Abstract 1326: Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer

作者: Lin, James C.L. ; Yang, Chih Ya ; Roy, Jaydeep ; Chow, Kronos ; Wong, Kwong F. ; Lin, Nga Tin ; Luk, John M.

AbstractBackground: Treatment for advanced gastrointestinal (GI) cancers has remained challenging, therefore, novel immunotherapies using chimeric antigen receptor (CAR)-T cells are being developed as a new hope for the patients. However, despite the clinical successes in hematologic malignancies, the overall safety and efficacy against solid tumors of CAR-T cells have yet to be established, and the manufacturing of autologous CAR-T cells requires lengthy cell expansion and viral transduction processes. In view of these technical challenges, we have developed ARB011, an RNA-based allogeneic CAR-NK therapy targeting GI cancers expressing cadherin-17 (CDH17), a promising therapeutic target with its overexpression associated with poor prognosis in patients with GI cancers. The use of allogeneic NK cells would mitigate the risk of graft-versus-host disease. Moreover, engineering NK cells with nonviral CAR-encoding mRNA would provide a rapid large-scale manufacturing platform with minimal risk of abnormal genetic alteration mediated by viral integration.Methods: To generate the ARB011 CAR-NK cells, ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were electroporated with mRNA encoding a humanized anti-CDH17 scFv fused with transactivation domains. Flow cytometry, in vitro cytotoxicity assay, and cytokine release assay were performed to evaluate the expression and functionality of ARB011. To examine the developability of ARB011 as an off-the-shelf cell product, cryopreservation and large-scale electroporation were also studied.Results: Flow cytometry analysis revealed efficient electroporation with a high level of CDH17 CAR expression (>80%) and optimal CAR-NK cell viability (60%-80%). Following electroporation, cytotoxicity of ARB011 was tested against gastric (AGS and NCI-N87), colorectal (DLD-1 and T84), and liver (MHCC-97H) cancer cells by co-culturing at 1:1 and 2:1 effector/target ratio for 12-24h. ARB011 showed significant cytotoxicity against CDH17+ but not CDH17- cancer cells. The CDH17-sepcific cytotoxicity was accompanied with elevated levels of CD107a or LAMP-1 expression on CAR-NK cells and IFN-γ and TNF-α releases. Interestingly, cryopreserved ARB011 maintained optimal cell viability, CAR expression, and potent CDH17 target-specific cytolytic activity. Lastly, large-scale electroporation showed the establishment of up-scaling process of ARB011 production for clinical application.Conclusions: Overall, the findings suggest that ARB011 could be a novel off-the-shelf, CAR-NK therapy that warrants further studies in animal models and in clinical trials.Citation Format: Jaydeep Roy, Nga Tin Lin, Kronos Chow, Chih Ya Yang, James C.L. Lin, John M. Luk, Kwong F. Wong. Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1326.

2024-03-22·Cancer Research

Abstract 3212: Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment

作者: Wong, Kwan Wa ; Wong, Po Yee ; Luk, John Moon ; Chow, Kronos ; Wong, Kwong Fai ; O, Chui Yee ; Wong, Dennis

