Combination treatment of VLA-4 targeted radionuclide therapy and immunotherapy in a mouse model of melanoma.

Very late antigen-4 (VLA-4; also known as integrin α4β1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radiotherapy. 177 Lu-DOTA-PEG4-LLP2A (177 Lu-LLP2A) shows high affinity for VLA-4 and had high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177 Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICI) in B16F10 tumor-bearing mice. Methods: B16F10 tumor cells (1 x 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 days, the mice were randomized into eight groups ( n = 8). 177 Lu-LLP2A was injected i.v. on day 8 or 9 (single dose), and ICI antibodies were administered i.p. in 3 doses. Tumor growth was monitored over time via calipers. TUNEL staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scramble LLP2A were injected in tumor-bearing mice, tumors were collected after 4 h post-injection, and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: 177 Lu-LLP2A alone showed comparable efficacy to combination anti-PD-1/PD-L1 + anti-CTLA-4 antibody treatment, while 177 Lu-LLP2A together with combination ICI significantly enhanced survival. TUNEL staining showed the highest levels of apoptosis were found in the 177 Lu-LLP2A + ICI groups. In addition to targeting tumor cells, 177 Lu-LLP2A also binds immune cells in the B16F10 tumor microenvironment. Based on flow cytometry data, the tumor consists of ~77 % tumor cells/fibroblasts (CD45-CD49d+) and ~23 % immune cells (CD45+CD49d+), and immune cells express 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 of 177 Lu-LLP2A and ICI targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.