Animals were housed and treated under an animal protocol approved by the institutional animal care and use committee. Female mice were purchased at age 6-8 weeks from Taconic (C57BL/6 and Fcγ receptor-deficient C57BL/6.129P2-Fcer1gtm1Rav N12), Harlan (NcR nude mice), and Jackson ( A/J and FasL deficient C57BL/6 Smn.C3-Tnfsf6gld/J).
B78, B16, NXS2, and Panc02 tumors were engrafted by subcutaneous flank injection of 2×106 tumor cells. For disseminated disease models 3.5×105 B16 cells were IV injected. Engraftment of SCC1-C was performed by subcutaneous flank injection of 3×106 cells = in 2:1 PBS:Matrigel (BD Biosciences).
Tumor size was determined using calipers and volume approximated as (width2 × length)/2. Mice were randomized immediately prior to treatment. The day of radiation was defined as “day 1” of treatment. IT injections were made by a single percutaneous needle puncture followed by injection of a 100 µL volume with needle redirection to distribute injected material around the tumor. IT injections of 50 µg hu14.18K322A, cetuximab, hu14.18-IL2, or control IgG were delivered daily on days 6-10. Anti-CTLA-4 or control IgG was administered by 200 µg intraperitoneal (IP) injection days 3, 6, and 9. For NK cell depletion, IP injections of 500 µg NK1.1 mAb (clone PK136, ATCC) were delivered days 1, 5, 10, and 15. Depletion of T cells was performed as previously described (22 (link)).
Treatment began when tumors were well established (~200 mm3), occurring ~ 5 weeks after tumor implantation for B78 melanoma. For “large” B78 tumors (~500 mm3), treatment began ~ 7 weeks after implantation. Animals were sacrificed when tumors exceeded a pre-determined dimension. Mouse experiments were repeated in triplicate. Final replicates are presented for tumor response and aggregate data for survival; number of animals (n) per group is indicated.
B78, B16, NXS2, and Panc02 tumors were engrafted by subcutaneous flank injection of 2×106 tumor cells. For disseminated disease models 3.5×105 B16 cells were IV injected. Engraftment of SCC1-C was performed by subcutaneous flank injection of 3×106 cells = in 2:1 PBS:Matrigel (BD Biosciences).
Tumor size was determined using calipers and volume approximated as (width2 × length)/2. Mice were randomized immediately prior to treatment. The day of radiation was defined as “day 1” of treatment. IT injections were made by a single percutaneous needle puncture followed by injection of a 100 µL volume with needle redirection to distribute injected material around the tumor. IT injections of 50 µg hu14.18K322A, cetuximab, hu14.18-IL2, or control IgG were delivered daily on days 6-10. Anti-CTLA-4 or control IgG was administered by 200 µg intraperitoneal (IP) injection days 3, 6, and 9. For NK cell depletion, IP injections of 500 µg NK1.1 mAb (clone PK136, ATCC) were delivered days 1, 5, 10, and 15. Depletion of T cells was performed as previously described (22 (link)).
Treatment began when tumors were well established (~200 mm3), occurring ~ 5 weeks after tumor implantation for B78 melanoma. For “large” B78 tumors (~500 mm3), treatment began ~ 7 weeks after implantation. Animals were sacrificed when tumors exceeded a pre-determined dimension. Mouse experiments were repeated in triplicate. Final replicates are presented for tumor response and aggregate data for survival; number of animals (n) per group is indicated.
