Abstract
BackgroundWhile immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.ResultsTo introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.ConclusionsA set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.Graphical
Highlights
While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treat‐ ment, only subgroups of patients show durable responses
Whole-body imaging would ideally be supplemented with an imaging modality suitable to assess the distribution of PD-L1 therapeutics at a cellular level, such as a fluorescent dye
The effect of isotype switching and site‐specific conjugation on the in vivo behavior of aPD‐L1 imaging agents First, using our molecular toolbox (Fig. 1A and B) we set out to determine the consequence of isotype switching of the PD-L1 targeting antibody from rIgG2a to mouse IgG1 (mIgG1) [27], as well as random versus sitespecific radiolabeling and additional functionalization with a fluorescent dye, on in vivo biodistribution of these compounds (Fig. 2A)
Summary
While immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treat‐ ment, only subgroups of patients show durable responses. Immunotherapy, in particular blockade of immune checkpoint molecules such as Programmed Death 1 (PD-1) and its ligand PD-L1 using antibodies, has revolutionized the field of cancer therapeutics due to unparalleled responses in a range of cancers [1,2,3]. Despite these successes, a large number of patients does not benefit from this treatment [4]. This allows microscopic imaging at high spatial resolution [17]
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