*2.2.2. Chemical conjugation*

Targeting RNase molecules to tumor cells was early carried out by chemical conjugation of both RNase A to transferrin (Tf) or mAb against Tf receptor (TfR) [148] as well as to the T‐cell antigen CD5 [149] and ONC to anti‐Tf receptor mAb 5E9 [150]. These studies showed that the antibody conjugates were more efficient than Tf conjugates and that ONC and RNase A conjugated to antibodies by a reducible disulfide bond were equally potent (IC50 values in the nM range). However, ONC conjugates showed increased efficiency likely due to the fact that ONC is not inhibited by the RI, while RNase A might not be able to saturate it even when conjugated to a particular cell driver [151]. In these primary studies also, ONC was conjugated to CD22‐specific mAb LL2 and RFB4, which resulted in a several thousand‐fold increase in cytotoxicity comparable to that of anti‐CD22 immunotoxin conjugated to plant or bacterial toxin cargoes [152]. These results confirmed that RNases are as potent as these toxins when properly targeted. Nevertheless, although these chemical conjugates reduced the tumor size in animal models while not showing appreciable toxicity, their lot‐to‐lot heterogeneity was a serious drawback for further development [153]. ONC was also conjugated to P‐glycoprotein (P‐gp) neutralizing mAb MRK16. This construct increased ONC cytotoxicity and at the same time sensitized the multidrug‐resistant cancer cells that overexpress MDR1 gene to vincristine [154]. These results may be explained by both the mAb binding to P‐gp that diminishes its drug‐expelling ability and its ability to internalize ONC.

More recently, the amino groups of Lys side chains of RNase A and variant Lys41Arg (Lys41 is an amino acid critical for the ribonucleolytic activity of the enzyme [155]) were randomly conjugated to folate since folate receptors are overexpressed on the surface of many types of cancer [156, 157]. However, the results showed an abolition of the catalytic activity of RNase A, while the variant Lys41Arg only retained 54 % of its catalytic activity. In the same work, a folate analogue was designed, produced, and used to specifically S‐alkylate Cys residues introduced by site‐directed mutagenesis at positions 19 or 88. Only those proteins modified at positions that endow them with the ability to evade the RI were able to diminish cell prolifer‐ ation. Thus, even in this case, enhanced internalization had to be accompanied by an RI evasion [158].

## *2.2.3. Fusion RNases and ImmunoRNases*

The progress attained in the technology of construction and production of recombinant fusion proteins particularly using antibodies [145, 146] has been applied to RNases. Several RNases have been used either as scaffold onto which a targeting domain is engineered or fused, including antibodies. In the latter case, several antibody constructs such as scFv, diabodies, scFv‐Fc, and F(ab)2 antibody fragments were used as a fusion partner [159]. Generally speaking, small antibody constructs are best suited to penetrate and distribute into solid tumors. However, the smaller the construct, the faster it disappears from blood circulation. Thus, a compromise in the molecular mass has been agreed, between 60 and 120 kDa for a therapeutic protein in order to ensure a good pharmacokinetics [160]. On the other hand, when choosing a particular RNase to be fused to an antibody or any targeting domain, it has to be taken into account the connecting linker and the orientation of the RNase relative to the carrier molecule in order not to alter some of the previously described RNase properties important for their cytotoxicity.

