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.

Chimeric antigen receptor modified T cells (CAR-T) therapy is an emerging immunotherapy against malignancies. However, only limited success was obtained in solid tumors. Polyinosinic-polycytidylic acid (poly I:C), ligand of TLR3, mediates innate immune and adaptive immune and shows broad antitumor effect on many types of cancer. In the present study, we combined EGFRvIII-targeted CAR-T cells with poly I:C treatment and evaluated the synergic antitumor effect in vitro and in immunocompetent mice bearing subcutaneous colon or orthotopic breast cancer xenografts. Poly I:C significantly promoted more IL-2 and IFN γ production as well as higher lytic activity of CAR-T cells. Upon systemic administration in vivo, CAR-T cells obviously suppressed tumor growth, and poly I:C significantly enhanced the suppression. Further study showed that poly I:C exerted antitumor effect dependent on type I IFNs. In addition, poly I:C decreased myeloid-derived suppressor cells (MDSC) number in peripheral blood and spleen, and attenuated the immunosuppressive activity of MDSC on proliferation and cytolytic function of CAR-T. Depletion of MDSC with anti-Gr1 Ab further increased the antitumor effect of CAR-T cells plus poly I:C treatment. In conclusion, CAR-T treatment combined with intratumoral delivery of poly I:C resulted in synergistic antitumor activity. We thus provide a rationale to translate this immunotherapeutic strategy to solid tumors.

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.

Epidermal growth factor receptor (EGFR) is frequently aberrantly expressed in cancer, and abnormal signalling downstream of this receptor contributes to tumour growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. Aberrant signalling downstream of this receptor contributes to tumour invasion. We previously reported that EGFRvIII can promote hepatocellular carcinoma (HCC) invasion. However, little is known concerning the mechanisms underlying EGFRvIII-mediated increases in cell motility and invasion in HCC. In this study, we observed that S100A11 was significantly upregulated in Huh-7 cells that overexpressed EGFRvIII. Moreover, S100A11 expression was elevated in HCC tissue samples (68.6%; 35/51), and this elevation was correlated with EGFRvIII expression (p = 0.0020; n = 20). Furthermore, the overexpression of S100A11 can promote HCC cell invasiveness, whereas siRNA against S100A11 can suppress the invasiveness of HCC cells stably transfected with EGFRvIII. Additionally, STAT3 inhibitors can block S100A11 expression and S100A11 promoter activity in HCC cells with stable overexpression of EGFRvIII. Furthermore, mutation in STATx binding sites could abolish the S1000A11 promoter activity stimulation by EGFRvIII. Taken together, the results demonstrate that the EGFRvIII-STAT3 pathway promotes cell migration and invasion by upregulating S100A11.

Chimeric antigen receptor (CAR) T cells targeting glypican-3 (GPC3) demonstrated early signs of therapeutic efficacy to hepatocellular carcinoma patients with a risk of cytokine release syndrome (CRS). Several adoptive cell therapies (ACTs) with T cells using the natural T cell receptor (TCR) signaling induced more efficient antitumor function and reduced cytokine production relative to CARs in solid tumors. To improve the efficacy and safety of GPC3-targeted ACTs, T cells were modified with anti-GPC3 single-chain fragment variable(sFv) linked to CD3ε, which could be incorporated into the entire TCR/CD3 complex to form chimeric sFv-CD3ε receptor (sFv-ε). sFv-ε T cells showed competitive antitumor activity and lower cytokine release compared to 28ζ or BBζ CAR T cells, which may be ascribed to moderately less activated Ca2+-calcineurin-NFAT signaling pathway. We further generated murine sFv-ε T cells with interleukin-7 co-expression (7sFv-ε) to promote T cell survival and to mobilize the endogenous immune system. In immunocompetent mouse models, 7sFv-ε T cells showed superior persistence, antitumor efficacy, and immunological memory while preserving the low production of cytokines associated with CRS compared to conventional sFv-ε T cells. These results indicate that GPC3-specific 7sFv-ε T cells could serve as a promising therapeutic strategy for solid tumors.

There are still unmet medical needs for the treatment of glioblastoma (GBM), the most frequent and aggressive brain tumor worldwide. EGFRvIII, overexpressed in approximately 30% of GBM, has been regarded as a potential therapeutic target. In this study, we demonstrated that CH12, an anti-EGFRvIII monoclonal antibody, could significantly suppress the growth of EGFRvIII+ GBM in vivo; however, PTEN deficiency in GBM reduced the efficacy of CH12 by attenuating its effect on PI3K/AKT/mTOR pathway. To overcome this problem, CH12 was combined with the mTOR inhibitor rapamycin, leading to a synergistic inhibitory effect on EGFRvIII+PTEN- GBM in vivo. Mechanistically, the synergistic antitumor effect was achieved via attenuating EGFR and PI3K/AKT/mTOR pathway more effectively and reversing the STAT5 activation caused by rapamycin treatment. Moreover, the combination therapy suppressed angiogenesis and induced cancer cell apoptosis more efficiently. Together, these results indicated that CH12 and rapamycin could synergistically suppress the growth of EGFRvIII+PTEN- GBM, which might have a potential clinical application in the future.

