Development and comparison of three 89Zr-labeled anti-CLDN18.2 antibodies to noninvasively evaluate CLDN18.2 expression in gastric cancer: a preclinical study

Abstract

Background: Isoform 2 of claudin 18 (CLDN18.2) is overexpressed in gastric cancer and may be a promising imaging target. In this study, we constructed three anti-CLDN18.2 antibodies and compared them in preclinical experiments.

Methods: Screening from anti-CLDN18.2 nanobody library, we constructed three antibodies, anti-CLDN18.2 VHH (recombinant single-chain antibody fused with poly-histidine-tag), anti-CLDN18.2 VHH-ABD (recombinant single-chain antibody fused fused with albumin binding domain), and anti-CLDN18.2 VHH-Fc (recombinant single-chain antibody fused with IgG1-Fc) and radiolabeled with ⁸⁹Zr. Affinity assay, in vitro stability, immunoactivity, blood pharmacokinetics, in vivo and ex vivo biodistribution study, specificity study, and immunohistochemical analysis were performed to assess these radiotracers.

Results: The EC50 were 12.21 nM, 2.48 nM, and 0.14 nM for anti-CLDN18.2 VHH, anti-CLDN18.2 VHH-ABD, and anti-CLDN18.2 VHH-Fc, respectively. ⁸⁹Zr-anti-CLDN18.2 VHH demonstrated the lowest tumor uptake in PET imaging. Both ⁸⁹Zr-anti-CLDN18.2 VHH-ABD and ⁸⁹Zr-anti-CLDN18.2 VHH-Fc demonstrated high tumor accumulation, with highest ID%/g of 25.78 ± 5.60 at 24 h post-injection with ⁸⁹Zr-anti-CLDN18.2 VHH-ABD and 49.43 ± 9.86 at 72 h post-injection with ⁸⁹Zr-anti-CLDN18.2 VHH-Fc. The specificity of ⁸⁹Zr-anti-CLDN18.2 VHH-Fc targeting CLDN18.2 was further confirmed by blocking study. The ex vivo biodistribution results were consistent with in vivo biodistribution data. For ⁸⁹Zr-anti-CLDN18.2 VHH-ABD, tumor uptake was 21.46 ± 1.78 ID%/g at 12 h and 13.73 ± 2.22 ID%/g at 108 h. For ⁸⁹Zr-anti-CLDN18.2 VHH-Fc, the tumor accumulation was 25.28 ± 3.83 ID%/g at 12 h and 40.13 ± 9.50 ID%/g at 108 h. Immunohistochemistry of the xenograft tissue revealed high and homogenous CLDN18.2 expression in CO-SNU620 tumor.

Conclusion: Both anti-CLDN18.2 VHH-ABD and anti-CLDN18.2 VHH-Fc can be efficiently and stably radiolabeled with ⁸⁹Zr for noninvasive imaging and quantification of CLDN18.2 expression in gastric cancer, of which ⁸⁹Zr-anti-CLDN18.2 VHH-ABD seems to be the optimal choice balancing tumor uptake and liver background. They can provide essential information to select patients who are likely to benefit from CLDN18.2-targeted treatment.

Keywords: CLND18.2; Gastric cancer; Nanobody; PET.