Primarily, HP‐RNase as well as other members of the same family [161] such as angiogenin (ANG) and other RNases such as eosinophil‐derived neurotoxin (EDN) and some engineered variants of them were tested in experimental sets of RNase‐antibody fusion targeting Tf. They showed about 103 times more potency than the respective chemically conjugated RNase‐ antibody (Section 2.2.2) [149, 162]. The main concern with these constructs was the host selected for production indicating that the final yield was dependent on the expression system used [154]. To overcome production concerns and to get more specific clinical targets, different immunoRNases have been produced directed to antigens expressed on certain types of leukemia but not in hematopoietic stem cells, such as CD22 and CD30 or the ErbB2 that showed significant advantages over the equivalent chemical conjugates [153]. As alternatives, ANG, HP‐RNase, or RapLR1 (*R. pipiens* liver RNase 1), a close relative of ONC, were fused to two CD22‐specific scFV antibody fragments generated either by reengineering the variable domain core structure of mAb LL2 or grafting the complementary‐determining regions (CDR) of the clinically established mAb RF4B into consistent human scFv scaffolds [163–165]. Some of them were successfully produced and exhibited potent cytotoxicity (IC50 in the nM range) [164] which drove to a second generation of anti‐CD22 immunoRNases in diabody format fusing LL2 or humRFB4 to ANG or RapLRI. Bivalent anti‐CD22 immunoRNases showed a superior cytotoxicity toward CD22+ tumor cells when compared to their monovalent counterparts due to antigen binding by avidity and enhanced internalization [163, 164]. Different CD30‐ targeting constructs have also been produced fusing HP‐RNase or ANG to CD30‐specific murine or human scFvs that inhibited tumor growth [166], but the entirely human bivalent scFv‐Fc‐HP‐RNase showed better properties and inhibited the growth of CD30+ Hodgkin lymphoma cells [167]. Even better results were obtained for immunoRNases resulting from the fusion of CD30‐specific scFv Ki4 to ANG [168]. Recently, a humanized anti‐epidermal growth factor receptor (EGFR) scFV was used to target ONC to EGFR‐expressing tumor cell lines [169]. Fusion was accomplished by a flexible linker (G4S)3, but the construct resulted in very poor cytotoxicity, likely due to endosomal accumulation and lysosomal degradation. To avoid this drawback, the authors substituted the linker by a peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. The modified immu‐ noRNase exhibited exceptionally high cytotoxicity toward EGFR‐expressing head and neck cancer cell lines without affecting specificity. More recently, the same research group con‐ structed a derived diabody fragment with the specificity of the clinically established mAb Cetuximab to deliver ONC to EGFR‐expressing tumor cells. The dimeric immunoRNase was several orders of magnitude more cytotoxic toward EGFR‐expressing tumor cell lines than its monomeric counterpart and exhibited significant antitumor activity in a murine A431 xenograft model, but in this case, the linker was (G4S)3 [170]. Thus, not only the linker between the ONC and the antibody fragment is important but the structure of the antibody moiety.

properly targeted. Nevertheless, although these chemical conjugates reduced the tumor size in animal models while not showing appreciable toxicity, their lot‐to‐lot heterogeneity was a serious drawback for further development [153]. ONC was also conjugated to P‐glycoprotein (P‐gp) neutralizing mAb MRK16. This construct increased ONC cytotoxicity and at the same time sensitized the multidrug‐resistant cancer cells that overexpress MDR1 gene to vincristine [154]. These results may be explained by both the mAb binding to P‐gp that diminishes its

More recently, the amino groups of Lys side chains of RNase A and variant Lys41Arg (Lys41 is an amino acid critical for the ribonucleolytic activity of the enzyme [155]) were randomly conjugated to folate since folate receptors are overexpressed on the surface of many types of cancer [156, 157]. However, the results showed an abolition of the catalytic activity of RNase A, while the variant Lys41Arg only retained 54 % of its catalytic activity. In the same work, a folate analogue was designed, produced, and used to specifically S‐alkylate Cys residues introduced by site‐directed mutagenesis at positions 19 or 88. Only those proteins modified at positions that endow them with the ability to evade the RI were able to diminish cell prolifer‐ ation. Thus, even in this case, enhanced internalization had to be accompanied by an RI

The progress attained in the technology of construction and production of recombinant fusion proteins particularly using antibodies [145, 146] has been applied to RNases. Several RNases have been used either as scaffold onto which a targeting domain is engineered or fused, including antibodies. In the latter case, several antibody constructs such as scFv, diabodies, scFv‐Fc, and F(ab)2 antibody fragments were used as a fusion partner [159]. Generally speaking, small antibody constructs are best suited to penetrate and distribute into solid tumors. However, the smaller the construct, the faster it disappears from blood circulation. Thus, a compromise in the molecular mass has been agreed, between 60 and 120 kDa for a therapeutic protein in order to ensure a good pharmacokinetics [160]. On the other hand, when choosing a particular RNase to be fused to an antibody or any targeting domain, it has to be taken into account the connecting linker and the orientation of the RNase relative to the carrier molecule in order not to alter some of the previously described RNase properties important

Primarily, HP‐RNase as well as other members of the same family [161] such as angiogenin (ANG) and other RNases such as eosinophil‐derived neurotoxin (EDN) and some engineered variants of them were tested in experimental sets of RNase‐antibody fusion targeting Tf. They

antibody (Section 2.2.2) [149, 162]. The main concern with these constructs was the host selected for production indicating that the final yield was dependent on the expression system used [154]. To overcome production concerns and to get more specific clinical targets, different immunoRNases have been produced directed to antigens expressed on certain types of leukemia but not in hematopoietic stem cells, such as CD22 and CD30 or the ErbB2 that showed significant advantages over the equivalent chemical conjugates [153]. As alternatives, ANG,

times more potency than the respective chemically conjugated RNase‐

drug‐expelling ability and its ability to internalize ONC.