Although Trastuzumab, an anti-HER2 antibody, benefits certain patients with HER2-overexpressing breast cancer, de novo or acquired trastuzumab resistance remains a haunting issue. EGFRvIII, co-expressing with HER2 in some breast tumors, indicates a poor clinical prognosis. However, the role of EGFRvIII in the function of trastuzumab is not clear. Here, we demonstrated that EGFRvIII overexpression contributed to de novo trastuzumab resistance and the feedback activation of STAT3 caused by trastuzumab also resulted in acquired resistance in EGFRvIII(+)HER2(+) breast cancers. CH12, a highly effective anti-EGFRvIII monoclonal antibody that preferentially binds to EGFRvIII, significantly suppressed the growth of EGFRvIII+HER2(+) breast cancer cells in vitro and in vivo. Importantly, CH12 in combination with trastuzumab had a synergistic inhibitory effect on EGFRvIII(+)HER2(+) breast cancers in vitro and in vivo via attenuating the phosphorylation of EGFR and HER2 and their downstream signal pathways more effectively and reversing STAT3 feedback activation. Moreover, the combination therapy suppressed angiogenesis and induced cell apoptosis significantly. Together, these results suggested a synergistic efficacy of the combination of trastuzumab with CH12 against EGFRvIII(+)HER2(+) breast cancers, which might be a potential clinical application in the future.

There are unmet medical needs for patients with lung squamous cell carcinoma (LSCC). Therefore, in this study, we explored the antitumor potential of third-generation glypican 3 (GPC3)-redirected chimeric antigen receptor (CAR)-engineered T lymphocytes (CARgpc3 T cells) in tumor models of LSCC. First, we demonstrated by immunohistochemistry (IHC) that GPC3 was expressed in 66.3% of LSCC samples and in 3.3% of lung adenocarcinoma (LAD) samples but not in normal lung tissues. In the presence of GPC3-positive LSCC cells, CARgpc3 T cells were highly activated and increased in number. CARgpc3 T cells could specifically lyse GPC3-positive LSCC cells in vitro. In two established LSCC xenograft models, CARgpc3 T cells could almost completely eliminate the growth of GPC3-positive cells. Additionally, the CARgpc3 T cells were able to persist in vivo and efficiently infiltrate the cancerous tissues. Taken together, these findings indicate that CARgpc3 T cells might be a novel potential therapeutic agent for the treatment of patients with LSCC.

A hostile tumor microenvironment is one of the major obstacles for the efficacy of chimeric antigen receptor modified T (CAR-T) cells, and combination treatment might be a potential way to overcome this obstacle. Poly(ADP-ribose) polymerase inhibitor (PARPi) has demonstrated tremendous potential in breast cancer. In this study, we explored the possible combination of the PAPRi olaparib with EGFRvIII-targeted CAR (806-28Z CAR) T cells in immunocompetent mouse models of breast cancer. The results indicated that the administration of olaparib could significantly enhance the efficacy of 806-28Z CAR-T cells in vivo. Interestingly, we observed that olaparib could suppress myeloid-derived suppressor cell (MDSC) migration and promote the survival of CD8+ T cells in tumor tissue. Mechanistically, olaparib was shown to reduce the expression of SDF1α released from cancer-associated fibroblasts (CAFs) and thereby decreased MDSC migration through CXCR4. Taken together, this study demonstrated that olaparib could increase the antitumor activities of CAR-T cell therapy at least partially through inhibiting MDSC migration via the SDF1α/CXCR4 axis. These findings uncover a novel mechanism of PARPi function and provide additional mechanistic rationale for combining PARPi with CAR-T cells for the treatment of breast cancer.

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.

