br Immunotoxin Therapy RIT Monotherapy and br Combination RI

Immunotoxin Therapy (RIT) Monotherapy and
Combination RIT-actinomycin D Therapy (P < .05)
Beginning on Day 15 (Indicated by *). Actinomycin D
Combination Therapy Is Significantly More Effective
those undergoing vehicle treatment or oxaliplatin alone, there was no difference in tumor volume between RIT alone versus combination therapy (Figure 6). Both the combination and RIT monotherapy group experienced reduction to 48% of the initial tumor volume and reached 300 mm3 tumor volume at 30 days in comparison to 6 days for vehicle treatment and 8 days for oxaliplatin alone. Furthermore, there were no complete regressions in the treatment groups (Figure 6).
Discussion
MSLN-targeted RIT therapy with SS1P has produced tumor regressions in patients with mesothelioma in some patients with epithelioid mesothelioma, and its current applications are being tested in phase II trials at the NCI in various MSLN-expressing gastrointestinal malignancies.11 Until recently, CRC was assumed to be a relatively poor target for this therapy.15 However, with the advent of more specific antibody testing for membranous MSLN expression, malignancies once thought to be immune to RIT therapy may in fact be susceptible.16 Given these findings, we attempted to create and explore MSLN-targeted RIT therapy in CRC. In vitro cytotoxicity experiments showed that not only were MSLN-expressing CRC cell lines susceptible to RIT therapy, but these cell lines responded to the same concentrations previously shown efficacious in KLM-1 pancreatic adenocarcinoma despite having a lower membranous expression of MSLN.
In vivo studies echoed these findings, with over 50% tumor regression in SW48 CRC tumors treated with LMB-100 mono-therapy. In KLM-1 pancreatic cells, LMB-164 (an ABD containing RIT) was able to produce complete regressions at the concentra-tions tested in our study.13 However, CRC cell lines were less susceptible, responding with tumor regression that was not statis-tically different than that of those tumors treated with LMB-100. The reason for the difference in response between these 2 cell 
Abbreviation: Act D ¼ actinomycin D.
lines is unclear at this time. It has been previously shown that MSLN shed from the membrane of tumor E-64 can have a sink effect, lowering the amount of RIT that is able to bind at the tumor cell membrane and halt protein uptake.21 Whether or not CRC cells shed MSLN at a higher rate than those of pancreatic adeno-carcinoma is unclear at this time and warrants further investigation. Another possibility lies in the fact that tumor stroma plays a role in inhibiting RIT diffusion into the tumor to malignant cell mem-branes, although the tumor stroma is also dense in pancreatic adenocarcinoma.22
Given these limitations, combination chemotherapy and RIT therapy warrants exploration. In fact, the unique mechanism of binding to EF-2 and halting protein synthesis of RITs provides a novel ability to reach both quiescent and active tumor cells. In our current study, we first explored the potential for combination with a current chemotherapeutic used in CRC therapy in oxaliplatin. Oxaliplatin, a platinum-based chemotherapy and DNA binding agent, has been previously shown to have additive or synergistic effects with fluorouracil and irinotecan in CRC in vitro and in vivo models.23,24 Furthermore, prior synergy screens performed in our lab showed moderate combination activity between cisplatin and MSLN-targeted RIT.18 Oxaliplatin has also been purported to
Clinical Colorectal Cancer Month 2019 - 5
LMB100 Targeting CRC
Figure 5 Spider Plots of Individual Tumor Sizes Per Treatment Group From a Representative Tumor Experiment. A, Control Group Treated With PBS. B, Mice Treated With 0.3 Mg/kg Actinomycin D. C, Mice Treated With 2.5 Mg/kg LMB-100. D, Mice Treated With 0.3 Mg/kg Actinomycin D and 2.5 Mg/kg LMB-100. The Combination-treated Group Was Noted to Have a 50% Complete Response Rate With a Significant Delay in Tumor Regrowth to 300 mm3 Following Initial 2 Cycles of Therapy
Abbreviations: CR ¼ complete response; PBS ¼ phosphate buffered-saline.
induce immunogenic cell death and activate the extrinsic pathway of apoptosis in CRC tumor cells in prior in vivo studies.25,26 However, our current testing in SW48 cells displayed no added benefit outside of slight growth delay in the combination of these 2 drugs. This may be owing to the inability to achieve high enough in vivo concen-trations of oxaliplatin to induce synergy with the RIT before first inducing toxicity.
Although actinomycin D is a chemotherapeutic agent largely used in the treatment of pediatric sarcoma, prior studies have shown an ability to synergize in vivo with RIT therapy in pancreatic cancer models.18 Actinomycin D has also been shown to activate the extrinsic pathway of apoptosis when combined with immunotoxin therapy in vitro.18 These findings led us to test actinomycin D in combination with MSLN-targeted RIT in our CRC model. Results from these experiments displayed not only significant synergy and tumor regression in vivo when compared with RIT monotherapy, but also showed 50% complete regression in tumors treated with