evasion [158].

for their cytotoxicity.

showed about 103

*2.2.3. Fusion RNases and ImmunoRNases*

146 Anti-cancer Drugs - Nature, Synthesis and Cell

An ONC variant with a modified putative N‐glycosylation site (Asn69Gln) has also fused to humanized antibody hRS7 raised against Trop‐2, a cell surface glycoprotein expressed in a variety of epithelial cancers [171]. The construct contained two ONC molecules fused to each of the N‐terminus of light chains of the antibody and was produced in stably transfected myeloma cells. The purified immunoRNase inhibited the proliferation of Trop‐2‐expressing cell lines with an IC50 in the nM range and was able to suppress tumor growth in a prophylactic model of nude mice bearing Calu‐3 human non‐small cell lung cancer xenografts with an increase of the median survival time from 55 to 96 days [172]. More recently, a second gener‐ ation of these immunoRNases has been produced by the dock‐and‐lock (DNL) method. This methodology consists in the use of a pair of distinct protein domains that are involved in the natural association between protein kinase A (PKA; cyclic AMP‐dependent protein kinase) and A‐kinase‐anchoring proteins. The dimerization and docking domain (DDD) found in the regulatory subunit of PKA and the anchoring domain (AD) of an interactive‐A‐kinase anchoring protein are each attached to a biological entity through specific linkers and the resulting derivatives, when combined, readily form a stably tethered complex of defined composition that fully retains the functions of individual constituents. That is, the docked complex can be made irreversible using a pair of linker modules that introduce Cys residues into both the DDD and the AD domains at strategic positions that facilitate the formation of disulfide bridges [173]. The integration of genetic engineering and conjugation chemistry of the DNL method has been used to get two constructs containing four ONC molecules linked to either the CH3 or CK C‐termini of hRS7 that have been evaluated as potential therapeutics for triple‐negative breast cancer (TNBC). Both constructs showed specific cell‐binding and rapid internalization in MDA‐MB‐486, a Trop‐2‐expressing TNBC, and displayed potent *in vitro* cytotoxicity against diverse breast cancer cell lines. In addition, both seemed well tolerated at clinically relevant concentrations. However, CK‐based construct exhibited superior Fc‐ effector functions *in vitro*, as well as improved pharmacokinetics, stability, and activity *in vivo*. Further studies are needed regarding their immunogenicity although they are potentially a new class of immunoRNases that warrant future research [174].

Not only animal RNases have been used to construct immunoRNases. For instance, the construct formed by two barnase molecules fused serially to scFv of humanized 4D5 antibody directed to the extracellular domain of epidermal growth factor receptor 2 (HER2 or ErbB2) was produced [175]. This scFv 4D5‐dibarnase showed cytotoxicity *in vitro* and significant *in vivo* inhibition of human breast cancer xenografts in nude mice without severe side effects [176].

The first entirely human immunoRNase was produced fusing HP‐RNase to an ErbB2‐specific scFv named Erbicin [177, 178]. The construct recognizes an epitope distinct to that of trastu‐ zumab and pertuzumab [179], the two humanized antibodies currently used to treat HER2+ metastatic mammary carcinomas [180, 181] and do not induce cardiac dysfunction as the other two do [182–184]. Although this immunoRNase was inhibited by the RI to an extent compa‐ rable to that of HP‐RNase, the quantity that entered the cell cytosol saturated the RI, and it exhibited a clear RNA degradation ability [185]. Due to this limitation, a second generation of immunoRNases was obtained by fusing an RI‐insensitive HP‐RNase variant (Arg39Asp/ Asn67Asp/Asn88Ala/Gly89Asp/Arg91Asp) [186] to ErbB2‐specific scFv showing resistance to RI inhibition and the ability to kill mammary ErbB2+ tumor cells more efficiently [187]. This variant does not show cardiotoxic effects *in vitro* and does not impair cardiac function in mouse models [188]. In addition, since bivalent immunoRNases are more powerful than monovalent ones, a dimeric variant of HP‐RNase was fused to two Erbicin molecules, one per subunit [189]. The new construct was able to bind to ErbB2‐positive cancer cell lines with an increased avidity with respect to the monovalent variant and was a more cytotoxic, likely due to an increased RI evasion.
