GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy

doi:10.21203/rs.3.rs-2750902/v1

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GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy

https://doi.org/10.21203/rs.3.rs-2750902/v1

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Hepatocellular carcinoma (HCC) patients are mostly diagnosed at advanced stages due to the insidious onset of the disease, making them ineligible for curative therapy. Chimeric antigen receptor (CAR)-T-cell therapy is a novel and revolutionary treatment that has become a mainstay of advanced cancer treatment. However, additional challenges limit the use of this therapy in solid tumors. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes, and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects.We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces coexpression of IL-7 at a moderate level (500 pg/mL) and CCL-19 at a high level (15000 pg/mL) and transduced it into human T cells. In vitro cell killing efficacy was validated by the xCELLigence RTCA system and the LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC cells and HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Peripheral blood mononuclear cells (PBMCs) and tumor, liver and other tissue samples were isolated from the mouse model and analyzed by flow cytometry. Finally, we initiated a phase 1 clinical trial in advanced HCC patients with GPC3 expression to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had approximately 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in the humanized NSG mouse xenograft model (highly expressing GPC3) treated with GPC3-7-19-CAR-T cells and in those treated with GPC3-CAR-T cells were 3/5 and 5/5, respectively, and the average tumor volumes were 0.065 cm³±0.061 vs. 0.50 cm³±0.381. Of note, the predominant cell subpopulations in the tumors, CD4⁺ T_EM and CD8⁺ T_EMcells, played a key role in enhancing the antitumor activity of GPC3-7-19-CAR-T cells. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cell (MDSC) and regulatory T (Treg) cell infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues than GPC3-CAR-T cells. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. In conclusion, all the data show that our engineered GPC3-7-19-CAR-T cells achieve antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4⁺ T_EM and CD8⁺ T_CM cell subsets, revealing a promising therapeutic strategy for advanced GPC3⁺ HCC patients in the clinic. Thus, CD4⁺ T_EM and CD8⁺ T_CMcells are a potential target CAR-T-cell treatment of solid tumors. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic.

Hepatocellular carcinoma

Chimeric antigen receptor

Glypican-3

Cytokines

Chemokines

Chimeric antigen receptor (CAR)-based immunotherapy has been recognized as a promising novel cancer modality since it was first reported and has shown impressive clinical efficacy in the treatment of hematologic malignancies^[1]. By 2021, four CAR-T-cell products had been approved by regulatory organizations in the United States, as had one each in Japan and Singapore. Despite the impressive achievements of CAR-T cells in hematologic malignancies, progress related to CAR-based immunotherapies for solid tumors is lacking. Lin Shen and colleagues published their phase I clinical trial data showing that the objective response rate of 18 advanced gastric cancer patients who received Claudin18.2-CAR-T-cell therapy was 61.1%, the disease control rate was 83.3%, and the progression-free survival (PFS) was 5.6 months^[2]. These results provide support for the use of this therapy and led us to actively look for useful CAR-T-cell therapy options for solid tumor therapy.

Due to the heterogeneity of solid tumors, it is first important for an ideal target for CAR-T-cell therapy to be identified. At present, tumor-associated antigens (TAAs) and viral antigens are the most common targets of CAR-T-cell immunotherapy, and such strategies have shown beneficial clinical efficacy in some patients^[3-4]. Regarding hepatocellular carcinoma (HCC), glypican 3 (GPC3) has been suggested to be a suitable target due to its strong positive expression in approximately 75% of HCC cells but lack of expression in most normal tissues^[5]. However, the efficacy of GPC3-CAR-T-cell therapy in HCC is not satisfactory in the clinic.

