Background Claudin 18.2 (CLDN18.2), a tetraspanin family member essential for the formation of tight junctions,1 is highly expressed in gastric, pancreatic and esophageal adenocarcinomas, with physiological expression restricted to gastric mucosal epithelial cells.2 CLDN18.2 is a validated therapeutic target; zolbetuximab, a CLDN18.2-directed monoclonal antibody, demonstrated clinical benefit in combination with chemotherapy in CLDN18.2-high gastric cancer.3 4 Similarly, CLDN18.2-targeting chimeric antigen receptor T cells and antibody-drug conjugates have shown efficacy in gastric cancers,5–7 and other modalities, including T cell-engagers (TCE), are also in clinical development. Here we describe AZD5863, a CLDN18.2 and cluster of differentiation 3 (CD3)-targeting TCE designed with bivalent high-affinity binding to CLDN18.2 and monovalent low-affinity binding to CD3 to reduce class-associated toxicities, such as cytokine release syndrome, while maintaining potent and specific antitumor activity
Methods Surface plasmon resonance and cell-binding assays were used to assess AZD5863 affinity and binding to CLDN18.2 and CD3. T cell-dependent cellular cytotoxicity (TDCC) assays were conducted by co-culturing human peripheral blood mononuclear cells (hPBMCs) with human tumor cell lines (hTCLs) expressing distinct levels of CLDN18.2. Bystander killing was determined by mixing CLDN18.2-high or CLDN18.2-knockout cells at different ratios as targets in TDCC assays. In vivo studies tested AZD5863 in both immunodeficient mice humanized with CD3/CD28-activated hPBMCs and implanted with hTCLs, and in immunocompetent human CD3 knock-in mice implanted with CLDN18.2-expressing MC38 cells. Cytokine release was assessed in supernatant and mouse plasma by enzyme-linked immunosorbent assay or multiplex immunoassays.
Results AZD5863 demonstrated specific binding to CLDN18.2 with high affinity, and to CD3 with low affinity. AZD5863-driven TDCC was demonstrated in vitro on a range of hTCLs, where EC50 values significantly correlated with levels of CLDN18.2 expression. Importantly, the cytotoxic activity of AZD5863 was associated with modest release of tumor necrosis factor alpha and interleukin 6. AZD5863 mediated concentration-dependent bystander killing of CLDN18.2-knockout cells at various CLDN18.2 +/- ratios. In humanized mouse models, AZD5863 significantly inhibited the growth of CLDN18.2-expressing gastric, pancreatic and esophageal tumors compared with isotype controls. AZD5863 also inhibited tumor growth in a human CD3 knock-in mouse model implanted with CLDN18.2- expressing MC38 tumor cells
Conclusions AZD5863, an affinity-optimized TCE targeting CLDN18.2 and CD3, demonstrated potent preclinical antitumor activity both in vitro and in vivo, and induced bystander killing of CLDN18.2-negative tumor cells. A phase 1 trial evaluating AZD5863 in gastric, pancreatic and esophageal adenocarcinoma, is ongoing.
Acknowledgements Medical writing support for the development of this abstract, under the direction of the authors, was provided by Eric Exner of Ashfield MedComms (Milwaukee, WI), an Inizio company, and was funded by AstraZeneca.
Suzuki H, et al. Crystal structure of a claudin provides insight into the architecture of tight junctions. Science. 2014;344:304–307.
Sahin U, et al. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development. Clin Cancer Res. 2008;14:7624–7634.
Shitara K, et al. Zolbetuximab + mFOLFOX6 as first-line (1L) treatment for patients (pts) with claudin-18.2+ (CLDN18.2+)/HER2− locally advanced (LA) unresectable or metastatic gastric or gastroesophageal junction (mG/GEJ) adenocarcinoma: Primary results from phase 3 SPOTLIGHT study. J Clin Oncol. 2023;41(4_suppl):LBA292.
Xu R, et al. Zolbetuximab + CAPOX in 1L claudin-18.2+ (CLDN18.2+)/HER2− locally advanced (LA) or metastatic gastric or gastroesophageal junction (mG/GEJ) adenocarcinoma: Primary phase 3 results from GLOW. J Clin Oncol. 2023;41(36_suppl):405736.
Qi C, et al. Claudin18.2-specific CAR T cells in gastrointestinal cancers: phase 1 trial interim results. Nat Med. 2022;28:1189–1198.
Wang Y, et al. First-in-human dose escalation and expansion study of SYSA1801, an antibody-drug conjugate targeting claudin 18.2 in patients with resistant/refractory solid tumors. J Clin Oncol. 2023;41(16_suppl):3016.
Xu R, et al. A phase 1a dose-escalation, multicenter trial of anti-claudin 18.2 antibody drug conjugate CMG901 in patients with resistant/refractory solid tumors. J Clin Oncol. 2023;41(4_suppl):352.
Ethics Approval This study was conducted in accordance with international, national, and institutional guidelines as well as all relevant laws and regulations regarding research involving animals. Studies performed in mice received IACUC approval under protocol numbers AUP-22–17 and AUP-22–25.
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