Required Immunoproteasome Subunit Inhibition Profile for Anti-Inflammatory Efficacy and Clinical Candidate KZR-616 ((2 S,3 R)- N-(( S)-3-(Cyclopent-1-en-1-yl)-1-(( R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)-3-hydroxy-3-(4-methoxyphenyl)-2-(( S)-2-(2-morpholinoacetamido)propanamido)propenamide).

Selective immunoproteasome inhibition is a promising approach for treating autoimmune disorders, but optimal proteolytic active site subunit inhibition profiles remain unknown. We reveal here our design of peptide epoxyketone-based selective low molecular mass polypeptide-7 (LMP7) and multicatalytic endopeptidase complex subunit-1 (MECL-1) subunit inhibitors. Utilizing these and our previously disclosed low molecular mass polypeptide-2 (LMP2) inhibitor, we demonstrate a requirement of dual LMP7/LMP2 or LMP7/MECL-1 subunit inhibition profiles for potent cytokine expression inhibition and in vivo efficacy in an inflammatory disease model. These and additional findings toward optimized solubility led the design and selection of KZR-616 disclosed here and presently in clinical trials for treatment of rheumatic disease.

Circulating Tumor Cells as Potential Biomarkers in Bladder Cancer.

PURPOSE: We explored the diagnostic use of circulating tumor cells in patients with neoadjuvant bladder cancer using enumeration and next generation sequencing. MATERIALS AND METHODS: A total of 20 patients with bladder cancer who were eligible for cisplatin based neoadjuvant chemotherapy were enrolled in an institutional review board approved study. Subjects underwent blood draws at baseline and after 1 cycle of chemotherapy. A total of 11 patients with metastatic bladder cancer and 13 healthy donors were analyzed for comparison. Samples were enriched for circulating tumor cells using the novel IsoFlux™ System microfluidic collection device. Circulating tumor cell counts were analyzed for repeatability and compared with Food and Drug Administration cleared circulating tumor cells. Circulating tumor cells were also analyzed for mutational status using next generation sequencing. RESULTS: Median circulating tumor cell counts were 13 at baseline and 5 at followup in the neoadjuvant group, 29 in the metastatic group and 2 in the healthy group. The concordance of circulating tumor cell levels, defined as low-fewer than 10, medium-11 to 30 and high-greater than 30, across replicate tubes was 100% in 15 preparations. In matched samples the IsoFlux test showed 10 or more circulating tumor cells in 4 of 9 samples (44%) while CellSearch® showed 0 of 9 (0%). At cystectomy 4 months after baseline all 3 patients (100%) with medium/high circulating tumor cell levels at baseline and followup had unfavorable pathological stage disease (T1-T4 or N+). Next generation sequencing analysis showed somatic variant detection in 4 of 8 patients using a targeted cancer panel. All 8 cases (100%) had a medium/high circulating tumor cell level with a circulating tumor cell fraction of greater than 5% purity. CONCLUSIONS: This study demonstrates a potential role for circulating tumor cell assays in the management of bladder cancer. The IsoFlux method of circulating tumor cell detection shows increased sensitivity compared with CellSearch. A next generation sequencing assay is presented with sufficient sensitivity to detect genomic alterations in circulating tumor cells.

Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay.

Circulating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates.