**2. How to endow a ribonuclease with selective antitumor activity**

In the last two decades, the knowledge gained on the cytotoxic mechanism of natively tumoricidal RNases, described in the previous section, as well as in the references therein, has been used to engineer more powerful and selective RNase variants able to kill cancer cells. From this knowledge, it is clear that RNases will be cytotoxic if they are able to reach the cytosol avoiding lysosomal degradation or nonproductive intracellular pathways and if, once in the cytosol, they can evade the action of RI (**Figure 2A**–**C**). Consequently, an RNase will be cytotoxic either if it can avoid the RI inhibition or it can efficiently reach the cytosol saturating all the RI present in this compartment (**Figure 2D**–**F**). Several approaches have been used to fulfill these requirements that will be reviewed in this section and are summarized in **Figure 3**.

#### **2.1. Engineered RNases that evade the RI**

In the literature two main approaches used to engineer noncytotoxic RNases to render them resistant to the RI and endow them with cytotoxicity are described: The most evident one consists of precluding RNase‐RI complex formation through steric hindrance or coulombic repulsion. The variant's design is based on the known 3D structure of the RI‐RNase A complex described by Kobe and Deisenhofer [83]. However, another approach is to hide RNases from the inhibitor. This is accomplished by targeting monomeric RNases to an organelle free of RI making needless neither the RI evasion nor the RI saturation.

#### *2.1.1. RNases in a monomeric form*

Apart from RNases of animal origin, it is worth mentioning that there are a vast array of RNases from fungal, bacterial, and plant origin that natively present remarkable cytotoxic properties. Among them we can mention mushroom RNases [72, 73]; microbial RNases such as α‐sarcin from *Aspergillus*; the two well‐known T1 ribonuclease members from *Bacillus*, binase (*B. intermedius*), and barnase (*B. amyloliquefaciens*); and RNase Sa (*S. aureofaciens*) [28, 74] as well as plant RNases from ginseng, wheat leaf, mung bean, black pine pollen, seeds of bitter gourd, tomato, and hop [12, 15, 75–79]. Although adverse effects due to immunogenicity and nonspecific binding [12] have been described for some of them, others are described to have a lower immunogenicity than ONC [28]. In addition, they have a remarkable resistance to RI, and in some cases, the cytotoxic effect is comparable to that of ONC. However, in terms of knowledge of their cytotoxic mechanism and clinical applications, they are still lagging behind when compared to the animal counterparts. In the last years, especially for binase, a significant advance has been attained in the understanding of its mechanism of cell intoxication. The cytotoxic effect of binase is effected via induction of both intrinsic and extrinsic apoptotic pathways [80], and evidence is provided that targets KIT and AML1‐ETO oncogenes in human leukemia Kasumi‐1 cells [81]. It has also shown a positive effect on the liver of tumor‐bearing mice, articulated as a tumor reduction in the volume of destructive changes in the live

parenchyma as well as of being effective in tumor growth suppression [82].

**2.1. Engineered RNases that evade the RI**

140 Anti-cancer Drugs - Nature, Synthesis and Cell

making needless neither the RI evasion nor the RI saturation.

**2. How to endow a ribonuclease with selective antitumor activity**

In the last two decades, the knowledge gained on the cytotoxic mechanism of natively tumoricidal RNases, described in the previous section, as well as in the references therein, has been used to engineer more powerful and selective RNase variants able to kill cancer cells. From this knowledge, it is clear that RNases will be cytotoxic if they are able to reach the cytosol avoiding lysosomal degradation or nonproductive intracellular pathways and if, once in the cytosol, they can evade the action of RI (**Figure 2A**–**C**). Consequently, an RNase will be cytotoxic either if it can avoid the RI inhibition or it can efficiently reach the cytosol saturating all the RI present in this compartment (**Figure 2D**–**F**). Several approaches have been used to fulfill these requirements that will be reviewed in this section and are summarized in **Figure 3**.

