# Design and Preclinical Validation of an Anti-B7-H3-Specific Radiotracer: A Non-Invasive Imaging Tool to Guide B7-H3-Targeted Therapies

## TL;DR

This paper describes the development of a radiotracer that can non-invasively detect B7-H3 in tumors, potentially improving targeted cancer therapies.

## Contribution

The novel contribution is the design and validation of a B7-H3-specific radiotracer using single-domain antibodies for non-invasive tumor imaging.

## Key findings

- Sixteen sdAbs bound specifically to B7-H3 with nanomolar affinities.
- Lead sdAb C51 radiolabeled with 99mTc showed high tumor contrast and uptake in xenograft models.
- The radiotracer supports broad applicability across different B7-H3 isoforms.

## Abstract

Background: B7-H3, an immunoregulatory protein of the B7 family, has been associated with both anti-cancer immunity and tumor promotion, with its expression commonly correlated with poor prognosis. Although it is frequently expressed across cancers, its heterogeneity may limit the effectiveness of B7-H3-targeted therapies. Consequently, a sensitive and non-invasive method is needed to assess B7-H3 expression for patient selection and stratification. Single-domain antibody fragments (sdAbs) offer a promising platform for developing such a diagnostic tool. Methods: To generate B7-H3 sdAbs, two Ilamas were immunized with the recombinant human B7-H3 protein. Positive clones were selected through Phage biopanning and characterized for thermal stability, binding specificity, and affinity to human and murine B7-H3 proteins. Selected sdAbs were radiolabeled with Technetium-99m (99mTc) and evaluated for B7-H3 detection in two xenograft tumor models using micro-SPECT/CT imaging and dissection studies. Results: Sixteen purified sdAbs bound specifically to recombinant B7-H3 proteins and cells expressing native B7-H3 antigens, with nanomolar affinities. The four best-performing sdAbs bound promiscuously to tested mouse and human B7-H3 isoforms. Lead sdAb C51 labeled with 99mTc displayed specific accumulation across two human B7-H3+ tumor models, achieving high contrast with a tumor-to-blood ratio of up to 10 ± 3.16, and a tumor uptake of up to 4.96 ± 1.4%IA/g at 1.5 h post injection. Conclusions: The lead sdAb enabled rapid, specific, and non-invasive imaging of human B7-H3+ tumors. Its isoform promiscuity supports broad applicability across cancers expressing different human B7-H3 isoforms. These results support further development for clinical translation to enable patient selection and improved B7-H3-targeted therapies.