PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired antigen-experienced anticancer T cells. However, the efficacy of current PD-L1-blocking antibodies is potentially reduced by ‘on-target/off-tumor’ binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity may induce a generalized activation of all antigen-experienced T cells which may explain the frequent occurrence of autoimmune-related adverse events during and after treatment.To address these issues, we constructed a bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and to more selectively reactivate anticancer T cells. Indeed, the IC50 of PD-L1xEGFR for blocking PD-L1 on EGFR+ cancer cells was ∼140 fold lower compared to that of the analogous PD-L1-blocking bsAb PD-L1xMock with irrelevant target antigen specificity. Importantly, activation status, IFN-γ production, and oncolytic activity of anti-CD3xanti-EpCAM-redirected T cells was enhanced when cocultured with EGFR-expressing carcinoma cells. Similarly, the capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-directed CD8+ effector T cells was enhanced when cocultured with EGFR-expressing CMVpp65-transfected cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in mice xenografted with EGFR-expressing cancer cells 111In-PD-L1xEGFR showed a significantly higher tumor uptake compared to 111In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, thereby promoting the selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies.

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1 þ tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/ CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1 þ tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 micro-SPECT/CT imaging could also distinguish between high and low PD-L1-expressing tumors. The presence of PD-L1 þ immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1-targeted therapy.

Immunotherapy with checkpoint inhibitors demonstrates impressive improvements in the treatment of several types of cancer. Unfortunately, not all patients respond to therapy while severe immune-related adverse effects are prevalent. Currently, patient stratification is based on immunotherapy marker expression through immunohistochemical analysis on biopsied material. However, expression can be heterogeneous within and between tumor lesions, amplifying the sampling limitations of biopsies. Analysis of immunotherapy target expression by noninvasive quantitative molecular imaging with PET or SPECT may overcome this issue. In this review, an overview of tracers that have been developed for preclinical and clinical imaging of key immunotherapy targets, such as programmed cell death-1, programmed cell death ligand-1, IDO1 and cytotoxic T lymphocyteassociated antigen-4 is presented. We discuss important aspects to consider when developing such tracers and outline the future perspectives of molecular imaging of immunotherapy markers.

Background While 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. Results To 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. Conclusions A 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 Abstract

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<div>Abstract<p>Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1⁺ tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1⁺ tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1–expressing tumors. The presence of PD-L1⁺ immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1–targeted therapy.</p></div>

<p>Supplemental Figure 3</p>

<p>Supplemental Table 1</p>