It has been shown that the tumor microenvironment (TME) plays a pivotal role in determining the therapeutic effects of anti-PD-1/PD-L1 antibody therapies in solid tumors^[6]. Reversal of the suppressive TME might greatly improve the efficacy of CAR-T-cell immunotherapy^[7]. One strategy is the introduction of proinflammatory cytokines or chemokines, such as IL-12, IL-18 or CCL20, into the CAR structure to recruit more immune cells and elicit desired antitumor responses; cells with such CAR constructs are known as "TRUCKs" (redirected T cells for general cytokine killing)^[8-9]. It should be noted that different chemokines usually recruit distinct subsets of immune cells via their cognate receptors and exhibit various effects on tumor progression. The 7-19-CAR-T cells designed by Koji Tamada showed significant antitumor effects on mastocytoma and pancreatic ductal adenocarcinoma growth in murine xenograft models^[10]. More studies have suggested antitumor efficacy of 7-19-CAR-T cells in solid tumors, but the detailed mechanisms have not been clarified. Although a large number of immune cells responsible for innate and adaptive immunity are found in liver tissue, immune tolerance obviously plays a role in removing exogenous antigens or pathogens in the liver. The unique composition of the TME leads to a relatively low response to immunotherapy drugs in HCC^[11-12]. Therefore, we constructed GPC3-CAR-T cells coexpressing IL-7 and CCL19 that can remodel the suppressive TME of HCC and elicit a robust antitumor response in GPC3-positive HCC.

GPC3-7-19-CAR-T cells exert superior cytotoxic effects on GPC3-positive HCC cells in vitro

To profile the characteristics of fourth-generation GPC3-CAR-T cells expressing the costimulatory molecules IL7 and CCL19 (GPC3-7-19-CAR-T cells), we constructed three other types of CARs in parallel in this study. Among them, Vector-CAR-T cells served as a negative control, while GPC3-CAR-T cells were used as conventional CAR-T cells specifically targeting GPC3, and 7-19-CAR-T cells expressed with IL7 and CCL19 but lacked GPC3 targeting (Fig. 1A). The GPC3-CAR-T cells were made to express an enhanced green fluorescence protein (EGFP) to enable evaluation of viral transfection efficiency. Most activated T-cell clusters expressed EGFP after 5 days of culture, as observed under a fluorescence microscope (Supplementary Fig. 1A, B). Approximately 48%-62% of CAR-positive T cells in each group were identified as positive by flow cytometry (Fig. 1A, B). To optimize the expansion efficiency of CAR-T cells, 10 ng/mL rhIL-15 was added to culture media with 10 ng/mL rhIL-2 beginning on the second day after T-cell stimulation. CAR-T cells proliferated rapidly with approximately 200-fold expansion within 12 days, which was much higher than that of the conventional T cells that were only given IL-2 (Supplementary Fig. 1C, D).

Moreover, GPC3-7-19-CAR-T cells secreted the highest levels of IL-7 and CCL-19 when cocultured with GPC3-positive HCC cell lines. Approximately 480 pg/mL IL-7 and 17000 pg/mL CCL-19 were detected when GPC3-7-19-CAR-T cells were cocultured with HepG2 cells. 7-19-CAR-T cells showed moderate levels of IL-7 (300 pg/mL) and CCL-19 (1500 pg/mL) secretion, whereas GPC3-CAR-T cells and vector-CAR-T cells secreted minimal amounts (Fig. 1D, E).

To assess the specific antitumor effects of GPC3-7-19-CAR-T cells against GPC3-positive HCC cells, we first evaluated GPC3 expression levels in several HCC cell lines by flow cytometry. GPC3 was primarily expressed in HepG2 and Hep3B cells (97.5% and 90.1% of cells with positive expression, respectively) and expressed at low levels in SMMC7721 cells (5.9% of cells with positive expression, Supplementary Fig. 1E, F).

Next, CAR-T cells from each group were incubated with HCC cells at an effector-to-target (E/T) ratio of 1:1. HepG2 cell growth was monitored continuously for 72 hours by measuring impedance using the RTCA SP xCELLigence system (Roche)^[13]. GPC3-7-19-CAR-T cells showed 20% (35% vs. 15%) higher cytotoxicity toward HepG2 cells than GPC3-CAR-T cells and other T cells (Fig. 2B, C).

To further verify the results, a nonradioactive LDH assay was employed. GPC3-7-19-CAR-T cells exhibited at least a 1.5-fold increased killing rate of HepG2 and Hep3B cells compared with GPC3-CAR-T cells at an E:T ratio of 1:1 or 2:1 (Fig. 1H, I), but there was no increased killing of SMMC7721 cells (Fig. 1J). As seen on optical microscopy, HepG2 cells were clearly surrounded by GPC3-7-19-CAR-T cells in clusters after a 6-hour coculture, suggesting that activated GPC3-7-19-CAR-T cells exerted strong and specific killing effects against GPC3-positive HCC cells (Fig. 1K).

Gpc3-7-19-car-t Cells Exhibit Excellent Activity Against Hcc Xenografts In Humanized Mouse Models

To better examine the in vivo safety and efficacy of GPC3-7-19-CAR-T cells, a subcutaneous xenograft model of HepG2-EGFP-Luci cells was established in humanized NSG mice, in which the microenvironment was similar to that of patients who have received CAR-T-cell infusion into peripheral blood^[14]. Each mouse received two doses of CAR-T cells at day 0 and day 4 via tail vein injection. The tumors and vital organs were collected at the endpoint to analyze cytokine secretion and tumor-infiltrating lymphocytes, and immunohistochemistry (IHC) staining was performed. A detailed schedule is shown in Fig. 2A.

To confirm the successful establishment of the humanized mouse model, baseline lymphocytes in the peripheral blood of all mice were tested by flow cytometry. The human CD45 population represented approximately 70% of all cells on average in each humanized mouse. Statistically, human pan-T cells (CD3⁺) represented approximately 19% of all cells, of which the percentages of human Th cells (CD3⁺CD4⁺), Ts cells (CD3⁺CD8⁺), natural killer (NK) cells (CD3⁻CD56⁺), dendritic cells (CD11c⁺) and monocytes (CD11b⁺) were approximately 30%, 54%, 3.6%, 3.0% and 7.9%, respectively (Fig. 2B-E).

Then, tumor growth was monitored and recorded every other day. On day 76, by intravital fluorescence imaging, the tumor volume of GPC3-7-19-CAR-T-cell-treated mice was significantly smaller than that of GPC3-CAR-T-cell-treated mice. Of note, 2 xenograft tumors in GPC3-7-19-CAR-T-cell-treated mice disappeared. In contrast, the largest subcutaneous tumor in GPC3-CAR-T-cell-treated mice was about 2 cm³, and the tumorigenesis rate was 60% in GPC3-7-19-CAR-T-cell-treated mice compared with 100% in GPC3-CAR-T-cell-treated mice (n = 5, Fig. 2F, G, H). Furthermore, the general condition and body weight of GPC3-7-19-CAR-T-cell-treated mice was better and higher than those of GPC3-CAR-T-cell-treated mice, indicating the safety of GPC3-IL7-CCL19-CAR-T-cell infusion (Fig. 2I). In summary, GPC3-7-19-CAR-T cells exhibited excellent antitumor effects against GPC3-positive HCC cells compared with conventional GPC3-CAR-T cells.

The CD4 ⁺ T-cell subset plays a dominant role in the enhanced antitumor activity of GPC3-7-19-CAR-T cells

To monitor the in vivo expansion of CAR-T cells in humanized mice, peripheral blood samples of mice were collected at days 0, 1, 4, 7, 14, 28, 56, and 73 to determine CAR copy numbers by q-PCR. As shown, the CAR copies of both GPC3-CAR-T and GPC3-7-19-CAR-T cells peaked around day 14. Then, there was a rapid decrease in CAR copies of GPC3-CAR-T cells and a sustained higher level of GPC3-7-19-CAR-T cells until day 56 (Fig. 3A). On day 28, higher levels of human IFN-γ and IL-2 but not TNF-α were observed in GPC3-7-19-CAR-T-cell-treated mice than in traditional GPC3-CAR-T-cell treated mice (Fig. 3B).

To explore the underlying mechanism of GPC3-7-19-CAR-T-cell-mediated antitumor effects in humanized NSG mice, human cell populations were analyzed in mouse peripheral blood, liver and tumor tissues by flow cytometry on day 76. A significant increase in human CD4⁺ T cells was detected in mouse liver and tumor tissues in GPC3-7-19-CAR-T-cell treated mice compared to GPC3-CAR-T-cell treated mice. Furthermore, two populations of human CD4⁺ and CD8⁺ T cells with central (T_CM cells, CD45RA⁻CCR7⁺) and effector (T_EM cells, CD45RA⁻CCR7⁻) memory phenotypes were further evaluated. The total CD4⁺ T cell and CD4⁺ T_EM cell subset levels in mouse liver and tumor tissues in GPC3-IL7-CCL19-CAR-T-cell treated mice were dramatically higher than those in GPC3-CAR-T-cell treated mice. Notably, the levels of both the CD8⁺ T_CM and CD4⁺ T_EM cell subsets, but not the CD8⁺ T_EM cell subset, in the tumor tissues of GPC3-IL7-CCL19-CAR-T-cell-treated mice were significantly elevated compared with those in GPC3-CAR-T-cell treated mice (Fig. 3C-F). Then, CD4⁺ T and CD8⁺ T cells in tumors were assessed once more by IHC and immunofluorescence (IF) staining, and more CD4⁺ T cells were found in tumor tissue in GPC3-7-19-CAR-T-cell-treated mice (Fig. 3G-J). All of the above results indicated that CD4⁺ T cells, especially CD4⁺ T_EM cell subsets, play a key role in the enhanced antitumor activity of GPC3-7-19-CAR-T cells in vivo. In addition, the increased level of CD8⁺ T_CM cells might also lead to a better tumor-suppressive effect of GPC3-7-19-CAR-T cells.

Gpc3-7-19-car-t Cells Remodel The Tumor Immune Microenvironment

Next, the tumor immune microenvironment (TIME) was evaluated after GPC3-7-19-CAR-T-cell infusion. First, bone marrow myeloid-derived suppressor cells (MDSCs) were assessed in xenograft tumors and mouse organs based on their surface markers outlined in Fig. 4A. Statistically, the percentages of MDSCs in all myeloid cells (CD45⁺CD11b⁺ HLA-DR⁻) in GPC3-IL7-CCL19-CAR-T-cell-treated mice were at least 2-fold lower than those in GPC3-CAR-T-cell-treated mice, especially in tumor and liver tissue (Fig. 4B).

In pedigree analysis, MDSCs can be classified into PMN-MDSCs (CD11b⁺ HLA-DR⁻, CD15⁺) and monocyte-like MDSCs (M-MDSCs, CD11b⁺ HLA-DR⁻, CD14⁺, Arg1⁺, Fig. 4A). We identified MDSC subsets in mice treated with CAR-T-cell therapy, and a 40% lower proportion of M-MDSCs was found in GPC3-7-19-CAR-T-cell treated mice, especially in the xenograft tumors and in most organs except the bone marrow. (Fig. 4C).

In the TME, Treg cells might also play an inhibitory role in adoptive CAR-T-cell therapy. Treg cells can be rapidly induced or recruited by TGF-β and IL-10 derived from MDSCs. In line with this, the levels of Treg cells in the peripheral blood, liver and tumor tissue of GPC3-7-19-CAR-T-cell-treated mice were significantly less than those of GPC3-CAR-T-cell-treated mice (Fig. 4D, E). All of the above data demonstrated that GPC3-7-19-CAR-T cells reversed the inhibitory TME by decreasing MDSC and Treg cell infiltration in tumors, thus achieving better therapeutic effects in HCC xenografts models.

Dcs Are Recruited To Assist The Killing Activities Of Gpc3-7-19-car-t Cells

CCL19 is thought to be a key chemokine that recruits CCR7⁺ immunocompetent cells, such as DCs, T and B cells, and macrophages, to tumor tissue. A remarkable increase in human DCs was found in tumor tissues in GPC3-7-19-CAR-T-cell-treated mice (Fig. 5F, G). To confirm the results, mature HLA-DR⁺CD11b⁺CD86⁺ DCs (Fig. 5A, B) were cocultured with either GPC3-7-19-CAR-T cells or GPC3-CAR-T cells in a Transwell system. The migrated DCs in the lower compartment were then counted (Fig. 5C). Statistically, the numbers of migrated DCs were 10% higher in the GPC3-7-19-CAR-T-cell coculture (Fig. 6D, E). IHC staining of DCs in humanized mouse liver and tumor tissue showed significantly higher DC infiltration in mouse liver and tumor tissue after GPC3-7-19-CAR-T-cell treatment compared with GPC3-CAR-T-cell treatment (Fig. 5F, G). Overall, these results confirmed that GPC3-7-19-CAR-T cells induce enhanced antitumor effects by recruiting DCs.

Potential Clinical Application Of Gpc3-7-19-car-t Cells As Treatment For Gpc3-positive Hcc Patients

After confirming the safety and efficacy of GPC3-IL7-CCL19-CAR-T cells in in vitro and in vivo mouse models, their utility in clinical application was further evaluated with primary HCC cells derived from 3 patients. Single primary GPC3-positive HCC cells were prepared by enzyme digestion and Ficoll-Paque separation. After 16–20 days, primary HCC cells were cocultured with CAR-T cells for 24 hours, and the killing efficiency was determined by flow cytometry. GPC3-IL7-CCL19-CAR-T cells killed approximately 70% of primary HCC cells, 25% higher than what GPC3-CAR-T cells achieved at an E/T ratio of 5:1 (Fig. 6A, B).

In addition, a 3D microfluidic tumor model was employed to evaluate the response of GPC3-IL7-CCL19-CAR-T cells to primary GPC3-positive HCC cells (Fig. 6C). In this system, before exerting their cytotoxicity on targets, CAR-T cells need to migrate from medium-filled channels (side) into a collagen-filled central region (tumor site). Over a 12-hour coculture, more CAR-T cells migrated into the tumor channel to kill tumor cells, and more dead cells in the central 3D matrix were observed, especially in the GPC3-7-9-CAR-T-cell group (Fig. 6D). Simultaneously, secreted factors were quantified in media channel supernatants. As expected, 100 pg/mL and 400 pg/mL IL-7 and 5000 pg/mL and 10000 pg/mL CCL19 were detected in the supernatants of GPC3-CAR-T cells and GPC3-7-19-CAR-T cells, respectively. In addition, 1300 pg/mL TNF-α and 4500 pg/mL IFN-γ were also detected in the supernatant of GPC3-7-19-CAR-T cells, which was 2-fold higher than those detected in GPC3-CAR-T-cell and 7–9 cell supernatants (Fig. 6E). In summary, GPC3-7-19-CAR-T cells displayed a higher migration ability and cytotoxic activity against primary HCC cells, indicating their potential in clinical applications in HCC patients.

