Background Glypican-3 (GPC3) is an oncofetal heparan sulfate proteoglycan and is associated with cell proliferation, differentiation, and mobility through Wnt, Yap, and other signaling pathways. GPC3 expression is highly restricted in adults, and aberrant overexpression of GPC3 is associated with several cancers, most prominently hepatocellular carcinoma (HCC). This expression profile makes GPC3 a promising target for CAR-T therapies, and several autologous anti-GPC3 CAR-T programs are currently being evaluated in early-stage clinical trials. Generating autologous CAR-T cells still poses significant logistical hurdles which may prevent patients’ timely access to critical therapies. Moreover, the HCC tumor microenvironment is strongly immunosuppressive. Allogeneic CRISPR/Cas9 gene-edited CAR-T cells incorporating potency edits to enhance anti-tumor activity in suppressive tumor microenvironments can address both of these challenges.

Methods We have generated allogeneic anti-GPC3 CAR-T cells using CRISPR/Cas9 to disrupt relevant genes and an AAV6 template to knock in the CAR construct containing a human single-domain antibody (HN3) targeting GPC3. These CAR-T cells incorporate TRAC disruption to reduce graft versus host disease (GvHD) and β2M disruption to reduce allogeneic host rejection. We also have made edits to disrupt TGFβR2 and Regnase-1 to boost our cells’ anti-tumor potency. These CAR T cells were assessed in vitro and in vivo using HCC-derived cell lines. We also evaluated the ability of a dominant negative TGFβR2 receptor to attenuate response to TGFβ signaling in comparison to the CRISPR knock-out.

Results Our allogeneic anti-GPC3 CAR-T cells showed significant cytotoxicity in vitro against GPC3-expressing cell lines. In a mouse xenograft model of HCC using the Hep3B cell line, the CAR-T cells induced significant tumor regressions, with the potency edits conferring even greater tumor control. Disruption of the endogenous TGFβR2 gene causes greater attenuation of TGFβ signaling compared to the knock-in of dominant negative TGFβR2 receptors in vitro.

Conclusions CRISPR-edited anti-GPC3 CAR-T cell therapies are a promising potential option for the treatment of HCC. We have shown that our allogeneic anti-GPC3 CAR-T cells have significant activity both in vitro and in vivo against HCC-derived cell lines, and this activity is further improved through the inclusion of potency edits.