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. 2014 Nov;13(11):2653-61.
doi: 10.1158/1535-7163.MCT-14-0132. Epub 2014 Sep 19.

Efficacy of RG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers

Christine Alewine  1 , Laiman Xiang  1 , Takao Yamori  2 , Gerhard Niederfellner  3 , Klaus Bosslet  3 , Ira Pastan  4
Affiliations

Efficacy of RG7787, a next-generation mesothelin-targeted immunotoxin, against triple-negative breast and gastric cancers

Christine Alewine et al. Mol Cancer Ther. 2014 Nov.

Abstract

The RG7787 mesothelin-targeted recombinant immunotoxin (RIT) consists of an antibody fragment targeting mesothelin (MSLN) fused to a 24-kD fragment of Pseudomonas exotoxin A for cell killing. Compared with prior RITs, RG7787 has improved properties for clinical development including decreased nonspecific toxicity and immunogenicity and resistance to degradation by lysosomal proteases. MSLN is a cell surface glycoprotein highly expressed by many solid tumor malignancies. New reports have demonstrated that MSLN is expressed by a significant percentage of triple-negative breast and gastric cancer clinical specimens. Here, panels of triple-negative breast and gastric cancer cell lines were tested for surface MSLN expression, and for sensitivity to RG7787 in vitro and in animal models. RG7787 produced >95% cell killing of the HCC70 and SUM149 breast cancer cell lines in vitro with IC50 < 100 pmol/L. RG7787 was also effective against gastric cancer cell lines MKN28, MKN45, and MKN74 in vitro, with subnanomolar IC50s. In a nude mouse model, RG7787 treatment (2.5 mg/kg i.v. qod ×3-4) resulted in a statistically significant 41% decrease in volumes of HCC70 xenograft tumors (P < 0.0001) and an 18% decrease in MKN28 tumors (P < 0.0001). Pretreatment with paclitaxel (50 mg/kg i.p.) enhanced efficacy, producing 88% and 70% reduction in tumor volumes for HCC70 and MKN28, respectively, a statistically significant improvement over paclitaxel alone (P < 0.0001 for both). RG7787 merits clinical testing for triple-negative breast and gastric cancers.

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Conflict of interest statement