Gpc3-7-19-car-t Cells Show Remarkable Antitumor Activity In One Patient With Hcc With Gpc3 Expression

Based on the preclinical results, we conducted a phase I clinical trial in advanced HCC patients with positive GPC3 expression to further explore the clinical safety and feasibility of GPC3-7-19-CAR-T-cell therapy. Patients were enrolled based on the inclusion and exclusion criteria. One patient was administered 5×10⁶ GPC3-7-19-CAR-T cells/kg (CAR-positive rate 69%, Supplementary Fig. 2B) after lymphodepletion according to the regimen. Metastatic lesions at baseline and follow-up were assessed by computed tomography (CT). The size of the metastatic retroperitoneal lymph nodule had decreased significantly (22.1×15.6 mm vs. 15.1× 11.7 mm) and the mediastinal lymph nodule had almost disappeared by day 56 (Supplementary Fig. 2A). The patient did not suffer any toxic effects, and CT-based staging evaluation revealed stable response (SD) according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Blood was collected for pharmacokinetic (PK) and pharmacodynamic (PD) analyses. At day 56, the proportion of CD8 + T cells was much higher than that at baseline (67.9% vs. 16.6%, Supplementary Fig. 2C). Furthermore, TNF-α and IFN-γ secretion was significantly increased (Supplementary Fig. 2D, E). It is worth noting that the tumor biomarkers serum AFP and CEA were decreased by approximately 50% (Supplementary Fig. 2F). In addition, hepatic dysfunction marker enzymes such as γ-GT, AST, ALT, and ALP were obviously decreased (Supplementary Fig. 2G). Every 7 days, the lymphocyte populations of the patient were profiled. Compared with that before GPC3-7-19-CAR-Tcell infusion, the level of CD3 + T cells was higher on day 56. CD8 + T-cell subsets were sustainably increased and CD4 + T-cell subsets were decreased after CAR-T-cell treatment (Supplementary Fig. 2H).

The success of engineered CAR-T-cell therapies in solid tumors has been limited, mainly due to the immunosuppressive microenvironment, tumor heterogeneity, on-target-off-tumor effects, T-cell exhaustion, etc.^[15–17]. In this regard, a novel CAR structure was recently developed that can induce the expression of different panels of cytokines to increase the proliferation, cytotoxicity and long-term survival of CAR-T cells in solid tumors^[18–19].

The liver is the most important metabolic organ in humans and has a unique immune function. Many kinds and numbers of immune cells are present in the liver and serve to mediate innate and adaptive immunity in the liver. However, immune tolerance rapidly develops after the removal of exogenous antigens and pathogenic agents^[20–21]. For this reason, a relatively lower response to immunotherapeutic drugs is observed in HCC patients. Constructs inducing the expression of IL-7 and CCL-19 were selected and inserted at the 2A locus in our anti-GPC3 CAR structure because of the relationship of these cytokines to T-cell viability and proliferation and TME remodeling.

In this study, we first established a highly efficient system to expand CAR-T cells in medium with the addition of IL2 and IL15, as well as IL7 secreted by GPC3-7-19-CAR-T cells. Numerous memory T cells (both cytotoxic T cells, CD3⁺ CD8⁺, and Th cells, CD3⁺ CD4⁺) were successfully generated and proliferated, which indicates a pivotal role of IL7 and IL15 in memory T-cell formation. More importantly, the safety and efficacy of GPC3-7-19-CAR-T cells were demonstrated in humanized NSG mouse models with GPC3-positive HCC xenografts.

High levels of inhibitory receptors such as PD1, LAG-3, CD244 and CD160 in activated Ts cells and progressive loss of CD4 + T cells might limit the long-term antitumor effect of CAR-T cells. Miao et al. confirmed that peripheral CD4⁺ T cells were significantly lower while CD8 + T cells and T_reg cells were significantly higher in HCC patients than in their healthy counterparts^[22–23]. This suggests that the CD4 + T-cell population might contribute greatly to the antitumor activity against HCC^[24].

Based on our HCC xenograft model in humanized NSG mice, T cells, especially CD4⁺ T_EM cells and CD8⁺ T_CM cells, were markedly infiltrated in tumor tissue, and robust and long-term antitumor effects were achieved after GPC3-IL7-CCL19-CAR-T-cell treatment. These results might imply that promoting the differentiation of naive T cells into memory T cells as well as antigen-specific T-cell expansion are key mechanisms by which GPC3-IL7-CCL19-CAR-T cells elicit a persistent immune response against HCC.

