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

Cheng Wang,Liangrong Wan,Lianghua Li,Gang Huang,Weijun Wei,Yan Sun,Haitao Zhao,Chao Wang,Yanling Yang,Jianjun Liu,Cheng Zhu

doi:10.1186/s12951-021-00785-9

Abstract

BackgroundAlthough 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).ResultsAmong 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.Conclusions68Ga-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.

Highlights

Immunotherapy has revolutionized treatment strategies for some types of cancers, most patients failed to respond or obtain long-term benefit
Synthesis and characterization of 68Ga‐NOTA‐SNA006 Precursors NOTA-SNA006a, c and d for radiolabel were prepared by condensation coupling of the amino groups of three different Nanobodies with the isothiocyanate group of p-SCN-Bn-NOTA, respectively (Fig. 2a)
68Ga-NOTA-SNA006 was produced at room temperature via the chelation of 68Ga and precursors in sodium acetate buffer, and impurities were further removed with PD-10 column

Summary

Introduction

Immunotherapy has revolutionized treatment strategies for some types of cancers, most patients failed to respond or obtain long-term benefit. Noninvasive elucidation of both systemic and tumorinfiltrating CD8+ T lymphocytes is of extraordinary significance for patients during cancer immunotherapy. A panel of 68Ga-labeled Nanobodies were designed and investigated to track human C D8+ T cells in vivo through immuno-positron emission tomography (immunoPET). Due to precise patient stratification and timely response assessment, CDx can improve the treatment efficacy while ensuring safety and reducing medical costs. One advanced modality of CDx is immuno-positron emission tomography (immunoPET), which delicately combines the extraordinary targeting specificity of monoclonal antibodies and the superior sensitivity of PET [3, 4]. Our previous studies have shown that immunoPET can visualize heterogeneous expression of tumor antigens in a noninvasive manner [5,6,7], and depict the whole process of immunotherapy dynamically and quantitatively [8]

Methods

Results

Discussion

Conclusion