In the literature two main approaches used to engineer noncytotoxic RNases to render them resistant to the RI and endow them with cytotoxicity are described: The most evident one consists of precluding RNase‐RI complex formation through steric hindrance or coulombic repulsion. The variant's design is based on the known 3D structure of the RI‐RNase A complex described by Kobe and Deisenhofer [83]. However, another approach is to hide RNases from the inhibitor. This is accomplished by targeting monomeric RNases to an organelle free of RI The first reported approaches to endow an RNase with the ability to evade the RI were carried out by Raines and coworkers, who introduced single or few amino acid changes in wild‐type noncytotoxic RNase A that created steric hindrance to decrease RI binding [84]. Replacement of Gly 88 of RNase A by bulky charged residues, like Arg or Asp, resulted in a variant with 104 ‐fold less affinity for the RI and which was only about 20‐fold less cytotoxic than ONC. Similar approaches were used on HP‐RNase [85, 86] or monomeric BS‐RNase [87, 88]. This first approach was concomitant or followed by the introduction of other changes that disturbed the electrostatic interaction between the RNase and the RI [89–92] creating new variants each time more cytotoxic. For instance, the RNase A variant Asp38Arg/Arg39Asp/Asn67Arg/ Gly88Arg had 5.9 × 109 ‐fold lower affinity for the RI keeping the activity and stability of the parental enzyme with a cytotoxicity equivalent to that of ONC [91]. Although this approach has attained success, in some cases the use of the same rationale has not worked to get variants with the expected properties [89, 93]. This is due to the fact that the replacement of some residues to disrupt the RNase‐RI interaction at the same time alters other factors important for the enzyme cytotoxicity, such as the catalytic activity or the stability of the enzyme that counterbalances the obtained gain on RI evasion. Nevertheless, one of the engineered RNases to evade the RI has reached clinical trials. The QBI‐139 RNase variant (Evade™ ribonucleases from Quintessence Biosciences Inc. (http://www.quintbio.com/) is now in Phase I of clinical

**Figure 3.** Strategies to create cytotoxic RNases or to improve its antitumor activity. Two groups of strategies are consid‐ ered: those allowing the RNase to avoid the inhibition by the RI (dark blue arrows) and those that improve the deliv‐ ery of the RNase into the cell (light blue arrows). Some of the indicated strategies can be included in both groups.

trials for the treatment of solid tumors [25]. On the other hand, different strategies have been carried out to avoid some of the non‐desired side effects. The Gly88Arg RI‐evading RNase A, described above, was engineered to introduce nonnative disulfide bonds to increase its conformational stability [94], resulting in a more cytotoxic variant. Also, an increase in stability has also been attained by the glycosylation of the protein. For instance, the production of ONC in *Pichia pastoris* yields a glycosylated protein more stable and 50‐fold more cytotoxic [95].

