Characterization of cetuximab- and panitumumab-based BiTE antibodies. Cross-reactivity of C- and P-BiTE antibodies between CHO cells transfected with human (A) and macaque antigen (B) was assessed by FACS analysis. Each panel shows FACS histograms analyzing binding of a control antibody with irrelevant specificity and respective BiTE antibodies at 5 μg/mL The x axis shows the mean fluorescence intensity. T cell activation in response to C-BiTE (C) or P-BiTE (D) was investigated by surface expression of immediate/early T cell activation surface marker CD69 on CD3⁺ cells using FACS analysis. A BiTE dose–response analysis testing three human T cell donors with C- or P-BiTE, or a control BiTE antibody to CD3 and to an irrelevant hapten antigen, was performed. Mean values and SDs of triplicate determinations are shown.

Antitumor activity of C-BiTE antibody and parental cetuximab in two human CRC xenograft mouse models Tumor cell doses were determined upfront, allowing a rapid and reproducible outgrowth of tumors from HCT116 (A) or HT29 human CRC cell lines (B) in NOD/SCID mice. HT-29 (2× 10⁶ cells per inoculum) or human colon carcinoma cell line HCT116 (5× 10⁶ per inoculum) were injected s.c. together with PBMCs from healthy human donors at an E:T cell ratio of 1:2 in the right dorsal flank of female NOD/SCID mice (n = 8 per group). As indicated, four different control conditions were tested for their influence on tumor outgrowth: vehicle in the absence, and vehicle, isotype IgG1, and a control BiTE antibody binding to CD3 and an irrelevant hapten antigen in the presence of human PBMCs. Cetuximab was intraperitoneally administered twice a week starting on d 1 at 50 mg/kg for 4 (HT29) or 5 wk (HCT116), whereas C-BiTE was daily administered by i.v. bolus starting on d 1 only for the first 8 d (HT29) or 10 d (HCT116). Tumor growth was determined by external caliper measurements, and tumor volumes were calculated using a standard hemiellipsoid formula (Eq. 2). Values represent mean tumor size (in cm³) ± SEM (n = 8 per group). Growth retardation by cetuximab in (A) was statistically significant for all time points labeled by an asterisk (P < 0.05). For HCT116 xenografts, all BiTE responses were highly significant, and at all four doses tested, all eight mice did not develop tumors. For HT29, all but the lowest dose of 0.005 mg/kg resulted in a statistically significant inhibition of tumor growth (Student t test).

Redirected lysis of human EGFR-expressing CHO cells and three human CRC cell lines by BiTE antibodies based on cetuximab and panitumumab. Dose-dependent redirected lysis of CHO cells transfected with human EGFR (A) or of the three human CRC cells lines SW480, HCT116, and HT29 (B) by C-BiTE (Left) and P-BiTE (Right) was assessed by an FACS-based cytotoxicity assay (A) or a chromium release assay (B). A control BiTE antibody binding to CD3 and an irrelevant hapten antigen was used in A and the left panel of B using HCT116 cells. For A, the three T cell donors were identical, whereas different T cell donors were used in B for C- and P-BiTE. The E:T ratio in all experiments was 10:1. Mean values and SDs of triplicate determinations are shown.

Validation of cynomolgus monkey as relevant animal models to assess the tolerability of C- and P-BiTE. PBMCs were prepared from nine cynomolgus monkey and three human donors and T cells therein tested for redirected lysis of EGFR-expressing human gastric carcinoma cell line KATO III in a FACS-based cytotoxicity assay at an E:T ratio of 10:1 and incubation period of 24 h. (A) Dose–response analysis was used to obtain EC₅₀ values for comparing the efficacy of individual donor T cells. Because of limited blood supply from monkeys, PBMCs were cultured and T cells therein expanded by anti-CD3/anti-CD28/IL-2 treatment for 3 d to obtain sufficient effector T cells for dose–response analyses. A control BiTE antibody binding to human and Cynomolgus CD3 and to an irrelevant hapten antigen was tested in the right panel. (B) EC₅₀ values determined in cytotoxicity assays for monkey and human T cells and mean values. (C) Serum concentrations of C-BiTE dose levels during continuous i.v. infusion up to the maximum infusion period of 3 wk. The lower limit of quantification was 0.5 ng/mL Animal test numbers and dose levels are shown in the insert. Sera from animals that did not receive the C-BiTE did not give a signal in the C-BiTE ELISA.