Very late antigen-4 (VLA-4; also known as integrin a4ß1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT).177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor–bearing mice. Methods: Tumor cells (1 · 106) were implanted subcutaneously in C57BL/6 mice. After 8–10 d, the mice were randomized into 8 groups.177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3–scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 1 anti-CTLA-4 or anti-PD-L1 1 anti-CTLA-4, whereas TRT 1 ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT 1 ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/ CD49d-positive) and about 23% immune cells (CD45-positive/ CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT 1 ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.
Choi, J., Beaino, W., Fecek, R. J., Fabian, K. P. L., Laymon, C. M., Kurland, B. F., ... Anderson, C. J. (2018). Combined VLA-4–targeted radionuclide therapy and immunotherapy in a mouse model of melanoma. Journal of Nuclear Medicine, 59(12), 1843-1849. https://doi.org/10.2967/jnumed.118.209510