#### *2.1.2. RNase dimerization or oligomerization*

The formation of oligomeric structures, such as the BS‐RNase dimers, has inspired the design and production of new RNase variants with the aim of precluding their binding to the RI by steric hindrance mimicking the way of action of BS‐RNase [96, 97]. The pursuit of dimeric or oligomeric variants is very attractive because they are more cationic proteins and can poten‐ tially strongly interact with the negative surfaces of cancer cells gaining selectivity and, at the same time, reducing kidney clearance due to the increase of molecular mass. As stated in Section 1.2, the current model for BS‐RNase cytotoxicity is that in the reducing conditions of the cell citosol, the unswapped isomer from (M=M) dissociates into monomers, which are strongly inhibited by the RI, whereas the swapped isomer (M×M) remains as a non‐covalent dimer able to evade the RI [64, 98]. In addition, analysis of the structure of the non‐covalent dimer of BS‐RNase [61] and different mutated forms [99] suggested that it adopts a compact quaternary structure that is critical for the RI interaction, explaining its trapping. One of the first approaches to get cytotoxic dimeric RNase variants was to reproduce the structural determinants of BS‐RNase swapping [62] in different members of the vertebrate‐secreted RNase family. Thus, different combinations of those residues identified as responsible of dimer formation (Cys31, Cys32, Leu28, Gly16, Ser80) of BS‐RNase were introduced in the sequence of either HP‐RNase or RNase A. Alternatively, the full N‐terminal hinge sequence (the peptide that links the N‐terminal α‐helix of V‐shaped RNase structure with the rest of the protein body) of RNase A was replaced by that of BS‐RNase [63] in order to endow RNase A with dimeri‐ zation abilities. These changes resulted in the formation of different ratios of swapped and unswapped forms, which was critical for their cytotoxicity [63]. Among these constructs it is remarkable that of a dimeric form of HP‐RNase containing the mutations Glu111Gly, Gln28Leu, Arg31Cys, Arg32Cys, and Asn34Lys that was more cytotoxic and selective than BS‐ RNase for cancer cells [100]. As another approach, covalent linkers to stabilize the dimeric structures have also been used. In this sense, first cytotoxic RNase A dimers [101, 102] and more recently higher oligomers cross‐linked with dimethyl suberimidate [103] were obtained. Although these constructs were cytotoxic, they presented heterogeneity, a drawback for their use as antitumor agents. The use of more specific cross‐linkers like the introduction of thioether bonds between different Cys residues of BS‐RNase [104] and RNase A [105], in some cases, allowed the production of variants with an increased cytotoxicity. Finally, an evaluation of cross‐linkers and selection of positions to introduce different Cys was carried out in the work of Rutkoski et al. [106]. In this case, some of the constructs were as cytotoxic as the RI‐evading RNases. However, as far as we know, none of the described constructs has reached clinical trials yet. An interesting and different way to get dimeric RNases consists in the fusion of two RNase genes using a linker to get a tandem RNase [107]. This construct although inhibited by the RI showed a cytotoxicity of the same order of that shown by BS‐RNase. Modeling studies of this tandem RNase bound to the RI revealed that the engineered enzyme binds the RI with a 1:1 stoichiometry, and the authors suggested that the cytotoxic effect was due to an improved endocytosis efficiency [108] likely due to a higher cationization (see below).

Finally, related with the formation of oligomeric structures, it is worth mentioning that RNase A can form 3D domain‐swapped multimers, ranging from trimers to hexamers [109, 110] and up to decatetramers [111]. These oligomers are enzymatically and biologically active [110, 112] and what is more interesting they exhibit cytotoxicity [99, 113]. The study of these oligomeric structures could reveal new scaffolds for the design of potential antitumor RNase variants [63].

#### *2.1.3. Targeting organelle RI-free*

trials for the treatment of solid tumors [25]. On the other hand, different strategies have been carried out to avoid some of the non‐desired side effects. The Gly88Arg RI‐evading RNase A, described above, was engineered to introduce nonnative disulfide bonds to increase its conformational stability [94], resulting in a more cytotoxic variant. Also, an increase in stability has also been attained by the glycosylation of the protein. For instance, the production of ONC in *Pichia pastoris* yields a glycosylated protein more stable and 50‐fold more cytotoxic [95].