On the other hand, not only CAR-T cells themselves but also other immune cells are necessary to exert antitumor activity in HCC patients. Indeed, mature DCs were extensively mobilized and had infiltrated in xenograft HCC tissues after GPC3-IL7-CCL19-CAR-T-cell treatment. This result was consistent with previous results, indicating that CCL19 is a pivotal chemotactic factor in recruiting CCR7⁺ cells^[25]. DCs were successfully recruited by GPC3-IL7-CCL19-CAR-T cells in our in vitro cell coculture system and in xenograft HCC tissue, as determined by staining. It is reasonable to believe that GPC3-IL7-CCL19-CAR-T cells induce antitumor effects with the help of DCs, although the specific subsets of DCs involved should be further explored in the future.

Tregs, TAMs and MDSCs in the TME induce various immunosuppressive effects by inhibiting T-cell proliferation, lymph node homing and the immune response, thus greatly compromising CAR-T-cell therapeutic efficacy^[26–28]. To verify the role of TME remodeling in humanized NSG mice, suppressive cell subsets were detected, and fewer MDSCs and Treg cells had infiltrated xenograft HCC tissues after GPC3-7-19-CAR-T-cell treatment. Similarly, in a meta-analysis of 1002 HCC patients, the more MDSCs that existed in tumor tissue, the worse the DFS and OS were^[29]. More interestingly, MDSCs and Treg cells have been reported to influence each other in vivo. For example, MDSCs promoted Treg cell expansion by inducing CD40 expression and TGF-β and IL-10 secretion in a TGF-β-dependent or TGF-β-independent manner, thereby reducing T-cell cytotoxicity, which was also confirmed in an in vivo assay^[30].

Therefore, the robust antitumor effects of GPC3-IL7-CCL19-CAR-T cells may at least partially be a result of reduced Treg cells and MDSCs in tumor tissue.

As shown in this study, 3D tumor models of primary HCC cells will be very helpful for predicting the potential of CAR-T cells in clinical therapy. However, more HCC patient-derived primary cells with or without GPC3 expression should be tested, and additional cytokines should be assessed. To date, although only one patient has received GPC3-7-19-CAR-T-cell treatment, it has shown good safety and promising clinical application prospects. All our results indicate that the clinical efficiency of GPC3-7-19-CAR-T cells should be tested in GPC3⁺ HCC patients.

Patients and sample collection

Nearly 8 mL of peripheral blood was collected from each HCC patient for T-cell culture. To establish a 3D tumor microfluidic model, 5 primary tumor tissue samples were collected from GPC3-positive HCC patients who had not received any adjuvant therapy before curative resection.

Construction Of Lentiviral Vectors Expressing Cars

To generate the GPC3-CAR lentiviral vector, a BamHI- and NheI-digested fragment encoding both the signal sequence of the human GM-CSFR alpha chain and the anti-GPC3 scFv was ligated into the lentiviral vector pLV-CAR under the EF1α promoter, which contains the hinge region and transmembrane portion of the human CD8α chain and the cytoplasmic portion of the human CD28, 4-1BB and CD3-ζ molecules. To evaluate the viral transfection efficiency, EGFP was cloned into the GPC3-CAR lentiviral vector preceded by the T2A peptide sequence. In the same way, human IL-7 and CCL19 constructs were inserted into the GPC3-CAR lentiviral vector with intervening T2A linkers to enable coexpression of these cytokines and GPC3-CAR.

Establishment Of Hcc Xenografts In Humanized Nsg Mice And Car-t-cell Treatment

For mouse model establishment, 5×10⁶ HepG2-EGFP-Luci cells/200 µL and 10 mg/mL Matrigel mixture were injected subcutaneously into the backs of humanized NSG mice. Tumor growth was monitored every 2 days, and tumor volume was calculated as 1/2 (length×width×height). When the tumor had grown to 0.5 cm³, 1×10⁷ CAR-T cells per kg were injected via the tail vein. On day 4, a second dose was administered in the same way. Nearly 10 µL peripheral blood were collected via a facial vein at days 0, 1, 4, 7, 14, 28, 56, and 73 to determine the CAR-T-cell expansion status. Meanwhile, mouse behaviors, weights and tumor sizes were recorded, and the mice were sacrificed for analysis at the endpoint of the experiment on day 76.

