Haitao Zhao1, Chao Wang2, Yanling Yang2,3, Yan Sun2, Weijun Wei1,4, Cheng Wang1, Liangrong Wan1, Cheng Zhu1, Lianghua Li1, Gang Huang1,5, Jianjun Liu6,7. 1. Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Rd, Shanghai, 200127, China. 2. SmartNuclide Biopharma Co. Ltd, 218 Xinghu St., BioBAY A4-202, Suzhou Industrial Park, Suzhou, China. 3. School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai, 264005, China. 4. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 1630 Dongfang Rd, Shanghai, 200127, China. 5. Shanghai Key Laboratory of Molecular Imaging, Shanghai University of Medicine and Health Sciences, Shanghai, 201318, China. 6. Department of Nuclear Medicine, Institute of Clinical Nuclear Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Rd, Shanghai, 200127, China. RJnuclear@126.com. 7. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 1630 Dongfang Rd, Shanghai, 200127, China. RJnuclear@126.com.
Abstract
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 68Ga-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 (KD) of 6.4 × 10-10 M and 4.6 × 10-10 M, respectively. 68Ga-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 68Ga-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, 68Ga-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: 68Ga-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 68Ga-NOTA-SNA006a make it precisely track the human CD8+ T cells in mice models, showing great potential for immunotherapy monitoring and efficacy evaluation.
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 68Ga-labeled Nanobodies were designed and investigated to track humanCD8+ 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 humanCD8 protein and CD8+ cells in vitro, with the equilibrium dissociation constant (KD) of 6.4 × 10-10 M and 4.6 × 10-10 M, respectively. 68Ga-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 68Ga-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-NPGmouse models, 68Ga-NOTA-SNA006a accumulated in both CD8+ tumors and specific tissues such as liver, spleen and lung where humanCD8 antigen was overexpressed or CD8+ T cells located during immunoPET imaging. CONCLUSIONS: 68Ga-NOTA-SNA006a, a novel Nanobody tracer targeting humanCD8 antigen, was developed with high radiochemical purity and high affinity. Compared with other candidates, the long retention time, low background, excellent TBRs of 68Ga-NOTA-SNA006a make it precisely track the humanCD8+ T cells in mice models, showing great potential for immunotherapy monitoring and efficacy evaluation.
Entities:
Keywords:
CD8+ T lymphocytes; Companion diagnostics; ImmunoPET; Immunotherapy; Nanobody
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