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Page 6 of 12 Ni et al. Hepatoma Res 2020;6:25 I http://dx.doi.org/10.20517/2394-5079.2020.14
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anti-tumor capacity and persistence than single-targeted T cells in two GPC3 ASGR1 HCC xenograft
models. Taken together, dual-targeted CAR-T cells may decrease the risk of on-target, off-tumor toxicity
[36]
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while maintaining relatively potent antitumor capacities in GPC3 ASGR1 HCC .
The combination of CAR-T cell therapy and knockout of endogenous inhibitory immune checkpoints
on T cells might be a promising immunotherapeutic approach for cancer treatment. The PD-1 gene in
-/-
GPC3-targeted CAR-T cells was knocked out via the CRISPR/Cas9 gene-editing system. In vitro, PD-1
GPC3-CAR-T cells demonstrated higher anti-tumor activity against PD-L1-expressing HCC cell PLC/
PRF/5 than WT CAR-T cells in a CAR-dependent manner. Moreover, PD-1 deficiency did not affect T
-/-
cell subsets and activation status of CAR-T cells. Notably, co-culture of PD-1 GPC3-CAR-T cells with
native PD-L1-expressing HCC did not induce CAR-T cell exhaustion. Furthermore, the knockout of PD-1
led to enhanced anti-tumor activity of CAR-T cells against HCC in vivo, and improved persistence and
infiltration of CAR-T cells in tumor-bearing NOD-SCID IL-2receptor gamma null (NSG) mice. These
findings suggest the improved anti-tumor capacity of PD-1 CAR-T cells in HCC and the potential of
-/-
[37]
precise gene editing on immune checkpoints to increase the efficacy of CAR-T cells . In another study,
Pan et al. introduced a fusion protein composed of a PD-1 extracellular domain and CH3 from IgG4 into
[38]
GPC3-specific CAR-T cells (GPC3-28Z). GPC3-CAR-T cells carrying the PD-1-CH3 fusion protein (sPD1)
specifically recognized and lysed GPC3 HCC cells, while secreting soluble PD-1 to impair PD-1/PD-
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L1 signaling. The incorporation of soluble PD1 protected CAR-T cells from exhaustion when combating
target cells. More importantly, GPC3-28Z-sPD1 T cells suppressed tumor xenograft growth significantly
when compared with GPC3-28Z T cells in two HCC tumor xenograft models. The treatment of mice with
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GPC3-28Z-sPD1 T cells resulted in a higher number of CD3 T cells in the circulation as well as in tumors,
enhanced granzyme B expression and reduced Ki67 expression in tumors. These findings suggest that
GPC3-targeted CAR-T cells carrying soluble PD-1 hold great potential for the treatment of HCC.

The combination of GPC3-CAR-T cells and targeted therapy (angiogenesis inhibitor) in the treatment of
HCC has been investigated. In an immunocompetent mouse model, mouse GPC3-CAR (mCAR) T cells
showed potent growth suppressive activity against small tumors, but did not show suppressive activity
against large, established tumors. Sorafenib (angiogenesis inhibitor), at a subpharmacologic but not a
pharmacologic dose, enhanced the anti-tumor activity of mCAR-T cells, partially by increasing IL-12
expression by TAMs and cancer cell apoptosis. Sorafenib, at both subpharmacologic and pharmacologic
doses, had a weak effect on the function of human CAR (huCAR) T cells in an immunodeficient mouse
model. However, huCAR-T cells and sorafenib together showed synergistic activities against tumor cells
in vivo. Collectively, these findings suggest the potential of combining sorafenib with GPC3-targeted
[39]
CAR-T cells in the treatment of HCC .
Other than GPC3, CD133 is another promising therapeutic target for CAR-T cells, for it is expressed by
stem cells of different cancer types. CD133-targeted CAR-T cells were prepared and its anti-tumor activity
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and toxicity were tested in a phase I clinical trial. In total, 23 patients with advanced and CD133 tumors (14
HCC, 7 pancreatic carcinomas, and 2 colorectal carcinomas) were enrolled and received CD133-CAR-T
cell infusion. Of 23 patients, 3 achieved partial remission, and 14 achieved stable disease. Repeat CAR-T
cell infusions might provide a longer period of disease stability, especially in patients who have achieved
tumor regression after the first ACT treatment. Of note, 91.3% of patients had not developed detectable
de novo lesions during treatment. CD133 cells were eliminated after CD133-CAR-T cell infusion and
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toxicity is well tolerated. This trial suggests that the infusion of CA133-CAR-T cells may be of value in
treating CD133 advanced cancers .
[40]
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NKG2D-NK group 2 member D (NKG2D) ligands (NKG2DL) are not expressed on the surface of normal
cells but are overexpressed on malignant cells, thereby providing targets for CAR-T therapy. The expression
levels of most NKG2DLs are higher in tumors than that in normal tissues. Due to this reason, NKG2D-