Tumor-associated Epithelial Cell Adhesion Molecule Research Articles

Engineering of a tumor cell–specific, cytosol-penetrating antibody with high endosomal escape efficacy

The main obstacles for practical uses of cytosol-penetrating peptides and proteins include their lack of cell- or tissue-specific targeting and limited cytosolic access owing to the poor endosomal escape ability. We have previously reported a cytosol-penetrating, human IgG1 antibody TMab4-WYW, generally referred to as a cytotransmab (CT), which reaches the cytosol of living cells but nonspecifically because it is endocytosed via a ubiquitously expressed receptor called heparan sulfate proteoglycan (HSPG). Here, our aim was to construct a next-generation CT with tumor cell specificity and improved endosomal escape efficiency. We first substantially reduced the HSPG-binding activity of TMab4-WYW and then fused a cyclic peptide specifically recognizing tumor-associated epithelial cell adhesion molecule (EpCAM) to the N terminus of the light chain for EpCAM-mediated endocytosis, while maintaining the endosomal escape ability in the light chain variable domain (VL), thus generating epCT05. Then, we separately engineered another CT, dubbed epCT65-AAA, with an endosomal escape ability only in the heavy chain variable domain (VH) but not in VL, by functional grafting of the endosomal escape motif of epCT05 VL to the VH. We finally combined the heavy chain of epCT65-AAA and the light chain of epCT05 to create epCT65 with endosomal escape capacity in both the VH and VL. epCT65 effectively localized to the cytosol of only EpCAM-expressing tumor cells and showed approximately twofold improved endosomal escape efficiency, as compared with CTs with endosomal escape motifs in either VH or VL. The full–IgG format CT, epCT65, with a tumor cell–specific cytosol-penetrating activity, has a great potential for practical medical applications, e.g., as a carrier for cytosolic delivery of payloads.

Abstract CT094: Novel treatment with intraperitoneal MOC31PE immunotoxin in colorectal peritoneal metastasis: Long-term outcome from the ImmunoPeCa phase I/II trial

Abstract Background. Peritoneal metastasis (PM) from colorectal cancer (CRC) is associated with poor outcome, but in patients with resectable disease long-term survival through cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) can be achieved. The dual rationale for applying intraperitoneal therapy in PM-CRC is dose intensification because of the peritoneal-plasma barrier and the conception of PM as a localized disease within the peritoneal cavity. Outcome following CRS-HIPEC is however highly variable and most patients will experience disease recurrence, illustrating the need for improved treatment. MOC31PE immunotoxin, composed of the monoclonal antibody MOC31 and pseudomonas exotoxin A (PE), was developed by researchers at the Norwegian Radium Hospital to rapidly kill cells expressing the tumor-associated epithelial cell adhesion molecule (EpCAM), which is highly expressed in CRC. In the ImmunoPeCa trial we investigated the use of intraperitoneal MOC31PE as a novel therapeutic principle to target PM-CRC. Methods. The ImmunoPeCa trial was a dose-escalating phase I trial to evaluate the safety and tolerability (primary endpoint), pharmacokinetic profile, and neutralizing antibody response (secondary endpoints) upon a single dose of intraperitoneal MOC31PE in patients with PM-CRC undergoing CRS-HIPEC. Overall survival (OS) and disease-free survival (DFS) were also examined (secondary endpoints). Fifteen patients received the study drug at four dose levels (2.5 (n=3), 5.0 (n=3), 7.5 (n=3) and 10 (n=6) µg kg). Additional 6 patients, constituting an expansion cohort, were treated on dose level 4 (10 µg/kg). Results. There was no major toxicity, and the maximum tolerated dose was not reached. The systemic drug exposure was low, and MOC31PE in peritoneal fluid samples retained cytotoxic capacity, suggesting that the drug is very stable under physiological conditions. With a median follow-up of 29 months (95% CI 22-35 months), the median OS was not reached and the estimated 3-year OS was 85%. Estimated median DFS was 20 months (95% CI 8-33 months) and the estimated 3-year DFS was 36%, with a median follow-up of 25 months (95% CI 19-31 months). Although very encouraging, the results may reflect the selection of the cohort, and investigation in a larger cohort would be necessary to study efficacy. Conclusions. Intraperitoneal, perioperative administration of MOC31PE was safe and well tolerated. The promising long-term outcome combined with the low systemic uptake and retained cytotoxic activity in peritoneal fluid samples support further clinical testing. Citation Format: Ida S. Froysnes, Yvonne Andersson, Stein Larsen, Ben Davidson, Janne-Merete T. Oien, Kari H. Olsen, Karl-Erik Giercksky, Lars Julsrud, Oystein Fodstad, Svein Dueland, Kjersti Flatmark. Novel treatment with intraperitoneal MOC31PE immunotoxin in colorectal peritoneal metastasis: Long-term outcome from the ImmunoPeCa phase I/II trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT094.