Claudin18.2 (CLDN18.2)-specific chimeric antigen receptor (CAR-T) cells displayed limited efficacy in CLDN18.2-positive pancreatic ductal adenocarcinoma (PDAC). Strategies are needed to improve the trafficking capacity of CLDN18.2-specific CAR-T cells. PDAC has a unique microenvironment that consists of abundant cancer-associated fibroblasts (CAFs), which could secrete stromal cell-derived factor 1α (SDF-1α), the ligand of CXCR4. Then, we constructed and explored CLDN18.2-targeted CAR-T cells with CXCR4 co-expression in treating immunocompetent mouse models of PDAC. The results indicated that CXCR4 could promote the infiltration of CAR-T cells and enhance their efficacy in vivo. Mechanistically, the activation of signal transducer and activator of transcription 3 (STAT3) signaling was impaired in CXCR4 CAR-T cells, which reduced the release of inflammatory factors, such as tumor necrosis factor-α, IL-6, and IL-17A. Then, the lower release of inflammatory factors suppressed SDF-1α secretion in CAFs via the nuclear factor κB (NF-κB) pathway. Therefore, the decreased secretion of SDF-1α in feedback decreased the migration of myeloid-derived suppressor cells (MDSCs) in tumor sites. Overall, our study demonstrated that CXCR4 CAR-T cells could traffic more into tumor sites and also suppress MDSC migration via the STAT3/NF-κB/SDF-1α axis to obtain better efficacy in treating CLDN18.2-positive pancreatic cancer. Our findings provide a theoretical rationale for CXCR4 CAR-T cell therapy in PDAC.

Epidermal growth factor receptor (EGFR), a well-known oncogenic driver, contributes to the initiation and progression of a wide range of cancer types. Aberrant lipid metabolism including highly produced monounsaturated fatty acids (MUFA) is recognized as a hallmark of cancer. However, how EGFR regulates MUFA synthesis in cancer remains elusive. This is the focus of our study.

IL12 is an immune-stimulatory cytokine for key immune cells including T cells and NK cells. However, systemic administration of IL12 has serious side effects that limit its clinical application in patients. Recently, synthetic Notch (synNotch) receptors have been developed that induce transcriptional activation and deliver therapeutic payloads in response to the reorganization of specific antigens. NK92 cell is a human natural killer (NK) cell line which has been developed as tools for adjuvant immunotherapy of cancer. Here, we explored the possibility of using synNotch receptor-engineered NK92 cells to selectively secrete IL12 at the tumor site and increase the antitumor activities of chimeric antigen receptor (CAR)-modified T cells. Compared with the nuclear factor of activated T-cells (NFATs) responsive promoter, which is another regulatory element, the synNotch receptor was better at controlling the expression of cytokines. NK92 cells transduced with the GPC3-specific synNotch receptor could produce the proinflammatory cytokine IL12 (GPC3-Syn-IL12-NK92) in response to GPC3 antigen expressed in cancer cells. In vivo GPC3-Syn-IL12-NK92 cells controlling IL12 production could enhance the antitumor ability of GPC3-redirected CAR T cells and increase the infiltration of T cells without inducing toxicity. Taken together, our results demonstrated that IL12 supplementation by synNotch-engineered NK92 cells could secrete IL12 in a target-dependent manner, and promote the antitumor efficiency of CAR-T cells. Local expression of IL12 by synNotch-engineered NK92 cells might be a safe approach to enhance the clinical outcome of CAR-T cell therapy.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with limited treatment options. Epidermal growth factor receptor (EGFR) is reported to be expressed in 50%-75% of TNBC patients, making it a promising target for cancer treatment. Here we show that EGFR-targeted chimeric antigen receptor (CAR) T cell therapy combined with radiotherapy provides enhanced antitumor efficacy in immunocompetent and immunodeficient orthotopic TNBC mice. Intriguingly, this combination therapy resulted in a substantial increase in the number of tumor-infiltrating CAR-T cells. The efficacy of this combination was independent of tumor radiosensitivity and lymphodepleting preconditioning. Cytokine profiling showed that this combination did not increase the risk of cytokine release syndrome (CRS). RNA sequencing (RNA-seq) analysis revealed that EGFR-targeting CAR-T therapy combined with radiotherapy increased the infiltration of CD8+ T and natural killer (NK) cells into tumors. Mechanistically, radiation significantly increased Icam1 expression on TNBC cells via activating nuclear factor κB (NF-κB) signaling, thereby promoting CAR-T cell infiltration and killing. These results suggest that CAR-T therapy combined with radiotherapy may be a promising strategy for TNBC treatment.

The claudin 18.2 (CLDN18.2) antigen is frequently expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). Although CLDN18.2-targeted CAR-T cells demonstrated some therapeutic efficacy in PDAC patients, further improvement is needed. One of the major obstacles might be the abundant cancer-associated fibroblasts (CAFs) in the PDAC tumor microenvironment (TME). Targeting fibroblast activation protein (FAP), a vital characteristic of CAFs provides a potential way to overcome this obstacle. In this study, we explored the combined antitumor activity of FAP-targeted and CLDN18.2-targeted CAR-T cells against PDAC.