Publications

Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded as an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers using PET. Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human CD44. The specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell–expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG1–fragment crystallizable (Fc). The scFv-Fc-CD44 was radiolabeled with 64Cu and 89Zr. The purified reagents were injected into athymic nude mice bearing CD44-positive human tumors (MDA-MB-231, breast cancer, triple-negative). Biodistribution studies were performed at different times after injection of [64Cu]Cu-NOTA-scFv-Fc-CD44 or [89Zr]Zr-DFO-scFv-Fc-CD44. PET/CT imaging was conducted with [89Zr]Zr-DFO-scFv-Fc-CD44 on days 1 and 7 after injection and compared with a scFv-Fc control antibody construct targeting glycophorin A. Results: Epitope mapping of the scFv binding site revealed a linear epitope within the extracellular domain of human CD44, capable of blocking binding to native hyaluronic acid. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor-to-blood ratios for [89Zr]Zr-DFO-scFv-Fc-CD44 between days 5 and 7. PET imaging confirmed excellent tumor specificity for [89Zr]Zr-DFO-scFv-Fc-CD44 when compared with the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64Cu or 89Zr, it demonstrated specific uptake in CD44-expressing MDA-MB-231 tumors. The high tumor uptake (∼56% injected dose/g) warrants clinical investigation of [89Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors.

Huntington’s disease (HD) is caused by a highly polymorphic CAG trinucleotide expansion in the gene encoding for the huntingtin protein (HTT). The resulting mutant huntingtin protein (mutHTT) is ubiquitously expressed but also exhibits the ability to propagate from cell-to-cell to disseminate pathology; a property which may serve as a new therapeutic focus. Accordingly, we set out to develop a monoclonal antibody (mAB) targeting a particularly exposed region close to the aa586 caspase-6 cleavage site of the HTT protein. This monoclonal antibody, designated C6-17, effectively binds mutHTT and is able to deplete the protein from cell culture supernatants. Using cell-based assays, we demonstrate that extracellular secretion of mutHTT into cell culture media and its subsequent uptake in recipient HeLa cells can be almost entirely blocked by mAB C6-17. Immunohistochemical stainings of post-mortem HD brain tissue confirmed the specificity of mAB C6-17 to human mutHTT aggregates. These findings demonstrate that mAB C6-17 not only successfully engages with its target, mutHTT, but also inhibits cell uptake suggesting that this antibody could interfere with the pathological processes of mutHTT spreading in vivo.

Background Kawasaki disease (KD) is the leading cause of childhood acquired heart disease in developed nations and can result in coronary artery aneurysms and death. Clinical and epidemiologic features implicate an infectious cause but specific antigenic targets of the disease are unknown. Peripheral blood plasmablasts are normally highly clonally diverse but the antibodies they encode are approximately 70% antigen-specific 1–2 weeks after infection. Methods We isolated single peripheral blood plasmablasts from children with KD 1–3 weeks after onset and prepared 60 monoclonal antibodies (mAbs). We used the mAbs to identify their target antigens and assessed serologic response among KD patients and controls to specific antigen. Results Thirty-two mAbs from 9 of 11 patients recognize antigen within intracytoplasmic inclusion bodies in ciliated bronchial epithelial cells of fatal cases. Five of these mAbs, from 3 patients with coronary aneurysms, recognize a specific peptide, which blocks binding to inclusion bodies. Sera from 5/8 KD patients day ≥ 8 after illness onset, compared with 0/17 infant controls (P < .01), recognized the KD peptide antigen. Conclusions These results identify a protein epitope targeted by the antibody response to KD and provide a means to elucidate the pathogenesis of this important worldwide pediatric problem.