135P Simultaneous noninvasive imaging of LAG-3 and PD-L1 for noninvasive immunotyping in lung cancers

Immunotherapy has significantly improved the survival of cancer patients, while its efficacy is limited by the reliance on single marker like PD-L1 and its spatiotemporal heterogeneity. To address this issue, the current study introduces a nuclear imaging approach for the simultaneous and noninvasive quantification of PD-L1 and lymphocyte activation gene-3 (LAG-3), aiming to improve immunotherapy response prediction. 99mTc-HYNIC-αLAG-3 and 99mTc-HYNIC-αPD-L1 probes were developed by using anti-human LAG-3 and PD-L1 antibodies, respectively. In vivo and vitro assays were conducted in LAG3+A549, H1975, LAG-3-A549, and LLC cells. Single photon emission computed tomography/computed tomography (SPECT/CT) imaging were performed in various lung cancer models. The correlation between tumor signals in region of interest (ROI), tumor uptake determined by ex vivo γ-counting, and immunohistochemistry (IHC) expression levels was analyzed. Additionally, αPD-L1 was labeled with 125I, combined with 99mTc-HYNIC-αLAG-3, to perform synchronous noninvasive nuclear imaging of immunotypes (LAG-3+PD-L1+, LAG-3-PD-L1+, LAG-3+PD-L1-, LAG-3-PD-L1-). 99mTc-HYNIC-αLAG-3/PD-L1 probes exhibited a RCP of over 95% and high stability in vitro within 24 hours. The specific binding of probes to LAG-3 or PD-L1 was confirmed. SPECT/CT imaging of 99mTc-HYNIC-αLAG-3 revealed tumor uptake of 15.5 ± 2.4, 5.3 ± 1.1, and 6.2 ± 2.1 %ID/g in LAG-3+A549, LAG-3-A549 models, and LLC-bearing LAG-3 humanized mice at 24 h, respectively. 99mTc-HYNIC-αPD-L1 had tumor uptake of 7.4 ± 0.8%ID/g in H1975 models and 2.1 ± 0.8 %ID/g in A549 models at 24 h. The tumor signals within the ROI for both probes demonstrated significant associations with ex vivo tumor uptake and IHC expression levels. Simultaneous SPECT/CT imaging of 99mTc-HYNIC-αLAG-3 and 125I-αPD-L1 at 24 h post-injection successfully differentiated various immunotypes. This study demonstrates the feasibility of SPECT imaging in visualizing LAG-3 and PD-L1. Moreover, this is the first report of simultaneous nuclear imaging to identify diverse immunotypes in lung cancers, offering new insights into forecasting immunotherapy response and selecting combination therapy.