br LMB Targeting CRC br membrane of ovarian cancer OVCAR
LMB100 Targeting CRC
membrane of ovarian cancer OVCAR-3 cells.4 Since then, MSLN has been studied extensively and found to be highly expressed on many malignancies, including pancreatic adenocarcinoma and mesothelioma.5-8 An RIT targeting MSLN named SS1P was con-structed by fusing the Fv portion of an anti-MSLN9 antibody to a 38 kDa fragment of PE.9 SS1P displayed a favorable safety profile in phase I trials and was investigated in a cohort of mesothelioma patients.10 However, efficacy was limited by the development of neutralizing 85-66-5 in 90% of treated patients.10 In a subse-quent study in which immunogenicity was mitigated with a lympho-depletive regimen prior to administration of RIT, major regressions lasting more than 20 months were observed in 3 of 10 patients with chemotherapy-resistant malignant mesothelioma.11
Another approach to prevent the production of anti-drug anti-bodies has been to remove or modify B cell epitopes in the RIT. In collaboration with Roche pharmaceuticals, RG7787, or LMB-100, was developed. In LMB-100, the mouse Fv is replaced by a humanized Fab,12 most of domain II is removed, and mutations introduced into domain III to inactivate B cell epitopes. LMB-100 has been found to be very cytotoxic to many human cancer cell lines expressing MSLN and causes regressions of several types of MSLN-expressing cancers.12 LMB-100 is currently in phase I/II trials at the National Cancer Institute (NCI) for mesothelioma and a variety of MSLN-expressing gastrointestinal malignancies. We have also developed an anti-MSLN RIT that contains an albumin binding domain that greatly increases half-life in the circulation.13 In the current study, we have determined if these 2 RITs are effective in killing colon cancer cells in culture and reducing the growth of colon cancer xenograft in mice.
Materials and Methods
Cell Culture and Reagents
HTB39, KLM-1, and SW48 cell lines were purchased from ATCC, and have been previously described.14 Identity of all cell lines was confirmed by STR testing. STR refers to short tandem repeat DNA profiling that identifies human cell lines derived from individual tissue, ensuring purity of cultures and lack of cross-contamination.
KLM-1 cells were cultured in RPMI 1640 medium (Gibco, Thermo Scientific) supplemented with L-Glutamine (2 mmol/L), penicillin (100 U), streptomycin (100 mg), and 10% fetal bovine serum (Hyclone, Thermo Scientific). HTB39 and SW48 cell lines were grown at 37 C with 5% CO2 in D-MEM medium (Gibco Life Technologies) supplemented with 100 U penicillin, 100 mg streptomycin (Gibco Life Technologies), and 10% fetal bovine serum (Thermo Scientific). LMB-100 was manufactured by Roche and provided for these studies through a Collaborative Research and Development Agreement. LMB-164 and LMB-12 were prepared in our laboratory as previously described.13 Actinomycin D was obtained through Sigma Pharmaceuticals. Oxaliplatin was obtained through the National Institutes of Health (NIH) DVR Animal Pharmacy and was manufactured by TEVA Pharmaceuticals.
MSLN Surface Expression Assay
CRC cell lines were assayed for membrane MSLN expression via flow cytometry. 20,000 cells per sample were analyzed on a FACSCalibur instrument (BD Bioscience) running CellQuest
software (BD Bioscience). Data processed using FlowJo software (Tree Star Inc). Cells were plated and grown for 48 hours in culture, then harvested with trypsin. Live cells were washed and fixed with FACS buffer, then incubated with mouse anti-MSLN antibody (BioXcell) for 30 minutes. Cells were subsequently washed again and incubated for 30 minutes in darkness with PE conjugated antibody Fragment Goat anti-mouse IgG (Jackson Immuno Research).
In Vitro Quantitative Cell Viability Assay
The viability of cells treated with RIT and chemotherapy was measured using Cell Counting Kit WST-8 Assay (Dojindo Molecular Technologies, Inc). All cell lines were plated at 5000 cells per well in 96-well plates and incubated at 37 C for 24 hours prior to treatment. RIT and chemotherapy treatments were diluted in complete medium and added to wells in indicated concentrations. Cells were incubated at 37 C for 72 hours, then treated with WST-8 assay reagent per manufacturer’s instructions. Plates were incu-bated at 37 C for 4 hours, and absorbance at 450 nm was measured. Values normalized between 0% viability for Staur-osporine treatment (Sigma Pharmaceuticals), which produces total cell killing, and medium alone for 100% cell viability. Data were then plotted in GraphPad Prism 6 for fit curve and interpolated half maximal inhibitory concentration (IC50) value determination.
Mouse Xenograft Tumor Model
All animal experiments were performed in accordance with NIH guidelines and approved by the NCI Animal Care and Use Committee. SW48 cells (4 106) supplemented with Matrigel (4.0 mg/mL) were injected subcutaneously into the right flank of 6-to 8-week-old female, athymic nude mice. Tumors were allowed to grow to an average of 100 mm3 prior to treatment initiation. Actinomycin D (0.3 mg/kg) was diluted in 0.2% human serum albumin (HSA) in Dulbecco’s phosphate-buffered saline (D-PBS) and injected intra-peritoneally on days indicated. All RITs were diluted in D-PBS and administered intravenously (IV) through the tail vein on days indicated. Oxaliplatin (4.2 mg/kg) was diluted in 5% dextrose solution and administered IV through the tail vein on days indicated. Tumor size was measured in 2 dimensions by digital calipers at least twice weekly, and volume was calculated using the formula: 0.4 width2 length. Animals were sacrificed before tumor volume reached 1000 mm3 or tumors developed necrotic ulcerations. Data were recorded, and tumor growth curves were plotted in Microsoft Excel and GraphPad Prism 6 for interpolation and statistical analysis.