GlyCLICK® Enhances Development of Immuno-SPECT/Fluorescent Tracer for PD-L1 in Preclinical Models
Scientists at Université de Bourgogne and collaborators have described the development of a bimodal tracer targeting PD-L1 in human and murine models for preclinical studies. Using a [111In]-DOTA-aza-BODIPY bimodal probe following conjugation onto PD-L1 monoclonal antibodies (mAbs) through either site-specific or random methods, the ability of the bimodal antibodies to selectively identify PD-L1+ tumors were assessed using both fluorescence and SPECT imaging. There were significant benefits observed with the probe generated using the GlyCLICK site-specific conjugation method compared to a random lysine conjugation approach!
Detection of biomarkers to diagnose, treat, and predict the efficacy of cancer therapies is a major clinical challenge. In particular, the programmed cell death protein 1 (PD-1) and its ligand PD-L1 constitute a critical regulatory pathway in maintaining immune homeostasis and preventing excessive immune activation. Currently, biomarkers such as PD-L1 are commonly detected from biopsies, but this approach does not take into account the spatiotemporal heterogeneity of their expression in tumors. A potential solution to this consists of conjugating mAbs targeting these biomarkers with multimodal imaging probes to improve both early diagnosis and non-invasive monitoring of patients during therapy.
The scientists here set out to create a bimodal agent combining fluorescence and SPECT imaging to provide a deeper understanding of biological mechanisms involving PD-1/PD- L1. The integration of both modalities within a single agent provides precise correlation between SPECT and fluorescence data, effectively minimizing any competition for the target. The GlyCLICK technology was used to generate site-specific conjugates combining this probe and PD-L1 mAbs, which were compared to conjugates generated through a random labelling method. The different conjugation methods were compared using both human and mouse models and assessed for their stability, affinity and efficacy, both in vitro and in vivo.
GlyCLICK is a three-step conjugation technology for site-specific and quantitative conjugation of IgG from several species and subclasses:
The stability of the different conjugates was tested under conditions close to in vivo, by incubation in human and murine sera for 72 hours. During this time, neither the generation of aggregates nor evidence of degradation were observed in any of the bioconjugates, suggesting that the molecules were similarly stable regardless of the conjugation method. However, when the affinity of the anti-PD-L1 mAbs to their targets were tested using biolayer interferometry (BLI) experiments, there were differences observed. Here, the GlyCLICK generated anti-PD-L1 mAbs exhibited lower KD values their randomly conjugated counterpart, with a much more significant decrease in affinity was observed with the murine mAbs.
The efficiency of the in vivo targeting of the different bioconjugates was also evaluated by fluorescence optical imaging and the results demonstrated that the tumor uptake of the GlyCLICK generated bimodal platform was much higher than what was observed with the randomly bioconjugated one, which corroborated the in vitro results. Tumor fluorescence quantifications showed a significant difference between the two bioconjugation methods with a much lower fluorescence signal in the tumor uptake of the random conjugates versus the site-specific ones at the tumors expressing PD-L1. In addition to poorer labelling specificity, random bioconjugates showed lower overall fluorescence levels, especially for the anti-mouse PD-L1 mAb. Furthermore, the uptake in elimination organs, such as the liver and kidneys, was also lower in the case of site-specific bioconjugates. GlyCLICK conjugated mAbs also exhibited a strong SPECT signal and efficient tumor uptake in PD-L1+ tumors.
In summary, this work highlights clear benefits of site-specific conjugation over random conjugation of mAbs and GlyCLICK has proved to be a valuable tool in the development of the first bimodal SPECT/fluorescent immuno-agent to target both human and murine PD-L1. Site-specific bioconjugation, using GlyCLICK, generated molecules with stronger affinity for both human and murine targets compared to randomly conjugated molecules. In addition, bioconjugates produced using a site-specfic approach showed superior in vivo efficacy with respect to fluorescence signal and tumor uptake with a lower uptake into elimination organs. This work allows the sequential imaging of PD-L1 expression in vivo following the injection of a unique bimodal radio-conjugate, which could be relevant in clinical settings based on whole-body SPECT, followed by fluorescence-guided surgery (FGS).
Reference
Privat et al., 2024. Development of an Immuno-SPECT/Fluorescent Bimodal Tracer Targeting Human or Murine PD-L1 on Preclinical Models. J Med Chem.
GlyCLICK – Site-specific conjugation of IgG