AbstractTrophoblast cell surface antigen 2 (Trop2) is considered as a potential therapeutic target because its over-expression in tumors is found to be associated with poor prognosis of patients with different cancers. Trop2-directed antibody-drug conjugates have been approved for the treatment of solid tumors, however, the expression of Trop2 in a broad range of epithelial tissues, even in low expression, would limit their clinical benefits. The present work generated a novel Trop2/CD3 bispecific antibody with its binding affinity towards Trop2 being well-modulated to mitigate the chance of on-target off-tumor effect. By linking a humanized anti-Trop2 monoclonal antibody with a CD3-binding scFv, a Trop2/CD3 bispecific antibody was produced. In order to adjust the bispecific antibody's affinity to the tumor target, ProABC-2 was used for in-silico paratope prediction (Ambrosetti F et al., 2020). A complementarity-determining residue (CDR) variant was produced and its binding to Trop2 antigen was analyzed using biolayer interferometry. The binding of both wild type (WT) and variant antibodies to Trop2-positive cancer cells, the activation of T cells upon target engagement and the redirection of T cell-mediated killing on cancer cells expressing high level of Trop2 were examined. The binding affinity of CDR variant to Trop2-expressing tumor cells was significantly reduced, which led to a slower induction of cytokines and T-cell proliferation, less acute T-cell activation, and a milder cytotoxic effect. This allowed the bispecific antibody to have a higher level of safety by restricting the cytotoxicity only to cancer cells that expressed high level of Trop2. Our novel Trop2/CD3 bispecific antibody not only showed potent anti-tumor activity, but also demonstrated a lower risk of on-target off-tumor toxicity. Monkey toxicity and other IND-enabling studies are beginning to further bring the drug candidate into clinical trial.Table showing EC50 values of Trop2/CD3 bispecific WT and CDR variant antibodies in different assays WT CDR variant Binding affinity (M) Trop2 4.62E-11 2.14E-10 Cytotoxicity (M) AsPC1 (low Trop2 level) 1.24E-11 No killing effect SW480 (medium Trop2 level) 1.25E-12 2.25E-11 NCI-H596 (high Trop2 level) 5.65E-12 5.89E-10 Receptor occupancy (%) Trop2 0.65 2.75 CD3 0.08 5.83 Cytokine induction (M) IL-2 4.82E-11 5.42E-11 IFNγ 6.46E-13 2.59E-11 T cell activation (M) CD25+ marker 2.02E-12 2.39E-10 CD69+ marker 3.81E-13 7.40E-11 T cell proliferation (M) Ki-67 marker 3.54E-12 8.80E-11Citation Format: Chui Yee O, Po Yee Wong, Kronos Chow, Kwan Wa Wong, Dennis Wong, John Moon Luk, Kwong Fai Wong. Development of a Trop2/CD3 bispecific antibody with modulated Trop2 binding affinity as a novel immunotherapy for advanced gastrointestinal cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3212.

2024-03-22·Cancer Research

Abstract 3213: A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers

作者: Wong, Po Yee ; Luk, John Moon ; Chow, Kronos ; Wong, Kwong Fai ; Wong, Kwan Wa ; O, Chui Yee ; Wong, Dennis

AbstractTargeting immune checkpoints like programmed death receptor 1 (PD-1) with monoclonal antibodies has achieved durable responses in a range of cancers, however, immune-related adverse events may occur in a subset of patients. As such, we developed a bispecific antibody targeting cadherin-17 (CDH17) and PD-1 with a hope to rejuvenate specifically the exhausted T cells within tumor microenvironment. In healthy individuals, CDH17 is expressed at a restricted level in intestinal epithelial junction that is not accessible to biologics, however, in gastrointestinal cancers, CDH17 is overexpressed and can be targeted by antibodies. Our new CDH17/PD-1 bispecific antibody would reactivate anti-tumor immunity with reduced risk of on-target off-tumor toxicity. Different bispecific antibody formats were designed, cloned, and expressed. Strong binders were first identified using CDH17 and PD-1 ELISA. The binding affinities to CDH17 and PD-1 of the selected candidate were examined using biolayer interferometry. The effect of these bispecific antibodies on blocking PD-1/PD-L1 axis in T cells was demonstrated in a cell-based reporter assay. T-cell mediated killing on CDH17-positive GI cancer cells and induction of T cell activation markers upon antibody treatment were also studied. A total of 7 CDH17/PD-1 bispecific antibodies of different formats were screened. Of these candidates, one, named ARB204, was selected because of its potent binding to both antigens in ELISA (i.e., EC50 values: CDH17, 1.6nM; PD-1, 0.4nM) and biolayer interferometry (i.e., KD values: CDH17, 2.4E-09M; PD-1, 6.02E-11M). ARB204 blocked PD-1/PD-L1 axis in the reporter assay with EC50 value 4.4nM. Notably, ARB204 redirected T cells to eradicate CDH17-positive pancreatic AsPC1 cells with an EC50 value of 3.5nM. No cytotoxicity against CDH17-negative cancer cells were seen. ARB204 effectively bound CDH17 on GI cancer cells and PD-1 on T cells. This bispecific design allowed selective T-cell mediated killing on cancer cells expressing CDH17, mitigating the risk of off-tumor toxicity. ARB204 warrants further studies on its immune checkpoint inhibition in animal models.Citation Format: Kwan Wa Wong, Po Yee Wong, Kronos Chow, Chui Yee O, Dennis Wong, John Moon Luk, Kwong Fai Wong. A novel CDH17/PD-1 bispecific antibody for treatment of advanced gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3213.

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