Transforming primary human hepatocytes into hepatocellular carcinoma with genetically defined factors.

Our understanding of human hepatocellular carcinoma (HCC) development and progression has been hampered by the lack of in vivo models. We performed a genetic screen of 10 oncogenes and genetic mutations in Fah-ablated immunodeficient mice in which primary human hepatocytes (PHHs) are used to reconstitute a functional human liver. We identified that MYC, TP53R249S , and KRASG12D are highly expressed in induced HCC (iHCC) samples. The overexpression of MYC and TP53R249S transform PHHs into iHCC in situ, though the addition of KRASG12D significantly increases the tumorigenic efficiency. iHCC, which recapitulate the histological architecture and gene expression characteristics of clinical HCC samples, reconstituted HCC after serial transplantations. Transcriptomic analysis of iHCC and PHHs showed that MUC1 and FAP are expressed in iHCC but not in normal livers. Chimeric antigen receptor (CAR) T cells against these two surface markers efficiently lyse iHCC cells. The properties of iHCC model provide a biological basis for several clinical hallmarks of HCC, and iHCC may serve as a model to study HCC initiation and to identify diagnostic biomarkers and targets for cellular immunotherapy.

Bispecific CAR-T cells targeting FAP and GPC3 have the potential to treat hepatocellular carcinoma.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated robust efficacy against hematological malignancies, but there are still some challenges regarding treating solid tumors, including tumor heterogeneity, antigen escape, and an immunosuppressive microenvironment. Here, we found that SNU398, a hepatocellular carcinoma (HCC) cell line, exhibited high expression levels of fibroblast activation protein (FAP) and Glypican 3 (GPC3), which were negatively correlated with patient prognosis. The HepG2 HCC cell line highly expressed GPC3, while the SNU387 cell line exhibited high expression of FAP. Thus, we developed bispecific CAR-T cells to simultaneously target FAP and GPC3 to address tumor heterogeneity in HCC. The anti-FAP-GPC3 bispecific CAR-T cells could recognize and be activated by FAP or GPC3 expressed by tumor cells. Compared with anti-FAP CAR-T cells or anti-GPC3 CAR-T cells, bispecific CAR-T cells achieved more robust activity against tumor cells expressing FAP and GPC3 in vitro. The anti-FAP-GPC3 bispecific CAR-T cells also exhibited superior antitumor efficacy and significantly prolonged the survival of mice compared with single-target CAR-T cells in vivo. Overall, the use of anti-FAP-GPC3 bispecific CAR-T cells is a promising treatment approach to reduce tumor recurrence caused by tumor antigen heterogeneity.

Disruption of CISH promotes the antitumor activity of human T cells and decreases PD-1 expression levels.

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of T cells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and T cell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in T cells remains unclear. In this study, we ablated CISH in T cells with CRISPR-Cas9 and found that the sensitivity of T cells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor T cells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient T cells in vitro and in vivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated T cells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

CD318 is a target of chimeric antigen receptor T cells for the treatment of colorectal cancer.

Colorectal cancer (CRC) currently has a poor prognosis with a 6.9-year median survival time; to relieve this malignant cancer, we proposed to establish CRC xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR)-T cells and accelerate the clinical translation of CAR-T cells for use against CRC. We first verified that CD318 had a higher expression level in primary human CRC tissues than in normal tissues based on hundreds of clinical samples. Then, we redirected CAR-T cells containing anti-CD318 single-chain variable fragment (anti-CD318 scFv), CD3ζ, CD28, and Toll-like receptor 2 (TLR2) domains. Next, we evaluated the function of these CAR-T cells in vitro in terms of surface phenotype changes, cytotoxicity and cytokine secretion when they encountered CD318+ CRC cells. Finally, we established two different xenograft mouse models to assess in vivo antitumor activity. The results showed that CAR318 T cells were significantly activated and exhibited strong cytotoxicity and cytokine-secreting abilities against CRC cells in vitro. Furthermore, CAR318 T cells induced CRC regression in different xenograft mouse models and suppressed tumors compared with CAR19 T cells. In summary, our work demonstrates that CAR318 T cells possess strong antitumor capabilities and represent a promising therapeutic approach for CRC.

