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Recently, various immuno-PET tracers based on monoclonal antibodies (mAbs), engineered scaffold proteins, and peptides were developed to target either programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1), showing promise in assessment of immune checkpoints. We sought to develop an immunotherapeutic agent based PET probe that enables real-time assessment of PD-L1 expression and evaluation of antibody drug biodistribution to select eligible candidates for anti-PD-1/PD-L1 immunotherapies. KN035, a 79.6 kDa size anti-PD-L1 domain antibody under analysis in clinical trials, was used to develop the immuno-PET probe, 89Zr-Df-KN035. Immuno-PET studies were performed to monitor PD-L1 levels in nude mice bearing LN229 xenografts with positive expression for PD-L1, and to evaluate the whole-body biodistribution in healthy non-human primates (NHPs). LN229 xenografts were markedly visualized from 24 h after injection of 89Zr-Df-KN035, with elevated accumulation persisting for up to 120 h. Tumor radioactivity was notably reduced in the presence of excess KN035. Mouse ex vivobiodistribution studies performed at 24 and 120 h revealed tumor-to-muscle ratios as high as 5.64 ± 0.65 and 7.70 ± 1.37, respectively. In the NHP model, PET imaging demonstrated low background. The liver and kidney showed moderate accumulation with the highest SUVmeanvalue of 1.15 ± 0.15 and 2.13 ± 0.10 at 72 h, respectively. The spleen, lymph nodes, and salivary glands were also slightly visualized. In conclusion, 89Zr-Df-KN035, a novel anti-PD-L1 domain antibody-based probe, shows the feasibility of noninvasive in vivoevaluation of PD-L1 expression. This work further provides a template for immunotherapeutic agent based imaging to evaluate human PD-L1 expression and to augment our understanding of therapeutic agent biodistribution, leading to better therapeutic strategies in the future.

•The human-derived and antibody-like C3b-targeted fusion protein CG001 may simultaneously and effectively block 3 complement pathways.•CG001 holds high potential of further development for clinical study based on the preclinical evaluation.

Accumulating evidence has suggested that the tumor microenvironment of nonsmall-cell lung cancer (NSCLC) may be impacted by chemotherapy, radiotherapy, or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). PD-L1 is an important biomarker in the tumor microenvironment that can predict patient response to immunotherapies. Therefore, it is highly desirable to achieve a real-time, noninvasive assessment of PD-L1 expression, which can provide critical information for recruiting patients as well as monitoring therapeutic efficacy. We herein studied the EGFR-TKI-induced effects on PD-L1 levels in NSCLC tumor models using immuno-PET imaging with 89Zr-Df-KN035, an imaging tracer previously established by our group. A549 human NSCLC xenografts were established in BALB/c nude mice and treated with different doses of an EGFR-TKI gefitinib. PET imaging with 89Zr-Df-KN035 was performed before and after the treatment to evaluate PD-L1 expression, which was further verified by immunohistochemical staining. Our results demonstrate that 89Zr-Df-KN035 can specifically evaluate PD-L1 levels in NSCLC tumor models. Compared to the untreated control, the high dose of gefitinib inhibited tumor growth and lowered the tumor uptake of 89Zr-Df-KN035. In comparison, the low dose of gefitinib did not affect tumor growth, although the extensive tumor necrosis also led to the lower uptake of 89Zr-Df-KN035. In conclusion, our results demonstrate that immuno-PET imaging with 89Zr-Df-KN035 is a promising tool to noninvasively monitor PD-L1 expression in NSCLC treated with EGFR-TKIs and can be used to optimize treatment plans for immunotherapy.