Timothy E. Weeden

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

Immunosuppressive activity of a novel peptide analog of alpha-melanocyte stimulating hormone (α-MSH) in experimental autoimmune uveitis

Autoimmune uveitis is an inflammatory disorder of the eye that can lead to pain and vision loss. Steroids and immunosuppressive drugs are currently the only therapeutics for uveitis and have serious ocular and systemic toxicities. Therefore, safer alternative therapeutics are desired. Alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide that suppresses effector T cell functions, induces regulatory T cells and has beneficial effects in certain autoimmune and transplant models. A novel d-amino acid peptide analog of native α-MSH (dRI-α-MSH) was produced that was protected from protease digestion and had increased selectivity for the melanocortin-1 receptor. Systemic delivery of the dRI-α-MSH analog dramatically suppressed disease progression and retained retinal architecture in the experimental autoimmune uveitis (EAU) model. Local delivery by periorbital injection was equally effective. Importantly, treatment with the novel dRI-α-MSH analog suppressed uveitis with a similar magnitude to the corticosteroid, dexamethasone. Data indicate that the novel dRI-α-MSH analogs show anti-inflammatory activities and have potential therapeutic use in uveitis and other autoimmune diseases.

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α‐melanocyte stimulating hormone (α‐MSH) is a tridecapeptide fragment of pro‐opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti‐inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro‐inverso (RI) sequence of α‐MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI‐α‐MSH exhibited a diminished binding affinity for MC1R compared to α‐MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non‐natural amino acids and substitution of the C‐terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical Ki as α‐MSH at MC1R and a lower EC50 in Mel‐624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L‐form α‐MSH, suggesting a significantly altered interaction with the MC1R. Copyright