The major goal of this work is to use human RNases and homologous RNases from other species instead of toxic plant and bacterial proteins in the construction of immunotoxins. Two of the major problems with the clinical use of immunotoxins is the toxicity and immunogenicity of the toxins. The use of human and humanized RNases addresses these problems. Isologous fusion proteins administered to mice are indeed less immunogenic and less toxic than fusion proteins made with plant or bacterial toxins. A human RNase based immunotoxin can prevent the growth of human glioma cells in athymic mice. Furthermore, both single-chain and intact-antibody RNase fusion proteins can be made in the milk of transgenic animals in amounts approaching 1 mg/ml. A homologous RNase (Onconase) from frog oocytes has inherent anti-tumor properties and is in clinical trials as an anti-cancer agent. Onconase has been administered to humans on a weekly basis for up to six months without causing immunological problems or serious toxicities, presumably because of its homology to human plasma RNases. The potency and specificity of Onconase as an anticancer agent can be improved by targeting. Onconase conjugated to the anti CD22 antibody LL2 inhibits the growth of disseminated human Daudi lymphoma cells in SCID mice, even when administred 7 days after tumor cell inoculation. Since the MST of untreated mice is 31 days, the conjugate is efficatious in treating fairly advanced cancer (MST conjugate treated mice 79 days). Based on these results a CRADA is being arranged between the NCI and industrial collaborators to begin preclinical evaluation of this conjugate including primate toxicology. Other studies of Onconase in combination with adriamycin to treat solid breast carcinomas are supported by another CRADA. Significant synergy is seen when adriamycin is combined with Onconase over either agent alone. The MST of athymic nude mice injected with MDA-MB-231 breast cancer cells is 55 days; adriamycin treated animals 70 days; Onconase treated animals 55 days; Onconase and adriamycin treated animals 179 days. These results mimic the effect of Onconase combined with drugs in human clinical trails. The mechanism of this synergy will be studied. To enable future generations of Onconase based drugs the gene has been cloned from Rana pipiens genomic DNA. Furthermore, Onconase has been humanized by making a hybrid protein comprised of sequences from human eosinophil RNase and frog Onconase.
