Breast and Colon Cancer - Immunotoxins targeted at LeYon 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 investigated the basis of this toxicity by preparing mutants of LMB-9 which should have diminshed nonspecific binding to cells in the liver by mutating basic residues in the Fv region. We have succeeded in making a mutant molecule which is approximately 2-fold less toxic to mice, yet retains full antitumor activity. 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, pancreatic cancer and some squamous cell and gastric cancers. We have carried out mutagenesis studies to increase the affinity of SS1(dsFv)-PE38 by mutating the CDRs and produced SS1(dsFv)-PE38. We have been able to reduce the nonspecific toxicity in mice of SS1(dsFv)-PE38 by mutating framework residues of the Fv that could be involved in nonspecific binding events. SS1(dsFv)-PE38 (SS1P)is now being evaluated in clinical trails at NIH and the University of Oklahoma. Glioblastomas - 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 have obtained an IND for a clinical trial to be carried out at Duke. Patients receive TGFa-PE38 by continuous perfusion into recurrent glioblastomas with the aim of establishing a safe dose and searching for possible efficacy. Because approximately 50% of glioblastomas produce a mutated form of the EGF receptor termed EGFRvIII, we have isolated an Fv fragment that specifically binds to this mutant receptor and have now used mutagenesis procedures to produce a mutant Fv (MR1-1) with an increase in affinity. This Fv has been used to make a recombinant immunotoxin that is very cytotoxic to glioblastoma cells. Preclinical studies show this recombinant immunotoxin should be developed for the treatment of glioblastomas. The RAID Program of NCI has agreed to produce MR1-1FvPE38 KDEL for a Phase 1 clinical trial. We have supplied the MR1-1 Fv to our collaborator, Dr. Darell Bigner at Duke. Investigators in his group have radiolabelled 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. In a Phase 1 trial with RFB4(dsFv)-PE38 (BL22) was shown to have excellent activity in drug resistant Hairy Cell Leukemia but low activity in CLL. To make a more active form of BL22, 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 increased affinity. Using this approach, a new Fv was produced that has a 5-fold increase in affinity and makes an immunotoxin (HA22) with a 5-10-fold increase in activity on cells from patients with CLL and HCL. A clinical lot of HA22 will be produced for a Phase 1 trial in CLL. CD30 is an antigen highly expressed in Hodgkin's disease and anaplastic large cell lymphoma. A series of antibodies to CD30 have been produced and several used to make immunotoxins with good cytotoxic activity against CD30 expressing cell lines. Non-Specific Toxicity and Immunogenicity - Immunotoxin therapy could be improved if non-specific toxicity and immunogenicity could be diminished or eliminated enabling a large dose to be given at repeated intervals. We have found that the non-specific toxicity of the immunotoxin anti-Tac(Fv)-PE38 (LMB-2) is due to uptake by Kupffer cells of the liver and the release of TNFa kills hepatocytes. This toxicity is blocked by the cycycloxygenase inhibitor indomethacin or by a protein that blocks TNFa action. To control immunogenicity we have used site-directed mutagenesis to introduce a cysteine into the peptide connecting the Fv to the toxin and have derivatized this cysteine with PEG. PEGylated molecules have been found to have markedly diminished immunogenicity and non-specific toxicity yet enhanced stability and antitumor activity. Other approaches to PEGylation are now being explored Radiolabelled Conjugates - We are collaborating with Dr. Jorge Carrasquillo (Clinical Center) and Dr. Michael Bishop (NCI) to develop a radioimmunotherapy for colon and breast cancer using monoclonal antibody B3 that binds to the LeY antigen. We have completed a Phase 1 study with a radiolabelled 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 radiolabelled ligand which specifically targets to antibody containing cells.
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