Flow Cytometry Analysis (Including Reagents And Antibodies)

The details of the antibodies, reagents, and methods are provided in the Supplemental Methods and Materials.

Statistical analysis

Statistical analysis was performed using SPSS, version 19.0 (IBM, Armonk, NY), except for special indications as noted. Data are presented as the mean ± standard deviation. The c² test, Fisher’s exact test, Student’s t test, and Mann‒Whitney U test were used as appropriate. A two-tailed P value < 0.05 indicated statistical significance.

Acknowledgments

We are grateful to the team of Professor Yan Li and Shanghai Model Organism for providing the humanized NSG mouse model and teaching us how to perform the animal experiments. We are also grateful to Zhengjun Zhou PhD for helping us establish the 3D microfluidic model of HCC. Finally, we would like to thank the surgeons who acquired the resected samples from patients during surgery.

Authors’ contributions

Weizhong Wu developed the study concept and supervised this project. Lili Lu performed the in-vitro essay and wrote the manuscript. Bin Lu designed CAR structure and generated the CAR plasmids. Jinjin Bai prepared lentivirus. Shuxiu Xiao made the animal essay and processed data. Zhiyuan Lin was responsible for statistical analysis. Zhengqing Song helped with the in vivo experiments. All authors read and approved the final manuscript. Wei Li and Yuhong Zhou were responsible for the clinical trail regimen and patient administration.

Funding

This project was supported by grants from the National Natural Science Foundation of China (82073216), the Projection of Shanghai Science and Technology Committee (20S11906300, 21ZR1477600), the youth project of Shanghai Municipal Health Commission ( 20204Y0229), Sailing Project of “Scientific and Technological Innovation Action Plan” of Shanghai Science and Technology Commission (20YF1406100). Shanghai Municipal Health Commission, Collaborative Innovation Cluster Project, 2019CXJQ02.

Ethics approval and consent to participate

The Ethics Committee of the Zhongshan Hospital Biomedical Research Department provided ethical approval (Approval No. B2020-211R), and informed consent for collecting and preserving samples and details was obtained from each patient.

Xenograft of humanized NSG mice experiments were approved by the Animal Experimentation Ethics Committee of Zhongshan Hospital, Fudan University (supplementary materials). And all the humanized mice owned qualification certificate (2017010014018) and production license.

The clinical trail has been registered at Chinese Clinical Trial Registery (www.chictr.org.cn) as ChiCTR2000036458.

Competing interests

The authors declare that they have no competing interests.

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GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy

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Abstract

Figures

Introduction

Results

GPC3-7-19-CAR-T cells exert superior cytotoxic effects on GPC3-positive HCC cells in vitro

Gpc3-7-19-car-t Cells Exhibit Excellent Activity Against Hcc Xenografts In Humanized Mouse Models

Gpc3-7-19-car-t Cells Remodel The Tumor Immune Microenvironment

Dcs Are Recruited To Assist The Killing Activities Of Gpc3-7-19-car-t Cells

Potential Clinical Application Of Gpc3-7-19-car-t Cells As Treatment For Gpc3-positive Hcc Patients

Gpc3-7-19-car-t Cells Show Remarkable Antitumor Activity In One Patient With Hcc With Gpc3 Expression

Discussion

Materials And Methods

Patients and sample collection

Construction Of Lentiviral Vectors Expressing Cars

Establishment Of Hcc Xenografts In Humanized Nsg Mice And Car-t-cell Treatment

Flow Cytometry Analysis (Including Reagents And Antibodies)

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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