Novel Treatment with Intraperitoneal MOC31PE Immunotoxin in Colorectal Peritoneal Metastasis: Results From the ImmunoPeCa Phase 1 Trial

MOC31PE immunotoxin was developed to rapidly kill cells expressing the tumor-associated epithelial cell adhesion molecule, which is highly expressed in colorectal cancer. Although cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) may offer long-term survival to patients with peritoneal metastasis from colorectal cancer (PM-CRC), most patients experience disease relapse and novel therapeutic options are needed. On this basis, MOC31PE is being developed as a novel therapeutic principle to target PM-CRC. This was a dose-escalating phase I trial to evaluate the safety and toxicity (primary endpoint), pharmacokinetic profile, and neutralizing antibody response (secondary endpoints) upon intraperitoneal administration of MOC31PE in patients with PM-CRC undergoing CRS-HIPEC with Mitomycin C. Fifteen patients received the study drug at four dose levels (3+3+3+6), administered intraperitoneally as a single dose the day after CRS-HIPEC. No dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. There was negligible systemic absorption of the study drug. Drug concentrations in peritoneal fluid samples were in the cytotoxic range and increased in a dose-dependent manner. MOC31PE recovered from peritoneal cavity retained its cytotoxic activity in cell-based assays. All patients developed neutralizing antibodies. Intraperitoneal administration of MOC31PE was safe and well tolerated, and combined with low systemic uptake, MOC31PE seems ideal for local intraperitoneal treatment. The drug will be further evaluated in an ongoing phase II expansion cohort.

MicroRNAs in Serum Exosomes as Potential Biomarkers in Clear-cell Renal Cell Carcinoma

BackgroundCirculating microRNAs (miRNAs) in exosomes are emerging as clinically useful tools for cancer detection. However, little is known about their diagnostic impact in clear-cell renal cell carcinoma (ccRCC). ObjectiveTo investigate whether miRNAs in serum exosomes can serve as biomarkers in ccRCC. Design, setting, and participantsSerum samples were obtained from 82 patients with ccRCC and 80 healthy volunteers. Exosomes were extracted and purified to selectively capture exosomes positive for tumor-associated epithelial cell adhesion molecule (EpCAM) via a magnetic bead technique. Total RNA was extracted and expression levels of miR-210, miR-1233, and miR-15a miRNAs were quantified and normalized to U6 levels. Outcome measurements and statistical analysisExpression levels were compared using a Mann-Whitney U-test, Friedman test, or Wilcoxon test. Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic value of exosomal miRNAs for differentiation between ccRCC patients and controls. Results and limitationsExpression levels of exosomal miR-210 and miR-1233 were significantly higher in ccRCC patients than in healthy individuals (both p<0.01). No significant difference was observed for exosomal miR-15a. Exosomal miR-210 and miR-1233 expression levels in different TNM stages were significantly higher than in the controls (all p<0.01). Exosomal miR-210 and miR-1233 expression levels were significantly lower in postoperative than in preoperative samples (both p<0.01). ROC analysis demonstrated that exosomal expression levels distinguished ccRCC patients from healthy individuals with 70% sensitivity and 62.2% specificity for miR-210, and 81% sensitivity and 76% specificity for miR-1233. The retrospective design and lack of other tumor subtypes are limitations of the study. ConclusionsSerum exosomal miRNAs might represent potential diagnostic biomarkers in ccRCC in the future. Patient summaryCirculating levels of exosomal microRNAs miR-210 and miR-1233 have potential as biomarkers for diagnostic and monitoring purposes in renal cancer in the future. These molecules can be measured in serum in so-called liquid biopsy.

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.