SAIL66, a next generation CLDN6-targeting T-cell engager, demonstrates potent antitumor efficacy through dual binding to CD3/CD137

Abstract

Background: Ovarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but displays aberrant upregulation in various malignancies, including ovarian cancer. Although several therapeutic modalities targeting CLDN6 are currently under investigation, there is still a need for more potent therapeutic options. While T-cell engagers (TCEs) hold substantial promise as potent immunotherapeutic agents, their current efficacy and safety in terms of target antigen selection and T-cell exhaustion due to only CD3 stimulation without co-stimulation must be improved, particularly against solid tumors. To provide an efficacious treatment option for ovarian cancer, we generated SAIL66, a tri-specific antibody against CLDN6/CD3/CD137.

Methods: Using our proprietary next-generation TCE technology (Dual-Ig), SAIL66 was designed to bind to CLDN6 with one Fab and CD3/CD137 with the other, thereby activating T cells through CD3 activation and CD137 co-stimulation. The preclinical characterization of SAIL66 was performed in a series of in vitro and in vivo studies which included comparisons to a conventional TCE targeting CLDN6 and CD3.

Results: Despite the high similarity between CLDN6 and other CLDN family members, SAIL66 demonstrated high specificity for CLDN6, reducing the risk of off-target toxicity. In an in vitro co-culture assay with CLDN6-positive cancer cells, we confirmed that SAIL66 strongly activated the CD137 signal in the Jurkat reporter system, and preferentially induced activation of both CD4⁺ and CD8⁺ T cells isolated from human peripheral blood mononuclear cells compared to conventional TCEs. In vivo studies demonstrated that SAIL66 led to a more pronounced increase in intratumor T-cell infiltration and a decrease in exhausted T cells compared with conventional CLDN6 TCE by contribution of CD137 co-stimulation, resulting in better antitumor efficacy in tumor-bearing mouse models.

Conclusion: Our data demonstrate that SAIL66, designed to engage CLDN6, CD3, and CD137, has the potential to enhance antitumor activity and provide a potent therapeutic option for patients with ovarian and other solid tumors expressing CLDN6. Clinical trials are currently underway to evaluate the safety and efficacy of SAIL66.

Keywords: Bispecific T cell engager - BiTE; Co-stimulatory molecules; Immunotherapy; Ovarian cancer; T cell.

Figure 1. SAIL66 is a potent CLDN6/CD3/CD137 tri-specific antibody. (A) SAIL66 has one CD3/CD137 dual-specific Fab and one anti-human CLDN6 Fab region. The Fc region is engineered to improve the efficiency of heavy chain heterodimerization whereas the binding to FcγRs and C1q is reduced. (B) The binding affinities of SAIL66 and conventional CLDN6 TCE to human and cynomolgus monkey CD3εγ, CD137, and CLDN6 were measured using surface plasmon resonance or kinetic exclusion assay. CLDN6, claudin-6; N.D., not determined; TCE, T-cell engager.

Figure 2. SAIL66 binds CLDN6 specifically and induces CD3 and CD137 signaling in T cells. (A) Jurkat-NFAT-luc reporter cells or CD137-expressing Jurkat-NF-κB-luc reporter cells were co-cultured with the CLDN6-expressing NIH:OVCAR-3 cell line or CLDN6-negative 5637 cell line in the presence of SAIL66 or control antibodies to assess activation of CD3 and CD137 pathways, respectively. Data are presented as mean+SD of fold changes in luciferase activity (n=3). (B) Outline figure of sequence differences in the extracellular domain between CLDN6 and CLDN9. (C) CLDN6-specific binding of SAIL66 was evaluated by flow cytometry using various CLDN-expressing Ba/F3 cell lines (n=1). CLDN, claudin; TCE, T-cell engager.

Figure 3. SAIL66 activates T cells. Human peripheral blood mononuclear cells from three healthy donors were co-cultured with the OV-90 cell line in the presence of SAIL66 at indicated concentrations. (A) Cytotoxicity was evaluated by LDH release assay (B) CD69 expression on CD8⁺ T cells and CD4⁺ T cells were analyzed by flow cytometry (C) IL-2 concentration in culture supernatant was measured. The data is represented in different colors for each donor and mean+SD are shown (n=3). LDH, lactate dehydrogenase; IL, interleukin.