The onco-embryonic antigen ROR1 is a target of chimeric antigen T cells for colorectal cancer.

Colorectal cancer is globally ranked second in both incidence and mortality rate. It usually develops during the middle or late stages of diagnosis, and is characterized by easy metastasis, poor prognosis, and a significant decline in postoperative quality of life. ROR1 is an excellent oncoembryonic antigen in numerous immunotherapy treatments for tumors. Additionally, it is overexpressed in colorectal cancer. To fill the void in CRC treatment with ROR1 as a target of CAR-T immunotherapy, we designed and prepared antiROR1-CART. This third-generation CAR-T cell can effectively inhibit the growth of colorectal cancer in vitro and in vivo.

The Chemokine Receptor CCR8 Is a Target of Chimeric Antigen T Cells for Treating T Cell Malignancies.

Chimeric antigen receptor (CAR) T cells have been successfully used in the therapy of B cell leukemia and lymphoma, but still have many challenges in their use for treating T cell malignancies, such as the lack of unique tumor antigens, their limitation of T cell expansion, and the need for third party donors or genome editing. Therefore, we need to find novel targets for CAR T cell therapy to overcome these challenges. Here, we found that both adult T-cell leukemia/lymphoma (ATLL) patients and ATLL cells had increased CCR8 expression but did not express CD7. Moreover, targeting CCR8 in T cells did not impair T cell expansion in vitro. Importantly, anti-CCR8 CAR T cells exhibited antitumor effects on ATLL- and other CCR8-expressing T-ALL cells in vitro and in vivo, and prolonged the survival of ATLL and Jurkat tumor-bearing mouse models. In conclusion, these collective results show that anti-CCR8 CAR T cells possess strong antitumor activity and represent a promising therapeutic approach for ATLL and CCR8+ tumors.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D.

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

Co-expression of a PD-L1-specific chimeric switch receptor augments the efficacy and persistence of CAR T cells via the CD70-CD27 axis.

Co-expression of chimeric switch receptors (CSRs) specific for PD-L1 improves the antitumor effects of chimeric antigen receptor (CAR) T cells. However, the effects of trans-recognition between CSRs and PD-L1 expressed by activated CAR T cells remain unclear. Here, we design a CSR specific for PD-L1 (CARP), containing the transmembrane and cytoplasmic signaling domains of CD28 but not the CD3 ζ chain. We show that CARP T cells enhance the antitumor activity of anti-mesothelin CAR (CARMz) T cells in vitro and in vivo. In addition, confocal microscopy indicates that PD-L1 molecules on CARMz T cells accumulate at cell-cell contacts with CARP T cells. Using single-cell RNA-sequencing analysis, we reveal that CARP T cells promote CARMz T cells differentiation into central memory-like T cells, upregulate genes related to Th1 cells, and downregulate Th2-associated cytokines through the CD70-CD27 axis. Moreover, these effects are not restricted to PD-L1, as CAR19 T cells expressing anti-CD19 CSR exhibit similar effects on anti-PSCA CAR T cells with truncated CD19 expression. These findings suggest that target trans-recognition by CSRs on CAR T cells may improve the efficacy and persistence of CAR T cells via the CD70-CD27 axis.

Human induced-T-to-natural killer cells have potent anti-tumour activities.

BACKGROUND: Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies. METHODS: Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies. RESULTS: We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission. CONCLUSIONS: The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

Chimeric CTLA4-CD28-CD3z T Cells Potentiate Antitumor Activity Against CD80/CD86-Positive B Cell Malignancies.

