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. Author manuscript; available in PMC: 2022 Apr 15.
Published in final edited form as: Nat Cancer. 2020 Dec 7;2(1):18–33. doi: 10.1038/s43018-020-00136-x

Figure 6: CL264-containing ISACs elicit tumor clearance in HER2 medium expressing xenografts and T cell mediated tumor clearance, immunologic memory, and epitope spreading in a syngeneic tumor model.

Figure 6:

(a) Chemical structures of T785 and CL264 adjuvants used for ISAC generation. (b) NSG mice implanted with the HCC1954 tumor cell line were randomized when the tumor volume reached 50 – 75 mm3. Mice were treated with once via intraperitoneal injection of 5 mg/kg trastuzumab, trastuzumab T785-ISAC or trastuzumab CL264-ISAC. Tumors were harvested 20 hours post administration, processed to a single cell suspension, and analyzed by flow cytometry to assess activation of tumor-infiltrating myeloid APCs. (c-d) NSG or Rag2/IL2rg double knockout mice were implanted with the indicated human tumor cell line and randomized when the tumor volume reached 50 – 75 mm3 (HCC1954) or 75 – 150 mm3 (JIMT-1). Mice were treated via intraperitoneal injection with 5 mg/kg of rituximab, trastuzumab, trastuzumab T785-ISAC, trastuzumab CL264-ISAC or the respective isotype-ISACs, every 5 days for a total of 6 treatments. (e) rHER2 expression was measured on CT26-rHER2 tumor cells in culture prior to implantation (left flow plot) and in tumors nine days post-implantation (right flow plot) by flow cytometry with fluorescently-conjugated anti-rHER2 antibody (red) or an isotype control (blue). (f) Balb/c mice were implanted with the CT26-rHER2 tumor cell line and were randomized when the tumor volume reached 50 mm3. Mice were then treated via intraperitoneal injection with 10 mg/kg of mouse anti-rat HER2 or mouse anti-rat HER2 CL264-ISAC every 5 days for a total of 6 treatments. Data shown are from 1 experiment with 8 mice per arm and are representative of 3 experiments. (g) Anti-rat HER2 CL264-ISAC treated mice that experienced complete tumor regression for >21 days after their last treatment were challenged with parental CT26 (left flank) and 4T1 cell lines (right flank), with or without CD4 and CD8 T cell depletion (n =3 each). Tumor naïve mice (n = 6) challenged with the parental CT26 and 4T1 cell lines were included as controls. (a-i) Data are shown from individual experiments with 3-6 mice per arm and are representative of 1-4 experiments with a minimum of 3 mice per arm in each experiment. Data are shown as mean with SEM and statistics are shown with *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.