In-depth cross-validation of human and mouse CD4-specific minibodies for noninvasive PET imaging of CD4+ cells and response prediction to cancer immunotherapy.

Increasing evidence emphasizes the pivotal role of CD4+ T cells in orchestrating cancer immunity. Noninvasive in vivo imaging of the temporal dynamics of CD4+ T cells and their distribution patterns might provide novel insights into their effector and regulator cell functions during cancer immunotherapy (CIT). Methods: We conducted a comparative analysis of 89Zr-labeled anti-mouse (m) and anti-human (h) CD4-targeting minibodies (Mbs) for in vivo positron emission tomography (PET)/magnetic resonance imaging (MRI) of CD4+ T cells in human xenografts, syngeneic tumor-bearing wild-type (WT), and human CD4+ knock-in (hCD4-KI) mouse models. Results: Both 89Zr-CD4-Mbs yielded high radiolabeling efficiencies of >90%, immunoreactivities of >70%, and specific in vitro binding to their target antigens. The specificity of in vivo targeting of 89Zr-hCD4-Mb was confirmed by PET/MRI, revealing ~4-fold greater 89Zr-hCD4-Mb uptake in subcutaneous hCD4+ hematopoietic peripheral blood acute lymphoblastic leukemia tumors (HPB-ALL) than in solid hCD4- diffuse histiocytic lymphomas (DHL) and 89Zr-mCD4-Mb uptake in hCD4+ HPB-ALL tumors. In a comparative cross-validation study in anti-programmed death ligand (αPD-L1)/anti-4-1BB-treated orthotopic PyMT mammary carcinoma-bearing hCD4-KI and WT mice, we detected 2- to 3-fold enhanced species-specific 89Zr-hCD4-Mb or 89Zr-mCD4-Mb uptake within CD4+ cell-enriched secondary lymphatic organs (lymph nodes and spleens). The 89Zr-hCD4-Mb uptake in the PyMT tumors was more pronounced in hCD4-KI mice compared to the WT control littermates. Most importantly, MC38 adenocarcinoma-bearing mice treated with a combination of αPD-L1 and anti-lymphocyte-activation gene 3 (αLag-3) antibodies exhibited ~1.4-fold higher 89Zr-mCD4-Mb uptake than mice that were not responsive to therapy or sham-treated mice. Conclusion: CD4 PET/MRI enabled monitoring of the CD4+ cell distribution in secondary lymphatic organs and the tumor microenvironment, capable of predicting sensitivity to CIT. Our imaging approach will provide deeper insights into the underlying molecular mechanisms of CD4-directed cancer immunotherapies in preclinical mouse models and is applicable for clinical translation.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.