Assessment of Novel Mesothelin-Specific Human Antibody Domain VH-Fc Fusion Proteins-Based PET Agents

Abstract

Mesothelin (MSLN) is a tumor-associated antigen found in a variety of cancers and is a target for imaging and therapeutic applications in MSLN-expressing tumors. We have developed high affinity anti-MSLN human VH domain antibodies, providing alternative targeting vectors to conventional IgG antibodies that are associated with long-circulating half-lives and poor penetration of tumors, limiting antitumor activity in clinical trials. Based on two newly identified anti-MSLN VH binders (3C9, 2A10), we generated VH-Fc fusion proteins and modified them for zirconium-89 radiolabeling to create anti-MSLN VH-Fc PET tracers. The focus of this study was to assess the ability of PETimaging to compare the in vivo performance of anti-MSLN VH-Fc fusion proteins (2A10, 3C9) targeting different epitopes of MSLN vs IgG1 (m912; a clinical benchmark antibody with an overlapped epitope as 2A10) for PET imaging in a mouse model of colorectal cancer (CRC). The anti-MSLN VH-Fc fusion proteins were successfully modified and radiolabeled with zirconium-89. The resulting MSLN-targeted PET-imaging agents demonstrated specific uptake in the MSLN-expressing HCT116 tumors. The in vivo performance of the MSLN-targeted PET-imaging agents utilizing VH-Fc showed more rapid and greater accumulation and deeper penetration within the tumor than the full-length IgG1 m912-based PET-imaging agent. Furthermore, PET imaging allowed us to compare the pharmacokinetics of epitope-specific VH domain-based PET tracers. Overall, these data are encouraging for the incorporation of PET imaging to assess modified VH domain structures to develop novel anti-MSLN VH domain-based therapeutics in MSLN-positive cancers as well as their companion PET imaging agents.

ImmunoPET imaging of TIGIT in the glioma microenvironment

“…Post 1 h incubation reaction mixture was purified using Cytiva 30 K centrifugal filters followed by PBS washes (3x). Following conjugation, radiolabeling was performed as previously described, with modifications, and was used to generate 89 Zr-αTIGIT 18 . Specifically, 500 µCi 89 Zr in 1 M oxalic acid (25 µL) and 1 M HEPES (175µL) were added per DFO-TIGIT solution (with a pH of 7–8), and the reactions were each incubated for 1 h at 37 °C.…”