**5. Ricin-Containing Immunotoxins**

Many researchers have tried to exploit the high cytotoxicity of ricin for medical purposes to eliminate pathological cells. Although ricin possesses highly e fficient cell killing mechanisms, it lacks selectivity towards cell targets. In order to increase selectivity, the possibility of linking ricin to carriers specific for targets on unwanted cells has been explored. The most widely used carriers are antibodies and the corresponding conjugates are referred to as immunotoxins (ITs).

The first IT, created in 1976 by Moolten and co-workers, was made by Ricin Toxin-A chain (RTA) linked to a rat tumor-specific antibody against a rat lymphoma, namely (C58NT)D (Figure 1) [107]. To date, a multitude of pre-clinical and clinical studies have shown the potential use of several ricin-ITs towards di fferent cancer types, from hematological to solid ones, and towards normal cells, unwanted due to them being responsible for a pathological state (reviewed in [108,109]). Di fferent approaches have been used, over time, to generate ITs. In the first strategy, ITs were composed by the antibody chemically linked to the entire RIP and they were used for in vitro studies showing high cytotoxicity [110]. Despite the high in vitro efficiency, the relevant non-specific toxicity reported in vivo, due to the characteristics of the lectin chain, brought researchers to sterically block, chemically modify, or remove the B chain, thus balancing toxicity and specificity. In 1985, Weil-Hillman et al. tested an anti-Mr 67,000 protein linked to either blocked-chain B ricin or RTA, reporting interesting results in vitro, but not in vivo in a nude mouse model [111]. The 1980s were years of grea<sup>t</sup> ferment for molecular biology and genetic engineering, paving the way for the second generation of ITs. Many researchers tried to improve the IT penetration in tumor mass by reducing the antibody size, using antigen-binding (Fab), or variable (Fv) fragments instead of entire antibodies. In 1988, Ghetie et al. created a new IT composed by Fab conjugated to chemically deglycosylated RTA (dgA) [112]. A few years later, they used an anti-CD122-dgA IT in SCID-Daudi mice, showing promising results since the IT was able to specifically kill tumor cells in vivo, extending the mean survival time up to 57.9 +/− 3.8 days [113]. Moreover, FitzGerald et al. described the antitumor activity of recombinant RTA (rRTA) linked to anti-mouse transferrin receptor in a nude mouse model of human ovarian cancer. Animals treated with IT had an extended life span from 45 (lower doses) to 70/80 days (higher doses) [114]. Finally, in 1997 the first ricin-containing recombinant immunotoxin (rIT) was obtained through the expression of a fusion gene composed by sequences encoding anti-CD19-FVS191 (single-chain Fv), cathepsin D proteinase digestion site, and rRTA. In this work, the authors compared the cytotoxicity of the rIT with the chemical linked IT, evidencing that only the latter was toxic in target cells [115]. About 20 ricin-ITs have been tested in Phase I, II, and III clinical trials to treat patients with tumors, either hematological or solid, transplant rejection, and GvHD. The first Phase I clinical trial, giving promising results, was conducted by Spitler et al. in 1987 (Figure 1), by treating 22 metastatic malignant melanoma patients with an IT composed by murine monoclonal anti-melanoma antibody coupled to RTA (XOMAZYME-MEL) [116–118]. Additionally, ITs were also exploited for the treatment of autoimmune diseases. Indeed, anti-CD5/RTA was the first IT used in clinical trials for therapy of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and insulin-dependent diabetes mellitus [119,120]. The advantages and limitations of ricin containing ITs for cancer therapy were recently discussed together with strategies for reducing the immunogenicity of recombinant ITs [121,122].

A di fferent new approach consists of nanoparticle construction, in which ricin is genetically fused to carrier peptides that are able not only to recognize specific cellular target, but also to auto assemble, as stable nanoparticles, thus increasing the toxin-concentration into the targeted site [109,123,124].