The formation of oligomeric structures, such as the BS‐RNase dimers, has inspired the design and production of new RNase variants with the aim of precluding their binding to the RI by steric hindrance mimicking the way of action of BS‐RNase [96, 97]. The pursuit of dimeric or oligomeric variants is very attractive because they are more cationic proteins and can poten‐ tially strongly interact with the negative surfaces of cancer cells gaining selectivity and, at the same time, reducing kidney clearance due to the increase of molecular mass. As stated in Section 1.2, the current model for BS‐RNase cytotoxicity is that in the reducing conditions of the cell citosol, the unswapped isomer from (M=M) dissociates into monomers, which are strongly inhibited by the RI, whereas the swapped isomer (M×M) remains as a non‐covalent dimer able to evade the RI [64, 98]. In addition, analysis of the structure of the non‐covalent dimer of BS‐RNase [61] and different mutated forms [99] suggested that it adopts a compact quaternary structure that is critical for the RI interaction, explaining its trapping. One of the first approaches to get cytotoxic dimeric RNase variants was to reproduce the structural determinants of BS‐RNase swapping [62] in different members of the vertebrate‐secreted RNase family. Thus, different combinations of those residues identified as responsible of dimer formation (Cys31, Cys32, Leu28, Gly16, Ser80) of BS‐RNase were introduced in the sequence of either HP‐RNase or RNase A. Alternatively, the full N‐terminal hinge sequence (the peptide that links the N‐terminal α‐helix of V‐shaped RNase structure with the rest of the protein body) of RNase A was replaced by that of BS‐RNase [63] in order to endow RNase A with dimeri‐ zation abilities. These changes resulted in the formation of different ratios of swapped and unswapped forms, which was critical for their cytotoxicity [63]. Among these constructs it is remarkable that of a dimeric form of HP‐RNase containing the mutations Glu111Gly, Gln28Leu, Arg31Cys, Arg32Cys, and Asn34Lys that was more cytotoxic and selective than BS‐ RNase for cancer cells [100]. As another approach, covalent linkers to stabilize the dimeric structures have also been used. In this sense, first cytotoxic RNase A dimers [101, 102] and more recently higher oligomers cross‐linked with dimethyl suberimidate [103] were obtained. Although these constructs were cytotoxic, they presented heterogeneity, a drawback for their use as antitumor agents. The use of more specific cross‐linkers like the introduction of thioether bonds between different Cys residues of BS‐RNase [104] and RNase A [105], in some cases, allowed the production of variants with an increased cytotoxicity. Finally, an evaluation of cross‐linkers and selection of positions to introduce different Cys was carried out in the work of Rutkoski et al. [106]. In this case, some of the constructs were as cytotoxic as the RI‐evading RNases. However, as far as we know, none of the described constructs has reached clinical trials yet. An interesting and different way to get dimeric RNases consists in the fusion of two RNase genes using a linker to get a tandem RNase [107]. This construct although inhibited by

*2.1.2. RNase dimerization or oligomerization*

142 Anti-cancer Drugs - Nature, Synthesis and Cell

The tumor cell nucleus is the final destination of multiple conventional antitumor drugs [114, 115] as well as a critical compartment for suicide gene therapy [116]. In addition, drugs that do not have a native tendency to accumulate in the cell nucleus have been conjugated/ engineered/encapsulated by different means to reach this compartment. Literature is full of examples, for instance, drugs that have been modified by the introduction of a nuclear localization signal (NLS) as a modular component of a construct [117–119] and that have been encapsulated in nanoparticles directed to the cell nucleus [120] or the viral‐based vectors, which are an elegant choice as vehicles to deliver DNA that encodes therapeutic proteins or RNAs to this organelle [121–123]. Based on this, an alternative strategy to bypass the RI action was to guide the RNases to the cell nucleus, which is described as free of RI [124] or at least the nucleolus [125]. Initially, an HP‐RNase variant was produced, namely PE5, that carries a noncontiguous extended bipartite NLS [31, 126]. Although this variant is inhibited by the RI, at the same time it is recognized by α‐importin [126] and cleaves nuclear but not cytoplasmic RNA *in vivo* [127]. At present, the mechanism by which the engineered HP‐RNase reaches the cell nucleus is different from the one described above for BS‐RNase (Section 1.2). It is postulated that the concentrations of RI and α‐importin are similar in the cytosol. Thus, the affinity of PE5 for each protein will determine to which it will mainly bind. However, those RNase molecules captured by the α‐importin will be released into the nucleus and, therefore, removed from the two competing equilibriums, and PE5 will progressively accumulate into the nucleus [128]. PE5 kills the cells by apoptosis mediated by the induction of p21WAF/CIP1 and inactivation of JNK and increases the number of cells in the S‐G2/M‐phases of cell cycle [129]. Moreover, the cytotoxic mechanism of PE5 is not prevented by a mutated p53 or a multidrug‐resistant (MDR) phenotype [129], and it is synergic with doxorubicin [130] on doxorubicin‐resistant NCI/ADR‐ RES cell line [130]. Very recently, using microarray‐derived transcriptional profiling, it has been shown that PE5 remarkably downregulates multiple genes that code for enzymes involved in the deregulated metabolic pathways in cancer cells [131], one of the hallmarks of cancer. In addition, new cytotoxic RNase variants directed to the cell nucleus, collectively named ND‐ RNases, have been engineered either by reverting some of PE5 changes to render the variant more similar to wild‐type enzyme or by the addition of an extra NLS to its N‐terminus. In the latter case, a tenfold more cytotoxic enzyme than PE5 [132] has been obtained. Due to their cytotoxic mechanism, which differs from that of RNases that exert its action on the cell's cytosol, ND‐RNases are very interesting antitumor agents that can cope with the complexity of cancer cell phenotype, and their multiple effects allow anticipating synergism with many currently clinically used antitumor agents. In *in vivo* studies with animal models, the ND‐RNases have shown very low toxicity (it has not been possible to calculate the maximum tolerated doses (MTD) but the maximum feasible dose (MFD) which is of 80 mg/kg) (Castro et al., results not published).