Figure 4. SAIL66 inhibits in vivo tumor growth by promoting intratumoral T-cell infiltration and suppression of exhausted T-cell population in a syngeneic mouse model. (A) Antitumor efficacy of a single administration of SAIL66 and conventional CLDN6 TCE at 1 mg/kg was measured in hCD3 tgm and hCD3/hCD137 KI mice bearing LLC1/hCLDN6 cells. Mean+SD of tumor volume is shown (n=5–7). P values were determined using the Wilcoxon rank-sum test. *p<0.05, **p<0.01. (B) CD8⁺ T-cell number and TOX expression as an exhaustion marker on CD8⁺ T cells in LLC1/hCLDN6 tumors were evaluated by flow cytometry at day 7 after single antibody administration (n=3–5). P values were determined by the Wilcoxon rank-sum test. *p<0.05. (C) Cytotoxic activity improvement of SAIL66 at 1 nmol/L by CD137 co-stimulation against Colon38/hCLDN6 cell lines was evaluated with T cells derived from hCD3 tgm and hCD3/hCD137 KI mice using xCELLigence. Cell growth inhibition ratio was calculated at 48 hours after SAIL66 and T cells addition. (D) Antitumor efficacy of SAIL66 and anti-PD-L1 antibody was evaluated in hCD3/hCD137 KI mice bearing LLC1/hCLDN6 cells. SAIL66 (0.2 mg/kg) was administered at day 6 and anti-PD-L1 antibody (10 mg/kg) was administered at day 6, 8, 10, 12, and 14 after tumor inoculation. Mean+SD of tumor volume is shown (n=5). P values were determined by the Wilcoxon rank-sum test. *p<0.05, **p<0.01. CLDN6, claudin 6; PD-L1, programmed cell death 1 ligand 1; TCE, T-cell engager.

Figure 5. SAIL66 inhibits in vivo subcutaneous and intraperitoneal tumor growth in humanized mouse model. (A) Antitumor efficacy of multiple dosages of SAIL66 was measured in humanized mice bearing OV-90 human ovarian cancer cells. Humanized mice were generated by human hematopoietic stem cell injection for NOG mice. SAIL66 was administered one time after OV-90 tumor establishment in humanized mice subcutaneously at indicated concentrations. Mean+SD of tumor volume is shown (n=6–7). P values were determined by Dunnett’s multiple comparison test. **p<0.01. (B) Antitumor efficacy of single administration of SAIL66 was measured against the OV-90 disseminated model. Human T cells which were derived and expanded from healthy donor peripheral blood mononuclear cells and SAIL66 at 5 mg/kg were injected intravenously 3 days after tumor intraperitoneal cavity implantation (n=9). P values were determined by log-rank test.

Figure 6. The use of dexamethasone eliminates CRS, but does not limit the antitumor efficacy of SAIL66. (A) The effect of dexamethasone (DEX) premedication on the plasma cytokine expression was evaluated at indicated time points after SAIL66 administration. DEX (33 mg/kg) was intraperitoneally administered 1 and 24 hours (DEX ×2), or 1 hour (DEX ×1) prior to SAIL66 (0.2 mg/kg) administration in humanized mice bearing OV-90 cells. (B) The antitumor efficacy of SAIL66 and DEX were indicated. Mean+SD are shown (n=6). P values were determined by the Wilcoxon rank-sum test. **p<0.01. (C) RNA profiling was conducted on RNA extracted from OV-90 tumors, which were dissected on day 7 following a single administration of SAIL66. This was done using the NanoString nCounter Human PanCancer Immune Profiling Panel. Data analysis was performed using the nSolver Analysis Software V.4.0, and the nCounter Advanced Analysis modules were used for Gene Set Analysis. This analysis compared differential expression in the vehicle versus the drug treatment group. The extent of differential expression in each gene set was summarized using a “global significance score”. The mean score of each group is displayed (n=3). CRS, cytokine release syndrome; IFN, interferon; IL, interleukin.

Figure 7. SAIL66 achieves superior efficacy over conventional TCE by enhancing T-cell infiltration and suppressing exhausted T-cell population in humanized mice model. (A) Antitumor efficacy of SAIL66 and conventional CLDN6 TCE was compared in humanized mice bearing NIH:OVCAR-3 cells. Humanized mice were generated by human hematopoietic stem cell injection for NOG mice. Antibodies were administered one time after NIH:OVCAR-3 tumor establishment in humanized mice subcutaneously at 1 mg/kg. Mean+SD of tumor volume is shown (n=5). (B) CLDN6 messenger RNA expression in tumor tissue was quantified at days 2, 7, and 14 after single administration (n=5). (C) Flow cytometry analysis of T-cell density and T-cell phenotype in tumor tissue were evaluated on days 2, 7, and 14 after single administration (n=5). (D) RNA expression of immune-related genes in NIH:OVCAR-3 tumors was measured using nCounter Analysis System. The expression levels of the indicated genes in tumors were quantified at 2-day, 7-day, and 14-day post-treatment and are shown as Z-scores. (E) Plasma IL-2 and CXCL10 concentrations were measured at the indicated time points after single administration (n=5). (F) Pathological analysis of tumor tissue by H&E staining and IHC from the tissues of OV-90 bearing huNOG model at day 2, 7, and 14 after SAIL66 injection at 1 mg/kg, and at day 2 after vehicle injection. P values in A, B, and C were determined by the Wilcoxon rank-sum test. P values of gene expression shown in D between the CLDN6 TCE-treatment and SAIL66-treatment group (n=5) were determined using Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. CLDN6, claudin-6; IHC, immunohistochemistry; IL, interleukin; LAG3, lymphocyte activation gene 3 protein; PD-1, programmed cell death protein-1; TCE, T-cell engager; Tim-3, T-cell immunoglobulin mucin receptor 3.