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

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.

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.

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.

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.