Disclosures and Potential Conflict of Interest: I.P. is an inventor on several patents on immunotoxins that have all been assigned to NIH (including US 8357783, US 20120263674, US 20140094417 and E-771-2013/1-US-01) and has a Cooperative Research and Development Agreement (#2791) with Roche Pharmaceuticals. All other authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
MSLN-targeted RITs in TNBC in vitro. (A) Structures of the MSLN-targeted RITs SS1P, SS1-LR and RG7787 are depicted in cartoon form to demonstrate the differences in the targeting and PE domain modules. Vh and VL domains (in blue) form the Fv anti-MSLN antibody region and are connected by engineered disulfide bond (gold bar). Vh, VL, CH1 and CK (in purple) form the humanized anti-MSLN Fab of RG7787. Domain III (in red) is the catalytic subunit of PE. Domain Ia (in yellow) is also important for enzymatic function. Residues 274–284 of domain II (green) contain the furin cleavage site that is critical for intracellular processing of PE. GGS is a recombinant linker that improves flexibility of the molecule following the large deletion of Domain II in the newer molecules. A slightly modified linker is used in RG7787. (B) Ribbon diagram depicting the predicted structure of RG7787. Position of point mutations that eliminate human B-cell epitopes are indicated. (C) Five different TNBC cell lines were tested for MSLN surface expression by FACS analysis. Live cells were incubated with anti-MSLN primary antibody, followed by secondary incubation with appropriate PE-conjugated antibody (detected as FL2-H). Gray filled area shows detection of PE-labeling in minimum of 20,000 cells. Black trace shows negative control where incubation with anti-MSLN antibody was omitted. Traces are representative runs from at least three separate experiments. (D) TNBC cell lines were treated with a physiologic dose of the MSLN-targeted immunotoxin SS1-LR for 72 hours. Cells were photographed prior to treatment (left), and then the same field rephotographed after 72 hours of SS1-LR exposure (left center). Cells were also photographed after 72 hours of treatment with vehicle (right center), or 10 μg/mL of the protein synthesis inhibitor cycloheximide as positive control (right). Photographs are representative images of at least duplicate fields taken in duplicate wells in at least two experiments. (E) HCC70 (above) and SUM149 (below) were treated with the indicated concentrations of SS1P or RG7787 and incubated for 72 hours before assessment of cell viability by WST-8 assay. Viability for vehicle-treated cells was normalized to 1.0, and that for cycloheximide (a positive control which reliably produces nearly 100% cell kill) was normalized to 0. Each data point represents measurements of six treated wells.
Figure 2
Figure 2
Effect of RG7787 on TNBC mouse xenograft tumor model. (A) Female athymic nude mice were innoculated in the intramammary fat pad with HCC70 cells at time 0. Intravenous treatment with RG7787 (2.5 mg/kg) or vehicle was begun on day 8 and continued every other day for a total of four doses. Each data point represents average of mean tumor volume for n = 7 animals treated with RG7787 or vehicle. Arrows indicate the days that treatment was administered. There is a statistically significant difference between the two groups beginning at day 10, with p < 0.0001 by day 14. Results shown are representative of three similar experiments. (B) Change in baseline weight for mice treated with the regimen described above is plotted over time. Weight dropped less than 5% in RG7787-treated animals over the course of therapy. (C) MSLN expression in tumors excised from vehicle and RG7787 treated mice was examined by IHC in formalin-fixed paraffin embedded tissue. Prominent staining was observed with anti-MSLN antibody (above) but not in control specimens labeled with isotype control antibody (below). Photographs were taken at 40X magnification.
Figure 3
Figure 3
Gastric cancer is a target for MSLN-targeted RITs. (A) Four different gastric cancer cell lines were assessed for surface expression of MSLN as described in Figure 3A. Traces are representative runs from at least duplicate experiments. (B) MKN28 or MKN45 were treated with the indicated concentrations of SS1P or RG7787 and incubated for 72 hours before assessment of cell growth inhibition by WST-8 assay. Viability for vehicle-treated cells was normalized to 1.0, and that for cycloheximide (a positive control which reliably produces nearly 100% cell kill) was normalized to 0. Each data point represents measurements of three treated wells. (C) Female nude mice were inoculated subcutaneously with MKN28 cells at time 0. Intravenous treatment with RG7787 (2.5 mg/kg) or vehicle was given on days 5, 7 and 9. Each data point represents average of mean tumor volume for n = 10 animals treated with RG7787 or n = 5 vehicle treated animals. Arrows indicate the days that treatment was administered. There is a statistically significant difference between the two group beginning on day 7. Results shown are representative of more than three similar experiments.
Figure 4
Figure 4
Pre-treatment with paclitaxel enhances RIT anti-tumor response. Female athymic nude mice were inoculated with tumor cells at time 0. Animals were treated with vehicle, RG7787 with IP vehicle injection, paclitaxel with IV vehicle injection, or paclitaxel and RG7787 combination. Paclitaxel (50 mg/kg) was administered by IP injection on days marked with long arrows. RG7787 (2.5 mg/kg) was administered IV on days indicated by short arrows. Mean tumor volumes are indicated by the markers. For vehicle control five mice were followed. Results of both experiments were confirmed by repeat. (A) HCC70 TNBC tumor xenografts. There is a statistically significant difference between paclitaxel and combination treatment beginning at day 13 (p ≤ 0.001, n = 6 per treatment group). (B) MKN28 gastric cancer tumor xenografts. There is a statistically significant difference between paclitaxel and combination treatment beginning at day 10 (p ≤ 0.001, n= 8 per treatment group).
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