The adoptive transfer of chimeric antigen receptor T (CAR T) cells have been recognized as a promising therapeutic strategy for the treatment of hematological malignancies; however, clinical success using CAR T cells for the treatment of solid tumors are still limited since the T-cell function is inhibited by negative signals in the microenvironment of solid tumors. CTLA4 is a well-known immune checkpoint molecule, thus we developed a novel CAR by converting this negative signal to positive signal. The CAR developed consists of the extracellular and transmembrane domains of CTLA4 and the cytoplasmic domains of CD28 and CD3z (CTLA4-CAR T). CTLA4-CAR T cells exhibited superior cytokine secreting activities and cytotoxic to tumor cells in vitro and in xenograft models. CTLA4-CAR T cells were found to accumulate in tumors and are toxic to myeloid-derived suppressor cells (MDSCs) without signs of severe GVHD and CRS in preclinical models. Thus, this chimeric CTLA4-CAR can enhance the antitumor activity of CAR T cells and shed light on the strategy of using armed CAR T cells to target the immunomodulatory tumor microenvironment.

Human Hyaluronidase PH20 Potentiates the Antitumor Activities of Mesothelin-Specific CAR-T Cells Against Gastric Cancer.

T cell infiltration into tumors is essential for successful immunotherapy against solid tumors. Herein, we found that the expression of hyaluronic acid synthases (HAS) was negatively correlated with patient survival in multiple types of solid tumors including gastric cancer. HA impeded in vitro anti-tumor activities of anti-mesothelin (MSLN) chimeric antigen receptor T cells (CAR-T cells) against gastric cancer cells by restricting CAR-T cell mobility in vitro. We then constructed a secreted form of the human hyaluronidase PH20 (termed sPH20-IgG2) by replacing the PH20 signal peptide with a tPA signal peptide and attached with IgG2 Fc fragments. We found that overexpression of sPH20-IgG2 promoted CAR-T cell transmigration through an HA-containing matrix but did not affect the cytotoxicity or cytokine secretion of the CAR-T cells. In BGC823 and MKN28 gastric cancer cell xenografts, sPH20-IgG2 promoted anti-mesothelin CAR-T cell infiltration into tumors. Furthermore, mice infused with sPH20-IgG2 overexpressing anti-MSLN CAR-T cells had smaller tumors than mice injected with anti-MSLN CAR-T cells. Thus, we demonstrated that sPH20-IgG2 can enhance the antitumor activity of CAR-T cells against solid tumors by promoting CAR-T cell infiltration.

Myeloid-derived suppressor cells promote lung cancer metastasis by CCL11 to activate ERK and AKT signaling and induce epithelial-mesenchymal transition in tumor cells.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune activities and facilitate cancer progression. Although the concept of immunosuppressive MDSCs is well established, the mechanism that MDSCs regulate non-small cell lung cancer (NSCLC) progression through the paracrine signals is still lacking. Here, we reported that the infiltration of MDSCs within NSCLC tissues was associated with the progression of cancer status, and was positively correlated with the Patient-derived xenograft model establishment, and poor patient prognosis. Intratumoral MDSCs directly promoted NSCLC metastasis and highly expressed chemokines that promote NSCLC cells invasion, including CCL11. CCL11 was capable of activating the AKT and ERK signaling pathways to promote NSCLC metastasis through the epithelial-mesenchymal transition (EMT) process. Moreover, high expression of CCL11 was associated with a poor prognosis in lung cancer as well as other types of cancer. Our findings underscore that MDSCs produce CCL11 to promote NSCLC metastasis via activation of ERK and AKT signaling and induction of EMT, suggesting that the MDSCs-CCL11-ERK/AKT-EMT axis contains potential targets for NSCLC metastasis treatment.

IL-6 trans-signaling promotes the expansion and anti-tumor activity of CAR T cells.

Chimeric antigen receptor (CAR) T cell therapies lead to high clinical response rates in B cell malignancies, and are under investigation for treatment of solid tumors. While high systemic interleukin- (IL-) 6 levels are associated with clinical cytokine release syndrome (CRS), the role of IL-6 trans-signaling within CAR T-cells has not been reported. We generated CAR T cells that constitutively express hyper IL-6 (HIL-6), a designer cytokine that activates the trans-signaling pathway. HIL-6-expressing CAR T-cells exhibited enhanced proliferation and antitumor efficacy in vitro and in xenograft models. However, HIL-6 CAR T cells caused severe graft-versus-host disease (GVHD). Transcriptomic profiling revealed that HIL-6 stimulation of CAR T cells upregulated genes associated with T cell migration, early memory differentiation, and IL-6/GP130/STAT3 signaling. Since IL-6 trans-signaling acts via surface GP130, we generated CAR T cells expressing a constitutively-active form of GP130 and found these retained improved antitumor activity without signs of GVHD in preclinical models of B-cell leukemia and solid tumors. Taken together, these results show that IL-6 trans-signaling can enhance expansion and antitumor activity of CAR T cells via the GP130/STAT3 pathway, and suggest that expression of GP130 within CAR T cells could lead to improved antitumor efficacy without systemic IL-6 trans-signaling.

