Immunotoxins in bone marrow transplantation

（申请人摘要）移植物抗宿主病（GVHD）是一门专业

DESCRIPTION: (Applicant's Abstract) Graft-versus-host-disease (GVHD) is a major complication in bone marrow (BM) transplantation and despite attempts at limiting it, GVHD still occurs in over 60% of matched transplants with high mortality rates. Over the past 15 years, we have constructed aggressive murine GVHD models based on this very grant and found that one of the best ways to target ongoing GVHD is with potent immunotoxins (IT), antibodies linked to catalytic toxins. We pursued conventional biochemically linked agents as far as possible, but studies revealed toxicity and caused us to conclude that the limitations of IT must be addressed with agents that can be genetically modified. Thus, we assembled a modifiable vector consisting of the anti-CD3 single chain Fv (smallest unit of antigen recognition) spliced to truncated diphtheria toxin. DT390anti-mCD3sFv protein was capable of potent anti-GVHD effects even in MHC disparate recipient mice, but its dose was limited by organ toxicity. In the last round of funding, we screened several potential modifications and used an aggressive mouse GVHD model to discover a modification that markedly decreased organ toxicity, increased tolerated dosage, creating a therapeutic window (TW) where none existed before. This was achieved by inserting a cysteine residue downstream of the sFv moiety at the c-terminus. This modification produced intermolecular disulfide bridging resulting in unique bivalent IT called MuSS2. In this round of funding, we plan to determine whether MuSS2 is successful in more aggressive models of animal GVHD that more closely approximate the manner in which we will be using it clinically. These include a therapy model of ongoing established GVHD designed to determine whether MuSS2 is effective against expanding GVHD-causing T cells. A second model will employ high dose irradiation conditioning since our first usage will be likely in clinical situations where patients have been conditioned aggressively. One advantage to allogeneic BM transplantation is a T cell-mediated graft-versus-leukemia (GVL) response. A third murine model established by our group will be used to address the important issue of whether anti-T cell GVHD effects induced by MuSS2 will interfere with advantageous T cell GVL responses. Our data indicate that it is the non-stabilized monomer that kills mice due to its filtration into kidneys. Therefore, stabilizing IT and preventing its breakdown may increase its efficacy even further widening the TW. Therefore, we will test 2 additional modifications designed to further enhance IT stability in vivo. One of these modifications will be directed at improving MuSS2 by introducing a stabilized intermolecular disulfide linkage specifically designed to curtail in vivo glutathione reduction of these bonds. A second, different approach will involve the insertion of Fc domain stabilizing sequences that have been shown to regulate antibody catabolism. If successful, these approaches would be applied to the final design of a human homologue currently under development.