Indeed, even heterologous RNases like Rana pipiens-derived ranpirnase (Onconase ® Alfacell, Inc) were safely administered into human patients and immunologically well-tolerated. 8 Moreover, immunogenicity issues and unspecific toxicity are not expected because human RNases are ubiquitously distributed all over the human body and reside in plasma and most tissues. 7 Intracellular microinjection of pancreatic RNase into Xenopus oocytes revealed cytotoxicity similar to those of ricin whereas high extracellular concentrations are well-tolerated. Particularly, ribonucleases (RNases) of the RNase A family have been investigated because they become cytotoxic only after translocation into the cytosol of target cells. To overcome immunogenicity and non-specific toxicity issues, human effector enzymes have been studied as alternative to heterologous toxins. Subsequent evaluation revealed that one compound showed tendency of aggregation after thawing and fatalities appeared only in patients with low number circulating tumor cells. 5 A Phase 1 study of refractory B cell lymphoma patients with a combination of two different IgG::deglycosylated ricin A chain immunotoxin conjugates (Combotox) resulted in three deaths at least two were directly related to the immunotoxin treatment. The ADC gemtuzumab ozogamicin (Mylotarg ®), approved by the US Food and Drug Administration for treatment of CD33+ acute myeloid leukemia was voluntarily withdrawn from the market because of increased occurrence of fatalities caused by hepato-occlusive disease upon treatment. 2, 3 Highly toxic drugs and heterologous toxins conjugated to antibodies have been extensively tested as so-called immunotoxins, 4 but this approach has been associated with severe adverse side effect, and even fatalities, due to unspecific toxicity and immunogenicity. 1 Another promising area is use of antibodies to internalizing tumor-associated antigens as vehicles to deliver cytotoxic payloads inside target cells, although the results to date have been mixed. Promising antibodies with novel mechanisms of action, such as programmed death 1 receptor antagonists lambrolizumab and nivolumab, are still in Phase 3 studies. Recombinant antibodies represent the largest class of biological therapeutics for treatment of cancer diseases, but, to date, clinical benefit of therapies with monoclonal antibodies has not met the initial anticipations and hopes. Taken together, human pancreatic RNase and variants did not prove to be generally suitable as effector component for a therapeutic antibody drug development platform. Similarly, RI evasive human pancreatic RNase variants mediated only small inhibiting effects on tumor cell growth at high concentrations, potentially reflecting inefficient cytosolic translocation. The introduction of different linkers containing endosomal protease cleavage sites into the IgG-RNase did not enhance cytotoxicity. Despite these promising properties, none of the IgG-RNases revealed significant inhibition of tumor cell growth in vitro even when targeting different antigens putatively employing different endocytotic pathways. Serum stability, cell binding and internalization of IgG-RNases were comparable to the parental IgGs. Antigen binding was comparable to the parental antibodies and RNase catalytic activity was retained even in the presence of 50-fold molar excess of human cytosolic RNase inhibitor (RI). Transient mammalian cell production was efficient and IgG-RNases were purified to homogeneity. We generated several fusion proteins consisting of tumor-specific human immunoglobulins (IgGs) and human pancreatic RNase. In this study, we investigated if human pancreatic RNase will be suitable as effector component in a therapeutic antibody development platform. Human antibody-ribonuclease (RNase) fusion proteins, referred to as immunoRNases, have been proposed as an alternative to heterologous immunotoxins, without their immunogenicity and unspecific toxicity issues.
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