PSCA is a target of chimeric antigen receptor T cells in gastric cancer.

BACKGROUND: Gastric cancer is a deadly malignancy and is a prognostically unfavorable entity with restricted therapeutic strategies available. Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein widely expressed in bladder, prostate, and pancreatic cancers. Existing studies have thoroughly recognized the availability of utilizing anti-PSCA CAR-T cells in the treatment of metastatic prostate cancer and non-small-cell lung cancer. However, no previous study has investigated the feasibility of using anti-PSCA CAR-T cells to treat gastric cancer, irrespective of the proven expression of PSCA on the gastric cancer cell surface. METHODS: We determined the expression of PSCA in several primary tumor tissues and constructed third-generation anti-PSCA CAR-T cells. We then incubated anti-PSCA CAR-T cells and GFP-T cells with target tumor cell lines at E:T ratios of 2:1, 1:1, 1:2, and 1:4 to evaluate the therapeutic efficacy of anti-PSCA CAR-T cells in vitro. We also assayed canonical T cell activation markers after coculturing anti-PSCA CAR-T cells with target cell lines by flow cytometry. The detection of a functional cytokine profile was carried out via enzyme-linked immunosorbent assays. We then evaluated the antitumor activity of anti-PSCA CAR-T cells in vivo by establishing two different xenograft GC mouse models. RESULTS: Anti-PSCA CAR-T cells exhibited upregulated activation markers and increased cytokine production profiles related to T cell cytotoxicity in an antigen-dependent manner. Moreover, anti-PSCA CAR-T cells exhibited robust anti-tumor cytotoxicity in vitro. Importantly, we demonstrated that anti-PSCA CAR-T cells delivered by peritumoral injection successfully stunted tumor progression in vivo. However, intravenous administration of anti-PSCA CAR-T cells failed to reveal any therapeutic improvements. CONCLUSIONS: Our findings corroborated the feasibility of anti-PSCA CAR-T cells and their efficacy against gastric cancer, implicating the potential of applying anti-PSCA CAR-T cells to treat GC patients in the clinic.

Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth.

BACKGROUND: Chimeric antigen receptor T cells (CAR-T cells) therapy has been well recognized for treating B cell-derived malignancy. However, the efficacy of CAR-T cells against solid tumors remains dissatisfactory, partially due to the heterogeneity of solid tumors and T cell exhaustion in tumor microenvironment. PD-L1 is up-regulated in multiple solid tumors, resulting in T cell exhaustion upon binding to its receptor PD-1. METHODS: Here, we designed a dominant-negative form of PD-1, dPD1z, a vector containing the extracellular and transmembrane regions of human PD-1, and a CAR vector against PD-L1, CARPD-L1z, a vector employs a high-affinity single-chain variable fragment (scFv) against human PD-L1. These two vectors shared the same intracellular structure, including 4-1BB and TLR2 co-stimulatory domains, and the CD3ζ signaling domain. RESULTS: dPD1z T and CARPD-L1z T cells efficiently lysed PD-L1+ tumor cells and had enhanced cytokine secretion in vitro and suppressed the growth of non-small cell lung cancer (NSCLC), gastric cancer and hepatoma carcinoma in patient-derived xenograft (PDX). However, the combination of anti-mesothelin CAR-T cells (CARMSLNz T) with dPD1z T or CARPD-L1z T cells did not repress tumor growth synergistically in PDX, as CARMSLNz T cells upregulated PD-L1 expression upon activation and were subsequently attacked by dPD1z T or CARPD-L1z T cells. CONCLUSIONS: In conclusion, we demonstrate CAR-T cells targeting PD-L1 were effective for suppressing the growth of multiple types of solid tumors in PDX models though their safety needs to be carefully examined.

