To prepare ⁹⁹Tc^m-hydrazinonicotinamide(HYNIC)-αCD8/Fab (⁹⁹Tc^m-αCD8/Fab), and explore the predictive value of ⁹⁹Tc^m-αCD8/Fab SPECT/CT imaging for the efficacy of anti-programmed death-1 (PD-1) immunotherapy.

The αCD8/Fab was modified with HYNIC-N-hydroxysuccinimide (NHS) and IRDye800-NHS to form HYNIC-αCD8/Fab and IRDye800-αCD8/Fab (Dye-αCD8/Fab), respectively. ⁹⁹Tc^m-αCD8/Fab was prepared in sodium bicarbonate buffer (pH=8.5), with SnCl₂ being used as the reducing agent. The labeling yield and radiochemical purity of ⁹⁹Tc^m-αCD8/Fab and its stability in PBS and fetal bovine serum (FBS) were tested in vitro. The mouse spleen and human peripheral blood lymphocytes were isolated for cell-specific binding and blocking experiments of ⁹⁹Tc^m-αCD8/Fab in vitro. SPECT/CT imaging was used to analyze the specific binding ability of the ⁹⁹Tc^m-αCD8/Fab probe in CT26 colon cancer mouse models (BALB/c). The near infrared fluorescence imaging and SPECT/CT imaging were performed to detect the intra-tumoral CD8⁺ T cell infiltration after anti-PD-1 therapy in tumor bearing mice, and the results were further verified by HE and immunofluorescence staining. CD8⁺ T cell depletion study was performed to determine the role of CD8⁺ T cells in the tumor responses to anti-PD-1 therapy. Two-way analysis of variance was used to compare the data difference.

The labeling yield of ⁹⁹Tc^m-αCD8/Fab was 90% with a high radiochemical purity (95%) and good stability in vitro (radiochemical purity still more than 80% after 720 min in PBS and FBS). ⁹⁹Tc^m-αCD8/Fab could specifically bind to mouse CD8⁺ T cells ((10.30±0.81) percent added radioactive dose (%AD)/10⁶ cells), compared with the binding ability in human peripheral blood lymphocytes group and CD8 antibody blocking group ((1.78±0.61) and (1.59±0.25) %AD/10⁶ cells; F=10.07, P<0.001). SPECT/CT imaging showed that ⁹⁹Tc^m-αCD8/Fab had markedly higher tumor uptake in the CT26 colon cancer mouse models. Near-infrared fluorescence imaging showed that the tumor uptake of ⁹⁹Tc^m-αCD8/Fab in the responsive group was significantly higher than in the nonresponsive group after anti-PD-1 treatment ((8.9±1.1)% vs (7.1±0.8)%; F=4.69, P=0.024), and SPECT/CT imaging found the similar result. HE and immunofluorescence staining of tumor and lymph nodes showed that the proportion of lymphocyte infiltration was higher in the responsive group. Furthermore, CD8⁺ T cell depletion significantly reversed the therapeutic effect of anti-PD-1 immunotherapy in tumor-bearing mice.

In this study, ⁹⁹Tc^m-αCD8/Fab was successfully obtained. CD8-specific SPECT imaging could sensitively visualize the tumor-infiltrating CD8⁺ T cells, suggesting the potential application value to predict and evaluate the efficacy of immunotherapy in the clinical settings.

Colorectal neoplasms; Lymphocytes, tumor-infiltrating; CD8-positive T-lymphocytes; Programmed cell death 1 receptor; Treatment outcome; Forecasting; Mice