PD-L1 has a heterogeneous and dynamic expression in gastric cancer with implications for immunoPET.

Introduction: This study aimed to investigate the dynamics of programmed death-ligand 1 (PD-L1) expression, spatial heterogeneity, and binding affinity of FDA-approved anti-PD-L1 antibodies (avelumab and atezolizumab) in gastric cancer. Additionally, we determined how PD-L1 glycosylation impacts antibody accumulation in gastric cancer cells. Methods: Dynamic PD-L1 expression was examined in NCIN87 gastric cancer cells. Comparative binding studies of avelumab and atezolizumab were conducted in gastric cancer models, both in vitro and in vivo. Antibody uptake in tumors was visualized through positron emission tomography (PET) imaging. PD-L1 glycosylation status was determined via Western blot analyses before and after PNGase F treatment. Results: Consistent findings revealed time-dependent PD-L1 induction in NCIN87 gastric cancer cells and spatial heterogeneity in tumors, as shown by PET imaging and immunofluorescence. Avelumab displayed superior binding affinity to NCIN87 cells compared to atezolizumab, confirmed by in vivo PET imaging and ex vivo biodistribution analyses. Notably, PD-L1 glycosylation at approximately 50 kDa was observed, with PNGase F treatment inducing a shift to 35 kDa in molecular weight. Tissue samples from patient-derived xenografts (PDXs) validated the presence of both glycosylated and deglycosylated PD-L1 (degPD-L1) forms in gastric cancer. Immunofluorescence microscopy and binding assays demonstrated enhanced avelumab binding post-deglycosylation. Discussion: This study provides an understanding of dynamic and spatially heterogeneous PD-L1 expression in gastric cancer. Anti-PD-L1 immunoPET was able to visualize gastric tumors, and PD-L1 glycosylation has significant implications for antibody recognition. These insights contribute to demonstrating the complexities of PD-L1 in gastric cancer, holding relevance for refining PD-L1 imaging-based approaches.

PDX Models in Theranostic Applications: Generation and Screening for B Cell Lymphoma of Human Origin.

This MIB guide briefly summarizes the generation of patient-derived xenografts (PDXs) and highlights the importance of validating PDX models for the presence of B cell lymphoma of human origin before their use in radiotheranostic applications. The use of this protocol will allow researchers to learn different methods for screening PDX models for Epstein-Barr virus (EBV)-infected B cell lymphoma.

In Vivo Biorthogonal Antibody Click for Dual Targeting and Augmented Efficacy in Cancer Treatment.

Antibody-drug conjugates (ADCs) have emerged as promising therapeutics for cancer treatment; however, their effectiveness has been limited by single antigen targeting, potentially leading to resistance mechanisms triggered by tumor compensatory pathways or reduced expression of the target protein. Here, we present antibody-ADC click, an approach that harnesses bioorthogonal click chemistry for in vivo dual receptor targeting, irrespective of the levels of the tumor's expression of the ADC-targeting antigen. Antibody-ADC click enables targeting heterogeneity and enhances antibody internalization and drug delivery inside cancer cells, resulting in potent toxicity. We conjugated antibodies and ADCs to the bioorthogonal click moieties tetrazine (Tz) and trans-cyclooctene (TCO). Through sequential antibody administration in living biological systems, we achieved dual receptor targeting by in vivo clicking of antibody-TCO with antibody-Tz. We show that the clicked antibody therapy outperformed conventional ADC monotherapy or antibody combinations in preclinical models mimicking ADC-eligible, ADC-resistant, and ADC-ineligible tumors. Antibody-ADC click enables in vivo dual-antigen targeting without extensive antibody bioengineering, sustains tumor treatment, and enhances antibody-mediated cytotoxicity.

PET and Optical Imaging of Caveolin-1 in Gastric Tumors.

Previous studies have suggested tumoral caveolin-1 (CAV1) as a predictive biomarker for the response to anti-HER2 antibody drug therapies in gastric tumors. In this study, radiolabeled and fluorescently labeled anti-CAV1 antibodies were developed and tested as an immunoPET or optical imaging agent to detect CAV1 in HER2-positive/CAV1-high NCIN87 gastric tumors. The expression of CAV1 receptors in NCIN87 gastric tumors and nontumor murine organs was determined by Western blot. Binding assays were performed to validate the anti-CAV1 antibody specificity for CAV1-expressing NCIN87 cancer cells. Subcutaneous and orthotopic NCIN87 xenografts were used for PET imaging and ex vivo biodistribution of the radioimmunoconjugate. Additional HER2-PET and CAV1-optical imaging was also performed to determine CAV1 in the HER2-positive tumors. 89Zr-labeled anti-CAV1 antibody was able to bind to CAV1-expressing NCIN87 cells with a Bmax value of 2.7 × 103 CAV1 receptors/cell in vitro. ImmunoPET images demonstrated the localization of the antibody in subcutaneous NCIN87 xenografts. In the orthotopic model, CAV1 expression was also observed by optical imaging in the HER2-positive tumors previously imaged with HER2-PET. Ex vivo biodistribution analysis further confirmed these imaging results. The preclinical data from this study demonstrate the potential of using CAV1-PET and optical imaging for detecting gastric tumors.

Immuno-PET Detects Antibody-Drug Potency on Coadministration with Statins.