#### **2.2. Engineered RNases that might saturate the intracellular RI and/or gain selectivity**

The efficiency of cell internalization is another important determinant of the cytotoxicity of the RNases because an RI‐sensitive RNase is still a potential danger provided that enough enzyme molecules reach the cell cytosol. The most basic strategy to increase the internalization of RNases is their cationization by chemical or genetic modification, that is, to make the RNases even more basic to increase their interaction with the anionic membranes of tumor cells. As stated above, this fact may also increase their selectivity for cancer cells [133]. Several examples of this approach can be found in the literature. The chemical modification of the carbonyl groups of RC‐RNase with a water‐soluble carbodiimide in the presence of nucleophiles or the amidation with ethylenediamine, 2‐aminoethanol, taurine, or ethylenediamine of HP‐RNase and RNase A increases their cytotoxicity [134–136]. The preparation of RNase A and noncy‐ totoxic cross‐linked dimers of RNase A, both covalently linked to polyspermine to increase their basicity, slightly increased their cytotoxicity [137]. In general, the higher cationic variants were more efficiently internalized into the cells. However, in some cases, the chemical modifications seriously compromised the ribonucleolytic activity of the modified enzymes [134, 135] and generated heterogeneous products difficult to use as antitumor drugs. RC‐RNase and RNase Sa variants were engineered substituting acidic residues by Asn, Gln, or Arg [138] or by positively charged residues [139, 140], respectively, showing antitumor activity and enhanced internalization. Gly38Lys‐BS‐RNase that bears an enforced cluster of positive charges at the N‐terminal surface also presented an increased cytotoxicity relative to its parental RNase and a higher membrane interaction capability [141]. Fuchs et al. [142] replaced two residues of a cytotoxic variant of RNase A to create a patch of Arg residues on its surface that rendered a threefold increase in cytotoxicity and added a protein translocation domain (nona‐arginine) to a previously cytotoxic RNase variant that increased their cytotoxicity [142, 143]. However, the same group has proposed that the internalization of pancreatic RNases by cationization can be counterbalanced by an increased affinity for the anionic RI in the cytosol [92]. Like for RI evasion, one has to be very cautious in the design of these variants in order to not counterbalance the increased internalization by the loss of other important characteristics responsible for the RNase cytotoxicity. In the same line but with a different approach, co‐ treating cells with a cationic 2 poly(amidoamine) dendrimer [144] increase the cytotoxicity of the RNase probably by increasing its translocation from the endosomes without affecting its ribonucleolytic activity or conformational stability observed upon cationization of some RNases.

In addition to merely increasing the positive charge of an RNase, other approaches that can enhance its delivery to the cytosol or to a specific organelle are related to the construction of targeted RNases either by chemical conjugation or fusion with a specific component that directs them to cancer cells. These procedures have been used with other drugs combining a targeting molecule, mainly antibodies, with an effector moiety getting what has been called immunotoxins (see below) [145, 146]. Small molecule drugs are still the modality of choice for addressing intracellular targets due to the barriers to cell entry that proteins have to face. Nevertheless, despite the considerable research efforts and advances attained, there remain many protein‐protein interactions that small molecules cannot modulate effectively [147], and proteins have a lower propensity for off‐targets. Thus, the strategies described below include both small molecules and proteins as drivers of payload RNases, including that nonsensitive to the RI.