[Distribution of ABO blood group in Tibetan population and their genetic relationship].

OBJECTIVE: To explore the distribution of ABO blood group in Tibetans population and their genetic relationship. METHODS: Data of ABO blood group of 28 Tibetan populations were collected from China and India. The gene frequencies were processed by Phylip3.68 and MEGA4.1 genetic analysis software pack, and the Nei's genetic distance was imported and the genetic relationship was analyzed. RESULTS: The distribution of ABO blood group among Gansu, Qianghai, Sichuan, Yunnan, Tibet, and India was O>A>B>AB. The nation index and genetic distance were 0.63-0.98 and 0-0.0072, respectively. CONCLUSION: There is probable historical relationship among the ancestors of these Tibetan populations, but the differentiation incident of Tibetan population living in different area in history was different, so that their distribution of gene frequencies is diversified.

DNAX-activating protein 10 co-stimulation enhances the anti-tumor efficacy of chimeric antigen receptor T cells.

Chimeric antigen receptor (CAR) T cell immunotherapies have shown remarkable efficacy in treating multiple types of hematological malignancies but are not sufficiently effective at treating solid tumors. NKG2D is a strong activating receptor for NK cells and a co-stimulatory receptor for T cells. NKG2D signal transduction depends on DNAX-activating protein 10 (DAP10). Here, we introduced the cytoplasmic domain of DAP10 into the second-generation CARs M28z and G28z to generate M28z10 and G28z10, which target mesothelin (MSLN) and glypican 3 (GPC3), respectively. T cells expressing M28z10 or G28z10 showed enhanced and prolonged effector function against MSLN+ lung cancer or GPC3+ hepatocellular carcinoma cell lines in culture and secreted elevated levels of cytokines, including IL-2, IFN-γ, granzyme B, and GM-CSF. In addition, M28z10 CAR-T cells showed greater anti-tumor activity than those expressing M28z in both A549 cell line xenografts and human lung cancer patient-derived xenografts (PDX). Similarly, G28z10 exhibited higher efficacy in causing tumor regression than did G28z in hepatocellular carcinoma PDX. Therefore, our results show that DAP10 signaling contributes to the function of CAR-T cells in both lung cancer and hepatocellular carcinoma and can enhance the efficacy of CAR-T cells.

Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer.

BACKGROUND: Gastric cancer (GC) is a common cancer in Asia and currently lacks a targeted therapy approach. Mesothelin (MSLN) has been reported to be expressed in GC tissue and could be targeted by chimeric antigen receptor (CAR) T cells. Mesothelin targeting CAR-T has been reported in mesothelioma, lung cancer, breast cancer, and pancreas cancer. However, the feasibility of using anti-MSLN CAR T cells to treat GC remains to be studied. METHODS: We verified MSLN expression in primary human GC tissues and GC cell lines and then redirected T cells with a CAR containing the MSLN scFv (single-chain variable fragment), CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. We evaluated the function of these CAR T cells in vitro in terms of cytotoxicity, cytokine secretion, and surface phenotype changes when they encountered MSLN+ GC cells. We also established four different xenograft GC mouse models to assess in vivo antitumor activity. RESULTS: M28z10 T cells exhibited strong cytotoxicity and cytokine-secreting ability against GC cells in vitro. In addition, cell surface phenotyping suggested significant activation of M28z10 T cells upon target cell stimulation. M28z10 T cells induced GC regression in different xenograft mouse models and prolonged the survival of these mice compared with GFP-transduced T cells in the intraperitoneal and pulmonary metastatic GC models. Importantly, peritumoral delivery strategy can lead to improved CAR-T cells infiltration into tumor tissue and significantly suppress the growth of GC in a subcutaneous GC model. CONCLUSION: These results demonstrate that M28z10 T cells possess strong antitumor activity and represent a promising therapeutic approach to GC.