The human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) are antibody-drug conjugates (ADC) clinically used to treat HER2-positive breast cancer, with the latter receiving clinical approval in 2021 for HER2-positive gastric cancer. Lovastatin, a cholesterol-lowering drug, temporally elevates cell-surface HER2 in ways that enhance HER2-ADC binding and internalization. Methods: In an NCIN87 gastric xenograft model and a gastric patient-derived xenograft model, we used the 89Zr-labeled or 64Cu-labeled anti-HER2 antibody trastuzumab to investigate the dosing regimen of ADC therapy with and without coadministration of lovastatin. We compared the ADC efficacy of a multiple-dose ADC regime, which replicates the clinical dose regimen standard, with a single-dose regime. Results: T-DM1/lovastatin treatment inhibited tumor growth, regardless of multiple- or single-dose T-DM1 administration. Coadministration of lovastatin with T-DM1 or T-DXd as a single dose enhanced tumor growth inhibition, which was accompanied by a decrease in signal on HER2-targeted immuno-PET and a decrease in HER2-mediated signaling at the cellular level. DNA damage signaling was increased on ADC treatment in vitro. Conclusion: Our data from a gastric cancer xenograft show the utility of HER2-targeted immuno-PET to inform the tumor response to ADC therapies in combination with modulators of cell-surface target availability. Our studies also demonstrate that statins enhance ADC efficacy in both a cell-line and a patient-derived xenograft model in ways that enable a single-dose administration of the ADC.

Metformin-Induced Receptor Turnover Alters Antibody Accumulation in HER-Expressing Tumors.

Metformin has effects beyond its antihyperglycemic properties, including altering the localization of membrane receptors in cancer cells. Metformin decreases human epidermal growth factor receptor (HER) membrane density. Depletion of cell-surface HER decreases antibody-tumor binding for imaging and therapeutic approaches. Here, we used HER-targeted PET to annotate antibody-tumor binding in mice treated with metformin. Methods: Small-animal PET annotated antibody binding in HER-expressing xenografts on administration of an acute versus a daily dose schedule of metformin. Analyses at the protein level in the total, membrane, and internalized cell extracts were performed to determine receptor endocytosis, HER surface and internalized protein levels, and HER phosphorylation. Results: At 24 h after injection of radiolabeled anti-HER antibodies, control tumors had higher antibody accumulation than tumors treated with an acute dose of metformin. These differences were temporal, and by 72 h, tumor uptake in acute cohorts was similar to uptake in control. Additional PET imaging revealed a sustained decrease in tumor uptake on daily metformin treatment compared with control and acute metformin cohorts. The effects of metformin on membrane HER were reversible, and after its removal, antibody-tumor binding was restored. The time- and dose-dependent effects of metformin-induced HER depletion observed preclinically were validated with immunofluorescence, fractionation, and protein analysis cell assays. Conclusion: The findings that metformin decreases cell-surface HER receptors and reduces antibody-tumor binding may have significant implications for the use of antibodies targeting these receptors in cancer treatment and molecular imaging.

Antibody-Targeted Imaging of Gastric Cancer.

The specificity of antibodies for antigens overexpressed or uniquely expressed in tumor cells makes them ideal candidates in the development of bioconjugates for tumor imaging. Molecular imaging can aid clinicians in the diagnosis of gastric tumors and in selecting patients for therapies targeting receptors with a heterogeneous intratumoral or intertumoral expression. Antibodies labeled with an imaging radiometal can be used to detect primary tumors and metastases using whole-body positron emission tomography (PET) or single photon emission computed tomography (SPECT), both during diagnosis and monitoring disease response. Conjugated with fluorescent dyes, antibodies can image tumors by targeted optical imaging. This review provides an overview of the most recent advances in the use of antibodies labeled with radiometals or conjugated with fluorescent dyes for gastric cancer imaging.

HER2-Targeted PET Imaging and Therapy of Hyaluronan-Masked HER2-Overexpressing Breast Cancer.

Human epidermal growth factor receptor 2 (HER2) is a biomarker in breast cancer, and its overexpression is required to initiate therapies using HER2-targeted antibodies. Although trastuzumab is one of the most effective therapeutic antibodies in HER2-overexpressing breast cancer, a significant number of patients do not benefit from this therapy due to inherent or acquired resistance mechanisms. One reported mechanism of resistance is the steric hindering effect caused by the polymeric complex formed between hyaluronan and CD44, thus preventing trastuzumab from binding to HER2. Hyaluronan/CD44 contributes as an obstacle for trastuzumab to bind HER2, but it is also involved in HER2 internalization. In this study, we used zirconium-89 (89Zr)-labeled trastuzumab immunoPET to investigate whether degradation of hyaluronan can resensitize HER2-overexpressing breast cancer cells to trastuzumab. Targeted degradation of endogenously produced hyaluronan and inhibition of its synthesis were achieved by treating trastuzumab-resistant JIMT1 breast cancer cells with hyaluronidase (HLX) and 4-methylumbelliferone (4MU). The 4MU/HLX treatment reduced HER2 internalization by depleting hyaluronan/CD44 and the caveolin-1 (CAV1) endocytic protein, resulting in enhanced membrane-bound 89Zr-labeled trastuzumab. 4MU/HLX enhanced trastuzumab tumor uptake, as evidenced by increased tumor binding of the 89Zr-labeled trastuzumab in JIMT1 tumor xenografts. In vitro mechanistic studies demonstrated a decrease in HER2-mediated oncogenic signaling upon cell treatment with 4MU/HLX. Importantly, 4MU/HLX enhanced trastuzumab efficacy in JIMT1 xenografts. These data showed the utility of 89Zr-labeled trastuzumab as a PET imaging agent to monitor the affinity of the antibody to HER2 during CD44/hyaluronan-specific inhibition with the overall goal of improving trastuzumab therapy.