To further investigate this aspect and given that the anti-EGFR EGa1 VHH single-domain antibody was isolated from a phage-displayed llama VHH library immunized with EGFR-positive human cells (14, 15), we studied here the impact of human EGFR expression on the liver in the 1D8N/CEGa1 toxicity profile in a liver-specific huEGFR-transgenic immunocompetent mouse (16)

To further investigate this aspect and given that the anti-EGFR EGa1 VHH single-domain antibody was isolated from a phage-displayed llama VHH library immunized with EGFR-positive human cells (14, 15), we studied here the impact of human EGFR expression on the liver in the 1D8N/CEGa1 toxicity profile in a liver-specific huEGFR-transgenic immunocompetent mouse (16). in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcR interactions in the major off-tumor?toxicities associated?with?IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols. and exhibits enhanced tumor penetration and powerful anti-tumor activity in immunocompetent mice bearing gene-modified CT26 colorectal carcinoma cells expressing human EGFR (10). In this model, the anti-tumor effect of the bispecific trimerbody was dependent on human EGFR expression (13), but the potential toxicity profile was dictated by the endogenous mouse EGFR. In this context, the 1D8N/CEGa1 trimerbody did not induce the systemic cytokine production and hepatotoxicity associated with IgG-based 4-1BB agonists (10). To further investigate this aspect and given that the anti-EGFR EGa1 VHH single-domain antibody was isolated from a phage-displayed llama VHH library immunized with EGFR-positive human cells (14, 15), we studied here the impact of human EGFR expression on the liver in the 1D8N/CEGa1 toxicity profile in a liver-specific huEGFR-transgenic immunocompetent mouse (16). In this model, systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and liver toxicity, whereas treatment with the EGFR-targeted 4-1BB-agonistic trimerbody lacked these immune-related side effects. Methods Mice C57BL/6 wild-type (WT) female mice and transgenic Alb-654C1186huEGFR (EGFR-tg) (16) littermates were housed in the animal facility of the Instituto de Investigaciones Biomdicas Alberto Sols (IIBm) (CSIC-UAM, Madrid, Spain). Animals were kept in controlled conditions of temperature (21 1C), humidity (50 5%), and 12?hours light/dark cycles. Manipulation was performed in laminar flow hood, when necessary, and sterilized water and food were available ad libitum All animal procedures conformed to European Union Directive 86/609/EEC and Recommendation 2007/526/EC, enforced in Spanish law under RD 1201/2005. Animal protocols were approved by the Animal Experimentation Ethics Committee of the IIBm, and the Animal Welfare Division of the Environmental Affairs Council Zolpidem of the Government of Madrid (66/14, 118/19). Cells and Culture Conditions HEK293 (CRL-1573) cells were obtained from the American Type Culture Collection and mouse CT26 cells (CRL-2638) expressing human EGFR (CT26huEGFR) or infected with the empty vector retrovirus (CT26mock) were provided by Dr M. Rescigno (European Institute of Oncology, Milan) (13). The cells were grown in complete Dulbeccos modified Eagles medium (DMEM) (Lonza) supplemented with 2 mM L-glutamine, 10% (vol/vol) heat-inactivated Fetal Calf Serum (FCS), and antibiotics (100 units/mL penicillin, 100 mg/mL streptomycin) (all from Life Technologies) referred as to DMEM complete medium Zolpidem (DCM), unless otherwise stated. The cell lines were routinely screened for mycoplasma contamination by PCR (Stratagene). Hepatocyte Isolation and Culture Hepatocytes were isolated as previously described following the two-step collagenase perfusion technique followed by isodensity purification in a Percoll gradient (17). Briefly, Zolpidem livers from 3 months-old mice were perfused with Hanks balanced salt solution supplemented with 10 mM Hepes and 0.2 mM EGTA for 5?min, followed by a perfusion (10C15 min) with Williams Rabbit Polyclonal to Cytochrome P450 2D6 E medium containing 10mM Hepes and 0.03% collagenase I (Worthington). Livers were further minced, and viable hepatocytes were selected by centrifugation in Percoll and seeded in collagen I-coated plates (5 g/sq cm) at a density of 28 x 103/cm2 in Dulbeccos modified Eagles medium/F-12 (1:1) supplemented with 10% serum. Expression and Purification of Recombinant Antibodies The 1D8N/CEGa1 trimerbody was produced in stably transfected HEK293 cells (10) cultured in complete DMEM with 500 g/mL G418 (all from Life Technologies), and conditioned medium purified using the (Twin-)value) were discriminated by Zolpidem applying a two-tailed, unpaired Students test assuming a normal distribution. values are indicated in the corresponding figures for each experiment. Results and Discussion The 1D8N/CEGa1 Trimerbody Binds to Human EGFR With a Higher Affinity Than to Mouse EGFR The EGa1 is a well characterized EGFR-specific VHH that was generated from a?phage-displayed?llama?VHH?library after immunizing and screening with EGFR-positive human cells (14, 18)..