IL-7 and CCL19-secreting CAR-T cell therapy for tumors with positive glypican-3 or mesothelin

1A recent study showed that IL-7 and CCL19 (7 × 19) secretion promotes the infiltration and survival of murine CAR-T cells in vivo. To further verify the effects of 7 × 19 secretion on human CAR-T cells, we constructed three CAR vectors targeting GPC3, MSLN, and CD20 (Fig. 1a), which all contained intracellular domains of CD28 and TLR2. As the cargo capacity of the lentiviral vector is limited, human IL-7 and CCL19 linked through the 2A peptide were inserted into a separate lentiviral vector (Fig. 1a). We then cotransduced T cells with CAR and 7 × 19 lentiviruses (Fig. 1b). IL-7 and CCL19 were detected in the supernatant of 7 × 19 CAR-T cell cultures (Fig. 1c). 7 × 19 augmented the chemotaxis and expansion of CAR-T cells (Fig. 1d, 1e), but did not influence the cytotoxicity and cytokine secretion of CAR-T cells (Additional file 1: Fig. S1a, S1b, S1c).

2We next adopted a GPC3+ HepG2 cell line-derived xenograft (CDX) mouse model to assess the antitumor efficacy of anti-GPC3-7 × 19 CAR-T cells. Anti-GPC3-7 × 19 CAR-T cells significantly suppressed HepG2 growth than anti-GPC3 CAR-T cells (Fig. 1f, 1g). In addition, the percentage of T cells in peripheral blood of anti-GPC3-7 × 19 group were higher than those in the anti-GPC3 group (Fig. 1h, Additional file 1: Fig. S2a). There were more infiltrated T cells in the tumors of the anti-GPC3-7 × 19 group, compared that in the anti-GPC3 group (Additional file 1: Fig. S2b). Similarly, anti-GPC3-7 × 19 CAR-T cells displayed superior antitumor efficacy in a HCC patient-derived xenograft (PDX) mouse model, compared to anti-GPC3 CAR-T cells (Additional file 1: Fig. S3). Moreover, anti-MSLN-7 × 19 CAR-T cells also displayed more potent antitumor capacity than anti-MSLN CAR-T cells in a MSLN+ AsPC-1 CDX mouse model (Fig. 1i, 1j, 1k, Additional file 1: Fig. S4).

3Based on the preclinical results, we conducted a phase I clinical trial in advanced hepatocellular carcinoma (HCC), pancreatic carcinoma (PC) and ovarian carcinoma (OC) patients with GPC3 or MSLN expression to further explore the clinical safety and feasibility of 7 × 19 CAR-T cell therapy. The protocol of this trial is described in the supplemental materials and is depicted schematically (Additional file 1: Fig. S5, S6). Patients were enrolled based on the patient inclusion and exclusion criteria (Additional file 1: Table S1). Six patients were administered anti-GPC3/anti-MSLN-7 × 19 CAR-T cells (Additional file 1: Table S2).

4Subject GD-G/M-001 is an advanced HCC patient with GPC3 expression (Additional file 1: Fig. S7a, S7b). Three metastatic lesions were chosen to perform computed tomography (CT) guided intratumor injection of CAR-T cells. Though the sizes of two lung nodules did not change significantly 60 days after CAR-T cells injection (Fig. 2a), the liver tumor lesion (1.2 × 1.3 cm) shrunk significantly on day 10 and completely disappeared on day 32 post anti-GPC3-7 × 19 CAR-T cells injection (Fig. 2b). The patient did not suffer any toxic effects and had staging evaluation revealing partial response (PR) according to standard Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 on CT.

5Subject GD-G/M-005 is an advanced PC patient with MSLN expression (Additional file 1: Fig. S7f). This subject progressed with a local lymph node metastasis (24 × 33 mm) (Fig. 2c). Anti-MSLN-7 × 19 CAR-T cells were first infused through hepatic artery with heavy fever on the night without cytokine release syndrome (CRS) or neurotoxicity. Subsequently, he received intravenous infusions of the anti-MSLN-7 × 19 CAR-T cells every 1–2 months. After 5 times of the CAR-T cells infusions, CT staging revealed complete response (CR) on day 240 with the lymph node measured 8.3 × 9.6 mm and no other enlarged lymph nodes visible (Fig. 2c). He remained in normal condition.

6The clinical course of the four other infused patients is shown in Additional file 1: Fig. S8 and described in Additional file 1: Table S3 and the Materials and Methods. Our current six-case cohort preliminary clinical study revealed no grade 2–4 adverse events or major complications; one PC patient (1/6, 16.7%) achieved CR; one HCC patient (1/6, 16.7%) achieved PR; and 2 (2/6, 33.3%) achieved steady disease (SD), demonstrating the huge therapeutic potential of 7 × 19 CAR-T cell therapy for advanced solid tumors with GPC3/MSLN expression. Due to the limited number of cases enrolled in this study, we admit this was not a comprehensive clinical trial. However, based on the safety and efficacy of 7 × 19 CAR-T cells shown by these results, we are preparing to conduct a formal phase I clinical trials of 7 × 19 CAR-T cells that specifically target liver cancer or pancreatic cancer.