoparticles show highly selective therapeutic e ffects, and ricin A-chain was highly active on target cells, significantly reducing the e ffect of leukemia cells on relevant organs.

#### *4.3. Vaccines against Ricin and Neutralizing Antibodies against Ricin*

Despite numerous medical applications in which ricin can be used, this toxin is among the most potent and lethal substances that are known [35,285]. Currently, no approved vaccine or therapeutics exist to protect against ricin intoxication. The idea to develop a preventive vaccine against ricin has grown over the last years mainly because of the increasing concern that crude ricin powder can easily be made and used as a bio-threat agent. Two of the leading vaccine antigen candidates, the closely related RTA-based subunit vaccines, RiVax ™ and RVEc ™, are now under development [39,286,287]. RiVax ™ is a full-length recombinant derivative of RTA whose enzymatic activity has been largely eliminated through a point mutation in a key active site residue (Y80A). RiVax ™ also contains a mutation in the site (V76M) attributed to the induction of the vascular leak syndrome (VLS) [286]. The VLS is the main side-e ffect of ricin-derived immunotoxins. It has a complex etiology involving damage to vascular endothelial cells [288]. Mutation in the vaccine to alter the VLS motif was introduced to eliminate

this toxicity. RVEc ™ is a truncated derivative of RTA that lacks the hydrophobic carboxy-terminal region (residues 199–267) as well as a small hydrophobic loop in the N-terminus (residues 34–43). RVEc ™ mutations do not directly influence the active site of RTA, but the removal of both regions causes that ricin present in this vaccine is inactive with reduced ability to cause the vascular leak syndrome [39,287]. Both candidate vaccines are under investigation in animal studies and Phase I clinical trials. Furthermore, the results of two Phase I clinical trials have indicated that RiVax ™ is safe and immunogenic in humans [289,290]. One obvious strategy to augmen<sup>t</sup> the overall immunogenicity of vaccines is the use of next-generation adjuvants. However, adjuvants themselves may not be su fficient to achieve maximal immunogenicity. Enhancing the immunogenicity of vaccines may require a structure-based redesign of the antigen itself. The resulting combinations of mutations led to the identification of derivatives of RiVax which are several times more e fficient [291].

In the late 1880s, Paul Erhlich and others first described the potential use of antibodies (Abs) to completely inactivate the toxin. Immunity to ricin is associated with the production of protective antibodies. Since those early studies, many studies of antisera and antibody preparations derived from di fferent animal species and tested on a diversity of cell types have been made. Anti-RTA and RTB antibodies were tested in rabbits and mice and displayed some neutralization action. Some results sugges<sup>t</sup> that antibodies neutralize ricin by perturbing toxin uptake and/or intracellular tra fficking without a ffecting the toxin attachment to cell surfaces. This confers passive immunity in vivo [37,291–293]. On the other hand, blocking ricin attachment to receptors on the cell surface is the mechanisms of action of other specific antibodies (24B11 and VHH D10/B7) [294]. Multiple studies revealed that there are three general classes of ricin-specific Abs; those that bind RTA, RTB and ricin holotoxin [286,295]. As part of an e ffort to engineer ricin antitoxin and immunotherapies, libraries of phage-displayed, heavy chain-only antibodies (V HHs) have been produced and well characterized [296]. It has been demonstrated that immunity against ricin is mediated by antibody. However, the specificity of particular epitopes involved in protective immunity remains unclear. The importance of toxin-neutralizing antibodies in protection against ricin is not questioned. However, the exact correlation between the structure of RTA and the induction of protective immunity must be more strictly evaluated. In just the past 10 years, several reports have been published that demonstrated that passive administration of a toxin-neutralizing antibody is su fficient to display mice protection to a lethal dose of ricin delivered by injection, ingestion or inhalation [292,297–301].
