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    r> Statistics
    Excel and GraphPad Prism 6 were used for all statistical calcu-lations and curve fitting. Errors are reported as standard deviations. The 2-tailed Student t test was used for statistical comparisons. Significance was established at a 0.01.
    MSLN Expression in CRC Cell Lines
    Although MSLN expression was previously analyzed in tissue micro-array data of CRC and considered relatively low, a recent analysis of the frequency in which MSLN protein can be detected in
    CRC samples is much higher, showing that almost two-thirds of CRC surgical specimens are positive.15,16 Further, the analysis of mRNA expression of MSLN in various carcinomas from the Cancer Cell Line Encyclopedia (CCLE) database (http://www. shows expression levels on par with those of ovarian cancer and gastric adenocarcinoma (Figure 1A). To identify CRC cell lines, which could be used in a model to test MSLN-targeted RITs, we examined surface MSLN expression in established CRC cell lines using FACS analysis with an antibody to MSLN.12 Figure 1B shows histograms of surface MSLN expression of 2 CRC cell lines, HTB39 and SW48. Both cell lines were uniformly positive, indicating they should be susceptible to MSLN-targeted RITs.
    Sensitivity of CRC Cell Lines to MSLN-targeted RITs
    We tested HTB39 and SW48 cell lines for RIT sensitivity using various anti-MSLN RITs: LMB-12, LMB-100, and LMB-164 (Figure 1C). LMB-12 consists of an anti-MSLN dsFv fragment fused to domain III of PE. LMB-100 consists of humanized Fab and mutations in domain III that silence many of B and some T cell epitopes to reduce immunogenicity.17 LMB-164 is based on
     Adam Cerise et al
    LMB-12 but has an albumin binding domain (ABD) that increases half-life.13 Sensitivity to these RITs was compared with that of KLM-1 pancreatic adenocarcinoma cells. The data in Figure 2 and Table 1 show that SW48 and HTB39 E-64 are sensitive to all 3 RITs. The IC50 on SW48 cells for LMB-12 is 0.37 ng/mL, for LMB-100 is 0.78 ng/mL, and for LMB-164 is 0.55 ng/mL. HTB39 cells are somewhat less sensitive than SW48 cells but are also killed by all 3 agents. The IC50 values are 2.7 ng/mL for LMB-12 and LMB-100; 5.7 ng/mL for LMB-164. No significant difference in RIT sensitivity was noted between SW48 cells and that of KLM-1 for any of the RITs. Immunotoxin LMB-11(HA22-Fab-LO10R456A) targeting CD22 antigen, which is not expressed in colon cancer or pancreatic cancer cell lines, was used as negative control. As expected, LMB-11 has no killing activity on these colon cancer cell lines at 1000 ng/mL concentration (Table 1).
    LMB-100 and LMB-164 Efficacy in CRC Mouse Xenograft Model
    Given the favorable sensitivity of SW48 cells to anti-MSLN RITs, the in vivo efficacy of LMB-100 and LMB-164 in an SW48 mouse xenograft model was evaluated. Athymic nude mice
    Figure 1 MSLN Expression and MSLN-targeted RITs. A, Expression of MSLN mRNA as Detected in Various Solid Organ Malignancies. Data Were Analyzed From Cancer Cell Line Encyclopedia (CCLE), Representing Gene Expression of 1036 Samples From 36 Tumor Types. B, Histogram of SW48 and HTB39 Colorectal Cancer Cell Lines Tested for Membranous MSLN Expression by Fluorescence-activated Cell Sorting Analysis. Live Cells Incubated With anti-MSLN Primary Antibodies Followed by Incubation With Appropriate Pseudomonas Exotoxin a (PE)-conjugated Antibody Detected as FL4-H. A Minimum of 20,000 Cells Were Used for MSLN Detection. C, Schematics of Different anti-MSLN RITs Used in this Manuscript. LMB100 Comprises Humanized SS1 Fab Linked to Furin Cleavage Site (Fur) Followed by Mutated Version of PE Toxin, LO10R456A. LMB-12 Comprises SS1disulfied-stabilized Fv Linked to Furin Cleavage Site (Fur) Followed by Mutated Version of PE Toxin, PE24. LMB-164 Comprises SS1disulfied-stabilized Fv Linked to an ABD Followed by Furin Cleavage Site (Fur) and a Mutated Version of PE Toxin, PE24
    Abbreviations: ABD ¼ albumin binding domain; ALL ¼ acute lymphoblastic leukemia; AML ¼ acute myeloid leukemia; CML ¼ chronic myeloid leukemia; MSLN ¼ mesothelin; NSC ¼ nonesmall-cell; RIT ¼ recombinant immunotoxin therapy.
    Clinical Colorectal Cancer Month 2019 - 3
    LMB100 Targeting CRC
    Figure 2 Activity of anti-MSLN Immunotoxin on Colon Cancer Cell Lines. A, KLM-1 Pancreatic Adenocarcinoma Cells Were Treated With Indicated Concentrations of LMB-12, LMB-100, or LMB-164 anti-MSLN Targeted RITs for 72 Hours Before WST-8 Assay of Cell Viability. Vehicle Treated Cells Were Normalized to 1, and Staurosporine Treated Cells Were Normalized to 0. Each Data Point Measures 6 Treated Wells. Identical Assay for SW48 CRC Cell Line (B) and HTB39 CRC Cell Line (C)