Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system. It was found that, in vitro, the CAR T cells with the deficient PD-1 showed the stronger CAR-dependent anti-tumor activity against native programmed death 1 ligand 1-expressing HCC cell PLC/PRF/5 compared with the wild-type CAR T cells, and meanwhile, the CD4 and CD8 subsets, and activation status of CAR T cells were stable with the disruption of endogenous PD-1. Additionally, the disruption of PD-1 could protect the GPC3-CAR T cells from exhaustion when combating with native PD-L1-expressing HCC, as the levels of Akt phosphorylation and anti-apoptotic protein Bcl-xL expression in PD-1 deficient GPC3-CAR T cells were significantly higher than those in wild-type GPC3-CAR T cells after coculturing with PLC/PRF/5. Furthermore, the in vivo anti-tumor activity of the CAR T cells with the deficient PD-1 was investigated using the subcutaneous xenograft tumor model established by the injection of PLC/PRF/5 into NOD-scid-IL-2Rγ-/- (NSG) mice. The results indicated that the disruption of PD-1 enhanced the in vivo anti-tumor activity of CAR T cells against HCC, improved the persistence and infiltration of CAR T cells in the NSG mice bearing the tumor, and strengthened the inhibition of tumor-related genes expression in the xenograft tumors caused by the GPC3-CAR T cells. This study indicates the enhanced anti-tumor efficacy of PD-1-deficient CAR T cells against HCC and suggests the potential of precision gene editing on the immune checkpoints to enhance the CAR T cell therapies against HCC.

Tumor metastasis is a leading cause of cancer-related death. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase recruited to integrin-mediated matrix attachment sites where FAK activity is implicated in the control of cell survival, migration, and invasion. Although genetic studies support the importance of FAK activity in promoting tumor progression, it remains unclear whether pharmacological FAK inhibition prevents tumor metastasis. Here, we show that the FAK inhibitor PND-1186 blocks FAK Tyr-397 phosphorylation in vivo and exhibits anti-tumor efficacy in orthotopic breast carcinoma mouse tumor models. PND-1186 (100 mg/kg intraperitoneal, i.p.) showed promising pharmacokinetics (PK) and inhibited tumor FAK Tyr-397 phosphorylation for 12 hours. Oral administration of 150 mg/kg PND-1186 gave a more sustained PK profile verses i.p., and when given twice daily, PND-1186 significantly inhibited sygeneic murine 4T1 orthotopic breast carcinoma tumor growth and spontaneous metastasis to lungs. Moreover, low-level 0.5 mg/ml PND-1186 ad libitum administration in drinking water prevented oncogenic KRAS- and BRAF-stimulated MDA-MB-231 breast carcinoma tumor growth and metastasis with inhibition of tumoral FAK and p130Cas phosphorylation. Although PND-1186 was not cytotoxic to cells in adherent culture, tumors from animals receiving PND-1186 exhibited increased TUNEL staining, decreased leukocyte infiltrate and reduced tumor-associated splenomegaly. In vitro, PND-1186 reduced tumor necrosis factor-α triggered interleukin-6 cytokine expression, indicating that FAK inhibition may impact tumor progression via effects on both tumor and stromal cells. As oral administration of PND-1186 also decreased experimental tumor metastasis, PND-1186 may therefore be useful clinically to curb breast tumor progression.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential utilities have not been explored in hepatocellular carcinoma (HCC). Glypian-3 (GPC3) is a rational immunotherapeutic target for HCC. In this study, we developed GPC3-specific NK cells and explored their potential in the treatment of HCC. The NK-92/9.28.z cell line was established by engineering NK-92, a highly cytotoxic NK cell line with second-generation GPC3-specific CAR. Exposure of GPC3 HCC cells to this engineered cell line resulted in significant in vitro cytotoxicity and cytokine production. In addition, soluble GPC3 and TGF-β did not significantly inhibit the cytotoxicity of NK-92/9.28.z cells in vitro, and no significant difference in anti-tumor activities was observed in hypoxic (1%) conditions. Potent anti-tumor activities of NK-92/9.28.z cells were observed in multiple HCC xenografts with both high and low GPC3 expression, but not in those without GPC3 expression. Obvious infiltration of NK-92/9.28.z cells, decreased tumor proliferation, and increased tumor apoptosis were observed in the GPC3 HCC xenografts. Similarly, efficient retargeting on primary NK cells was achieved. These results justified clinical translation of this GPC3-specific, NK cell-based therapeutic as a novel treatment option for patients with GPC3 HCC.

Our previous study indicated that GPC3-targeted chimeric antigen receptor (CAR) T cell therapy has a high safety profile in patients with hepatocellular carcinoma (HCC). However, the response rate requires further improvement. Here, we analyzed the combined effect of GPC3-CAR T cells and sorafenib in both immunocompetent and immunodeficient mouse models of hepatocellular carcinoma. In immunocompetent mouse model, mouse CAR (mCAR) T cells induced regression of small tumors (approximately 130 mm 3 tumor volume) but had no effect on large, established tumors (approximately 400 mm 3 tumor volume). Sorafenib, at a subpharmacologic but not a pharmacologic dose, augmented the antitumor effects of mCAR T cells, in part by promoting IL12 secretion in tumorassociated macrophages (TAMs) and cancer cell apoptosis. In an immunodeficient mouse model, both subpharmacologic and pharmacologic doses of sorafenib had limited impacts on the function of human CAR (huCAR) T cells in vitro and showed synergistic effects with huCAR T cells in vivo, which can at least partially be ascribed to the upregulated tumor cell apoptosis induced by the combined treatment. Thus, this study applied two of the most commonly used mouse models for CAR T cell research and demonstrated the clinical potential of combining sorafenib with GPC3-targeted CAR T cells against HCC.

Adoptive immunotherapy based on chimeric antigen receptor–modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived–armored glypican-3 (GPC3)–specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12–armored GPC3–CAR-T cells could broaden the application of CAR-T–based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

There is limited success in using chimeric antigen receptor (CAR) T cells to treat solid tumors. One of the major reasons is the suppressive tumor microenvironment that impairs the infiltration, expansion and persistence of CART cells in tumor tissues. In this study, we engineered CLDN18.2 specific CART cells to co-express cytokines IL-7 and CCL21 (7×21 CART). Our studies indicated that IL-7 and CCL21 can enhance the survival of CART cells and infiltration of T cells and dendritic cells in tumor tissues. 7×21 CART cells can efficiently destroy different solid tumors with or without preconditioned lymphodepletion chemotherapy and suppress the tumor growth with heterogenous antigen expression in vitro and in vivo. These findings suggest that 7×21 could boost CART cells antitumor activity and may be served as a potential therapy strategy for solid tumors.

Incorporation of inverted cytokine receptor (ICR) such as interleukin (IL)-4 vs. IL-7 (4/7) ICR is one strategy to improve the antitumor activities of chimeric antigen receptor (CAR) modified T (CAR-T) cells facing immunosuppressive cytokines. Here we report a novel interleukin (IL)-4 vs. IL-21 ICR (4/21 ICR) that enhanced CAR-T cell potency in IL-4 + tumor milieu via a different working-mechanism from 4/7 ICR. Upon IL-4 stimulation, 4/21 ICR activated the STAT3 pathway and promoted Th17-like polarization and tumor-targeted cytotoxicity in CAR-T cells in vitro . Furthermore, 4/21 ICR-CAR T cells persisted and eradicated established IL-4 + tumors in vivo . Thus, 4/21 ICR is a promising clinical CAR-T cell therapeutics for solid tumors rich in IL-4.

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