Patients
Sixteen consecutive cancer patients undergoing abdominal tumor resection at the University Hospital Frankfurt, Germany, were enrolled in this study (mean 66.8 years) between March 2019 and November 2019. Only tumor indications were selected known to be EpCAM-positive such as advanced colon cancer, cholangiocarcinoma, esophageal cancer; ovarian cancer; pancreatic cancer, bile duct cancer, rectum cancer and perihilar cholangiocellular carcinoma.
The clinical study protocol was approved in December 2018 by the Ethics Committee of the University Hospital Frankfurt (number 325/18) and each patient was provided with informed consent. All patients were aware of the procedure and were informed that the shed blood was collected for research purposes and would not be transfused back to them.
Catuvab medical device and Catumaxomab
Catuvab was used in combination with mechanical auto-transfusion devices, which are part of the operating equipment and remove leukocytes from ECs as standard. The medical device Catuvab kit consists of the following components: Syringe containing 10 µg antibody (Catumaxomab) in 100 µl buffer (aseptically filled) with cannula, sterilized and sterile packed, Conformité Européenne (CE)-marked; 6R vial with 5.7 mL 0.9% NaCl solution (aseptically filled); 2 pieces of 2 mL syringe with 100 µL graduation with cannula (21G x 1½”, 40 mm), sterilized and sterile packed, CE-marked two sterile LDFs, pore size 40 μm, with silicone hose and standardized connection. The manufacturer of the investigational medical device Catuvab is LINDIS Blood Care GmbH, Neuendorfstr. 20b, Henningsdorf, Germany. Catumaxomab is a biologically engineered, intact, trifunctional bispecific EpCAM x CD3 binding monoclonal antibody consisting of a mouse immunoglobulin G (IgG)2a chain and a rat IgG2b chain [21,24,25].
EpCAM is strongly expressed in squamous cell carcinomas derived from epithelial tissue and can be found in various tumors of epithelial original (gastric carcinoma, ovarian carcinoma, pancreatic carcinoma, colon / rectal carcinoma, non-small cell lung cancer, or peritoneal carcinomatosis) [25-27]. In the past, Catumaxomab has been developed as a targeted therapy for intraperitoneal treatment of malignant ascites and epithelial cancers expressing the EpCAM antigen (e.g. bladder, ovarian, pancreatic, lung and gastric cancer). In the lead indication treatment of malignant ascites due to epithelial cancers, the European Medicines Agency (EMA) approved Catumaxomab in 2009. For commercial reasons, the product was withdrawn in 2017.
The primary mode of action of Catumaxomab in the Catuvab device consists of the physical aggregation of tumor cells and lymphocytes/accessory cells and the subsequent removal of the cell aggregates by centrifugation and filtration as part of a machine autotransfusion. Simultaneous binding ex vivo of the antibody to lymphocytes in the patient’s intraoperative blood (via the CD3-specific region of the antibody), tumor cells and FcγR-positive accessory immune cells ultimately leads to the formation of larger cell aggregates.
Technical procedure and Blood Sampling
Three parameters were investigated in intraoperative blood, during processing and in the final product after filtration with a leukocyte depletion filter (LDF)(figure 1):
- Detection and quantification of EpCAM positive tumor cells in patient blood, the EC and EC after LDF
- Detection and quantification of cytokines IL-6, IL-8 in the reservoir, in EC and in EC after LDF (probe sampling 1, 2 and 3)
- Detection and quantification of Catumaxomab in EC after LDF (probe sampling 3).
Blood and purge solvent accumulated during surgery was collected in a reservoir containing a bone splinter filter (ATR 120 reservoir, Fresenius Kabi, figure 1). The blood and purge solvent mixture collected in the reservoir was centrifuged and washed using a IBS machine (C.A.T.S.+, Fresenius Kabi, AT3 Autotransfusionsset, Fresenius Kabi), resulting in an erythrocyte concentrate (EC). The erythrocyte concentrate (EC) was filtered using a 40 µm Leukocyte depletion filter (LDF, RS1, Haemonetics) (LDF sample 3). All samples for analysis were extracted via an output connection/an outlet and collected in sterile tubes. The samples collected for antibody analysis were frozen within 1-2h (-20°C). The samples were sent immediately to Trion Research GmbH. The blood samples for cytokine analysis were centrifuged, the supernatant (plasma) collected, frozen within 1-2h (-20°C). The samples were sent immediately to the analysis laboratory on dry ice.
Catumaxomab was first diluted and a defined amount of the diluted antibody (2,5 µg or 5 µg antibody) was supplied to the blood mixture via a port on the reservoir, using a syringe. Antibodies were distributed within intraoperative blood and aggregates of tumor and immune cells developed within approximately 30 minutes. During the usual washing and concentration process of the IBS, cell aggregates with a relatively lower density could be separated from the red blood cells in the total mixture by centrifugation. A second filtration step (LDF filter of Catuvab) removed any remaining cell aggregates.
Detection and quantification of EpCAM positive tumor cells, Catumaxomab and IL-6, IL-8
Detection and quantification of EpCAM positive tumor cells were performed by immunofluorescence staining using the tumor marker EpCAM and cytokeratin. Density gradient centrifugation applying Ficoll-Paque was used as separation medium for lymphocytes and tumor cells that were stained after centrifugation on cytospin preparations. These cytospins were analyzed for the presence of EpCAM-positive tumor cells using the antibody BER-EP4. Quantification of tumor cells was performed by immunofluorescence microscopy with integrated digital image analysis (Applied Imaging) [28].
Detection and quantification of EpCAM binding Catumaxomab in EC before and after LDF
Catumaxomab concentrations were measured by an established two-site ELISA. Briefly, catumaxomab was captured by an anti-rat IgG light chain-specific antibody (LA1B12, TRION Research, Munich, Germany). Bound catumaxomab was then detected via an anti-mouse
IgG2a-specific biotin-labeled detection antibody (BD Pharmingen, San Diego, CA). Then, streptavidin-b-galactosidase and its corresponding substrate, chlorphenolred-b-D-galactopyranosid (Roche Diagnostics, Mannheim, Germany), were added, and the colorimetric reaction was measured at 570 nm. Catumaxomab concentrations were calculated by interpolation on a standard curve. The lower limit of quantification (LLOQ) of the assay was determined to be 125 pg ml-1; the upper limit of quantification was 4000 pg ml-1. All samples were diluted 1:2 before measuring in duplicate [21,28].
Detection and quantification of cytokines IL-6, IL-8 in the reservoir, in EC and in EC after LDF
As Catumaxomab is well known to activate different types of immune cells, the aim of the measurements was to determine a potential increase of proinflammatory cytokines during the Catuvab procedure [29]. Samples were sent to Synlab MVZ laboratory (Munich, Germany) and cytokines were determined by Luminex ® Corporation Multiplex technology using magnetic microsphere beads. The Multiplex ELISA is based on unique fluorescent signature coated microbeads binding specific cytokines which are subsequent measured by laser technology.
Statistics
Due to the exploratory character and the low number of patients of this pilot study only descriptive statistics using mean values were performed.