ImmunoPET imaging of human CD8(+) T cells with novel (68)Ga-labeled nanobody companion diagnostic agents

BACKGROUND: Although immunotherapy has revolutionized treatment strategies for some types of cancers, most patients failed to respond or obtain long-term benefit. Tumor-infiltrating CD8(+) T lymphocytes are closely related to the treatment outcome and prognosis of patients. Therefore, noninvasive elucidation of both systemic and tumor-infiltrating CD8(+) T lymphocytes is of extraordinary significance for patients during cancer immunotherapy. Herein, a panel of (68)Ga-labeled Nanobodies were designed and investigated to track human CD8(+) T cells in vivo through immuno-positron emission tomography (immunoPET). RESULTS: Among the screened Nanobodies, SNA006a showed the highest binding affinity and specificity to both human CD8 protein and CD8(+) cells in vitro, with the equilibrium dissociation constant (K(D)) of 6.4 × 10(−10) M and 4.6 × 10(−10) M, respectively. (68)Ga-NOTA-SNA006 was obtained with high radiochemical yield and purity, and stayed stable for at least 1 h both in vitro and in vivo. Biodistribution and Micro-PET/CT imaging studies revealed that all tracers specifically concentrated in the CD8(+) tumors with low accumulation in CD8(−) tumors and normal organs except the kidneys, where the tracer was excreted and reabsorbed. Notably, the high uptake of (68)Ga-NOTA-SNA006a in CD8(+) tumors was rapid and persistent, which reached 24.41 ± 1.00% ID/g at 1.5 h after intravenous injection, resulting in excellent target-to-background ratios (TBRs). More specifically, the tumor-to-muscle, tumor-to-liver, and CD8(+) to CD8(−) tumor was 28.10 ± 3.68, 5.26 ± 0.86, and 19.58 ± 2.70 at 1.5 h, respectively. Furthermore, in the humanized PBMC-NSG and HSC-NPG mouse models, (68)Ga-NOTA-SNA006a accumulated in both CD8(+) tumors and specific tissues such as liver, spleen and lung where human CD8 antigen was overexpressed or CD8(+) T cells located during immunoPET imaging. CONCLUSIONS: (68)Ga-NOTA-SNA006a, a novel Nanobody tracer targeting human CD8 antigen, was developed with high radiochemical purity and high affinity. Compared with other candidates, the long retention time, low background, excellent TBRs of (68)Ga-NOTA-SNA006a make it precisely track the human CD8(+) T cells in mice models, showing great potential for immunotherapy monitoring and efficacy evaluation.