Background. Breast cancer is the leading cause of death in women, with over 2.2 million cases recorded in 2020 worldwide.^[1] Among all identified cases, 10-20% are determined as triple negative breast cancer (TNBC) which is defined as human epidermal growth factor receptor 2 (HER2) negative, estrogen receptor (ER) negative and progesterone negative (PgR).^[2]  When positive for at least one of these receptors breast cancer has a generically good treatment prognosis. However, triple negative breast cancer lacks these druggable receptors and therefore available targeted therapies. Surgical removal of cancer lesions is the standard of care but unfortunately, it is connected to potential residual disease that ultimately can lead to reoccurrence.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) has emerged as a novel target over recent years for the treatment of several cancers.^[3] As an essential kinase during fetal maturation, postpartum tissue levels expression is low to non-detectable in humans.^[4] A broad range of cancers however do express ROR1 and accumulating evidence shows that the expression of ROR1 in TNBC is associated with a more aggressive disease.^[5] Zilovertamab is a clinically used humanized IgG1 antibody targeting ROR1 that binds to the Frizzled domain, blocking the downstream signaling of the Wnt family ligands.

In this study, we aim to develop fluorescent probes based on Zilovertamab, Zilovertamab-FNIRtag and Zilovertamab-IR800cw, and to evaluate their suitability for the imaging of triple negative breast cancer for intra-operative applications.

Methods. Zilovertamab was conjugated to IR800cw-NHS or FNIRtag-NHS to obtain a degree of labeling (DOL, antibody-dye-ratio) of 1 and 3, respectively, to determine best tumor-to-background ratios (TBR). In vivo studies were conducted using female nude mice bearing MDA-MB231 and MDA-MB-468 subcutaneously injected xenografts (5 ´ 10⁶ cells). Small animal fluorescence imaging with Zilovertamab-FNIRtag and Zilovertamab-IR800cw (20 µg/mouse) was conducted on an IVIS instrument and Quest surgical camera system. Accompanying blocking study using excess unlabeled Zilovertamab (20-fold) were conducted to demonstrate the specificity of the novel fluorescent tracers.

Results. Native Zilovertamab was successfully conjugated to IR800cw-NHS or FNIRtag-NHS and resulted in Zilovertamab-FNIRtag and Zilovertamab-IR800cw as novel fluorescent agents. Intravenous administration of the tracers showed tumor uptake peaking at 72 h with best TBR of 2.5 for ZV-FNIR (DOL1). Epifluorescence images show a clear delineation of the tumor from tissue as early as 24 h. Using the surgical Quest camera system, tumor lesion could be visualized and showed demarcation from surrounding tiussue.

Conclusion. Our results demonstrated that the developed ROR1-targeting fluorescent agents allow the delineation of tumor from surrounding tissue in triple negative breast cancer with high tumor-to-background ratio, seting the stage for the future development of potential intra-operative imaging agents.

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Image/Figure Caption:

Figure 1. A) Scheme for the exemplary synthesis of Zilovertamab-FNIRtag (ZV-FNIR). B) Tumor-to-background ratios achieved with Zilovertamab-IR800cw and Zilovertamab-FNIRtag at indicated timepoints based on IVIS epifluorescence. C) Epifluorescence images of ZV-FNIR recorded on an IVIS using MDA-MB-231 tumor bearing mice after indicated timepoints. D) Parallel recorded Quest camera images showing near infrared channel (NIR) and overlay at indicated timepoints (bottom).

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