Dual Targeting of Mesothelin and CD19 with Chimeric Antigen Receptor-Modified T Cells in Patients with Metastatic Pancreatic Cancer

Abstract

B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress T cell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin to target tumor cells along with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDAC received 1.5 g/m² cyclophosphamide prior to separate infusions of lentiviral-transduced T cells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3 × 10⁷/m²) and chimeric anti-CD19 immunoreceptor (CART-19, 3 × 10⁷/m²). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7-10 days post-infusion, and remained undetectable for at least 28 days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence.

Keywords: B cells; CD19; chimeric antigen receptor; mesothelin; pancreatic cancer.

Graphical abstract

Figure 1

B Cell Infiltration in Human Pancreatic Ductal Adenocarcinoma In (A)–(C), human PDAC tissues were stained for CD3, CD19, and CK19 by multiplex immunohistochemistry. (A) Representative images showing four patterns of CD19⁺ B cell infiltration in PDAC. Scale bars, 100 μm. (B) Quantification of CD19 and CD3 expression in human PDAC tissues (n = 11). Statistical significance was evaluated using a Mann-Whitney test. (C) Correlation plot of number of CD3⁺ cells per mm² versus number of CD19⁺ cells per mm². Data points represent quantification of individual 5X fields captured across stained human PDAC tissues (n = 11). Spearman correlation was performed for r and p value (two-tailed). (D) Correlation plot showing gene expression of CD3E and CD19 in human pancreatic adenocarcinoma (PAAD) patient samples (n = 179) downloaded from The Cancer Genome Atlas (TCGA) dataset. Pearson correlation was performed for r and p value (two-tailed).

Figure 2

Peripheral Blood B Cell Clone Expansion after CART-Meso Cell Infusion in Patients with Advanced Solid Cancers B cell clones were detected in the peripheral blood by DNA sequencing of the IgH CDR3. (A and B) Shown is the number and fold change of expanded B cell clones found in patients at day 28 after beginning treatment (compared to baseline) with (A) RNA CART-Meso or (B) lentiviral CART-Meso cells on prior clinical studies.

Figure 3

CART Cell Expansion, Persistence and Activity (A) Frequency of B cells detected in the peripheral blood of patients with PDAC at baseline (Pre) and at day 28 after receiving a 1:1 mixture of CART-19 and CART-Meso cells. (B) Frequency of CART-19 cells and B cells in blood over time for indicated patients. (C and D) Shown is the copy number of CART-Meso/μg DNA and CART-19/μg DNA detected in the blood at (C) peak expansion and (D) the indicated time points. Statistical significance in (A) and (C) was determined with unpaired Mann Whitney test (two-tailed). ∗p < 0.05.

Figure 4

Cytokine Release after CART Cell Infusion (A–F) Serum cytokines, including (A) IL-10, (B) IL-12, (C) IL-6, (D) IFN-γ, (E) IP-10 (CXCL10), and (F) MIG (CXCL9) were detected in the peripheral blood at defined time points after CART cell infusion. Shown in each left panel is comparison of cytokine levels detected pre-infusion (Pre) and at day 10 post-infusion. In right panel, fold change in cytokine levels are depicted over time. Statistical significance was determined by paired t test. ∗p < 0.05.