Breast and Colon Cancer - Immunotoxins targeted at LeY on epithelial cancers have been constructed, and one of these (LMB-9) that is stabilized by a disulfide bond engineered into the framework region is now in clinical trials. Nonspecific toxicity of recombinant immunotoxins in mice is manifest by damage to the liver. We have begun to investigate the basis of this toxicity by preparing mutants of LMB-9 which should have diminshed nonspecific binding to cells in the liver by mutating residues in the Fv region. We have succeeded in making a mutant molecule which is approximately 2-fold less toxic to mice and are now characterizing its properties further. Ovarian Cancer - We used DNA immunization combined with phage display to produce ss(Fv)-PE38, a recombinant immunotoxin that kills cancer cells expressing mesothelin. These include cancers of the ovary, mesotheliomas, and some squamous cell and gastric cancers. We have carried out mutagenesis studies to increase the affinity of SS(dsFv)-PE38 by mutating the CDRs. We have now prepared a variety of mutants which have increased cytotoxic activity. These mutants are now being characterized for their activity in cell culture and in animal models with the aim of identifying one molecule that could be carried forth into the clinic. In addition, we ahve carried out studies to reduce the nonspecific toxicity of one of these recombinant immuntoxins, SS1(dsFv)-PE38 by mutating framework residues that could be involved in nonspecific binding events. These molecules are currently being characterized. Glioblastomas ? During the past year, we have prepared a clinical lot of TGF alpha-PE38 which is very cytotoxic to glioblastoma cells. These cells almost uniformly overexpress EGF receptors. We are currently completing toxicology studies and preparing an IND for a clinical trial to be carried out at Duke and UCSF. The current plan is to administer TGF alpha-PE38 by continuous perfusion into recurrent glioblastomas with the aim of establishing a safe dose and searching for possible efficacy. Approximately 50% of glioblastomas produce a mutated form of the EGF receptor termed EGFRvIII. We have previously isolated an Fv fragment that specifically binds to this mutant receptor termed MR1 and have now used mutagenesis procedures to produce a mutant Fv with an increase in affinity termed MR1-O12. This Fv has been used to make a recombinant immunotoxin that has increased cytotoxic activity towards glioblastoma cells. We are now carrying out preclinical studies to evaluate whether or not this recombinant immunotoxin could be developed for the treatment of glioblastomas. We have supplied MR1(Fv)O12 to our collaborator, Dr. Darell Bigner at Duke. Investigators in his group have radiolabeled the Fv and shown that it specifically targets glioblastomas. Hematopoietic Tumors - RFB4(dsFv)-PE38 is a recombinant immunotoxin that binds to CD22 and kills CD22 expressing tumor cells. A Phase I trial with RFB4(dsFv)-PE38 (BL22) is currently being conducted by Dr. Robert Kreitman. Our goal is to make a more active form of BL22. To accomplish this we have used molecular modeling and DNA sequence analysis to identify regions in the CDRs of BL22 that can be mutated and are likely to lead to Fvs with incresed affinity. An initial round of mutageneis has been perfomed and a new Fv produced that has a 4-fold increase in cytotoxic activities. We plan to continue mutagenesis studies in order to find a mutant with a 10-fold increasee in activity. Another antigen that is prevalent on leukemia cells is CD19. We have prepared a recombinant immunotoxin targeting CD19. We plan to increase the activity of this molecule by mutating CDR residues as described above. Osteosarcomas - We have obtained recombinant Fv fragments of two antibodies that react with osteosarcomas from Dr. Bruland. We have prepared recombinant immunotoxins from these antibodies and shown that TP3(dsFv)-PE38 has good cytotoxic activity against osteosarcoma cell lines. Radiolabeled Conjugates - We are collaborating with Dr. Jorge Carrasquillo to develop a radioimmunotherapy for colon and brest cancer using monoclonal antibody B3 that binds to the LewisY antigen. We have completed a Phase I study with a radiolabeled conjugate which indicates that the antibody effectively targets tumors expressing LeY. We are now trying to improve targeting by using a pretargeting method in which unlabeled antibody is first administered to patients followed by a short-lived radiolabeled ligand which specifically targets antibody containing cells.
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