ImmunoPET Imaging of CTLA-4 Expression in Mouse Models of Non-small Cell Lung Cancer

Abstract

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on the surface of activated T cells and some tumor cells, and is the target of the clinically-approved monoclonal antibody ipilimumab. In this study, we investigate specific binding of radiolabeled ipilimumab to CTLA-4 expressed by human non-small cell lung cancer cells in vivo using positron emission tomography (PET). Ipilimumab was radiolabeled with 64Cu (t1/2 = 12.7 h) through the use of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to formulate 64Cu-DOTA-ipilimumab. CTLA-4 expression in three non-small cell lung cancer (NSCLC) cell lines (A549, H460, and H358) was verified and quantified by Western blot and enzyme-linked immunosorbent assays (ELISA). A receptor binding assay was utilized to monitor the binding and internalization of 64Cu-DOTA-ipilimumab in the NSCLC cell lines. Next, the biodistribution of 64Cu-DOTA-ipilimumab was mapped by longitudinal PET imaging up to 48 h after injection. Ex vivo biodistribution and histological studies were employed to verify PET results. By in vitro analysis, CTLA-4 was found to be expressed on all three NSCLC cell lines with A549 and H358 showing the highest and lowest level of expression, respectively. PET imaging and quantification verified these findings as the tracer accumulated highest in the A549 tumor model (9.80 ± 0.22 %ID/g at 48 h after injection; n=4), followed by H460 and H358 tumors with uptakes of 9.37 ± 0.26 %ID/g and 7.43 ± 0.05 %ID/g, respectively (n=4). The specificity of the tracer was verified by injecting excess ipilimumab in A549 tumor-bearing mice, which decreased tracer uptake to 6.90 ± 0.51 %ID/g at 48 after injection (n=4). Ex vivo analysis following the last imaging session also corroborated these findings. 64Cu-DOTA-ipilimumab showed enhanced and persistent accumulation in CTLA-4-expressing tissues, which will enable researchers further insight into CTLA-4 targeted therapies in the future.

Tracers for non-invasive radionuclide imaging of immune checkpoint expression in cancer

“…Polymerase chain reaction (PCR)-analysis on CT26 tumor tissues from BALB/c and T cell lacking BALB/c nu/nu indicated that the CTLA-4 expression was T cell dependent and therefore the tracer could be used to image CTLA-4 positive T cells. In 2017, Ehlerding et al reported uptake of 64 Cu-labeled ipilimumab by CTLA-4 expressing human NSCLC xenografts (A549, H460, and H358) (Ehlerding et al 2017). In vivo tumor tracer uptake correlated with in vitro CTLA-4 expression levels of these tumor cells, with the highest uptake in the A549 cell line 48 h post infusion.…”

Cell Tracking in Cancer Immunotherapy

“…A detailed understanding of the tumor microenvironment, including the identification and quantification of different immune cell subsets, their spatial context, and the expression of these immune checkpoint markers is obviously required to go further with these new therapies (107). Changes in immune cell infiltration and biomarker expression before and after therapeutic intervention are critical parameters for clinical development (108).…”

“…Immuno-detection using antibodies labeled with zirconium-89 or copper-64 for PET, as well as indium-111 for SPECT, has been used to assess the CTLA-4 and PD-1 status of TIL in vivo and the expression of PD-L1 by tumor cells in order to predict the therapeutic efficacy of the administration of immune checkpoint inhibitors in mice and in human (79,(109)(110)(111). This approach was also proposed in the context of anti CTLA-4 therapy (107). Based on tumor biopsies, it appears that some patients with PD-L1-negative tumors show clinical benefit of anti-PD-L1 treatment.…”

Interactions Between Tumor Biology and Targeted Nanoplatforms for Imaging Applications

“…The cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) is a marker of immune T cells and also some lung cancer cells. The Ehlerding group162 used 64 Cu‐radiolabeled ipilimumab (anti‐CTLA‐4 mAb) for PET imaging of human NSCLC cells. In vivo results showed the radiolabeled Ab effectively accumulated in CTLA‐4 + NSCLC.…”