*2.2.4. Delivery strategies*

Although RNases have reached clinical trials, one important aspect that researchers have still to cope with is to improve their tissue delivery. This means, to enhance the RNases circulating half‐live in the blood and to avoid a high glomerular filtration rate. These factors contribute to an optimal pharmacokinetics and biodistribution. Related to these issues, some formulations have been carried out. One of the ways to increase persistence in circulation of small proteins such as RNases is PEGylation. Early, RNase A‐PEG conjugates were randomly made [190– 194]. However, although they presented increased persistence in circulation, they showed a significant reduced catalytic efficiency due to modification of the critical catalytic residue Lys 41 that abolished their cytotoxic properties. More recently, previously acylated RNase A [195] or HP‐RNase [196] has been PEGylated at specific positions (Gly88Cys in RNase A and Gly89Cys in HP‐RNase). Although the conjugates show a significantly reduced cytotoxicity *in vitro*, they are effective in inhibiting tumors in xenograft mouse models, likely because the diminished renal clearance *in vivo* compensates the potential loss of cytotoxicity due to the PEG moiety. Due to the efficacy of this approach, mono‐PEGylation of RNase A has been studied using two chemicals, N‐hydroxysuccinimide ester of S‐acetylthioacetic acid (SATA) and 2‐iminothiolane (IT). Both react with primary amino groups to introduce thiol groups, a process followed by PEGylation using maleimide chemistry. Interestingly, by thiolation, the original positive charges of RNase A can be conserved, an important feature in order not to lose the cationic residues. In addition, in both cases the enzymatic activity of the RNase A was essentially maintained [197].

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

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

Although RNases have reached clinical trials, one important aspect that researchers have still to cope with is to improve their tissue delivery. This means, to enhance the RNases circulating half‐live in the blood and to avoid a high glomerular filtration rate. These factors contribute to an optimal pharmacokinetics and biodistribution. Related to these issues, some formulations have been carried out. One of the ways to increase persistence in circulation of small proteins such as RNases is PEGylation. Early, RNase A‐PEG conjugates were randomly made [190– 194]. However, although they presented increased persistence in circulation, they showed a

a new class of immunoRNases that warrant future research [174].

148 Anti-cancer Drugs - Nature, Synthesis and Cell

RI evasion.

*2.2.4. Delivery strategies*

Another method to increase the half‐life of a protein in the blood is its conjugation with bovine serum albumin (BSA) [198]. However, depending on the way used to get the conjugate, albumin can decrease the enzymatic activity of RNase A. Thus, different strategies have been described to prepare RNase A‐BSA conjugates to keep the bioactivity of the enzyme, although the pharmacokinetic and pharmacodynamic properties still need to be determined [199].

In an attempt to get an ONC that can circumvent renal clearance, improve tumor cell targeting, and gain endosomal escape, a modular construct has recently been described. ONC was fused to human serum albumin (HSA) through its C‐terminus, and this former construct was also C‐terminus appended to scFv 4D5MOCB, which targets epithelial cell adhesion molecule (EpCAM), a validated target for anticancer therapy [200] (construct Onc‐HSA‐4D5MOCB). In addition, in the same work, the link between ONC and the rest of the construct was also carried out through a cleavable disulfide linker (construct Onc‐SS‐HSA‐4D5MOCB) that potentially enables the release of ONC from its carrier after endocytosis and avoids HSA inactivation of ONC catalytic activity. Although both constructs overcame most of the *in vitro* barriers, *in vivo* toxicity studies with animal models showed that they increased liver toxicity while ONC is described to produce renal toxicity [32]. Unfortunately, only the construct Onc‐SS‐ HSA‐4D5MOCB showed a reduction of tumor growth, but it was similar to that of ONC alone, and the tumor started to regrowth when treatment was discontinued [201]. Nevertheless, this all‐in‐one drug delivery system may inspire other constructs that can accomplish the pursued goal.

The genetic delivery of ONC using oncolytic adenovirus has just been tested. A combination of viral oncolysis with intratumoral genetic delivery of an EGFR‐binding scFv antibody fragment fused to ONC (ONCEGFR) has demonstrated feasible. ONCEGFR expression by oncolytic viruses is possible with an optimized, replication‐dependent gene expression strategy. Very interestingly, virus‐encoded ONCEGFR induced a potent and EGFR‐dependent bystander killing of tumor cells. That is, some of the non‐transformed cells die by the entry of ONCEGFR released from transfected cells. Thus, ONCEGFR‐encoding oncolytic adenovirus showed dramatically increased cytotoxicity specifically to EGFR‐positive tumor cells *in vitro* and significantly enhanced therapeutic activity in a mouse xenograft tumor model. The authors claim that this virus‐antibody therapy platform can be further developed for personalized therapy by exploiting antibody diversity to target further established or emerging tumor markers or combination of thereof [202].

Finally, to avoid cerebellar neuronal toxicity while affecting glioma cells, ONC has been encapsulated in biodegradable poly(ricinoleic‐co‐sebacic acid) for local controlled delivery in the parietal lobe of the brain [203]. In this way ONC was released in a controlled manner and was cytotoxic against 9L glioma cells xenograft into the brain while evading neurotoxicity in the cerebellum.
