In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB)

Abstract

Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-α (FRα) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRα-specific scFv (MOv19) coupled to the T-cell receptor CD3ζ chain signaling module alone (MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBζ). Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FRα(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CAR mediated tumor regression in immunodeficient mice bearing large, established FRα(+) human cancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FRα CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FRα T-cell targeting strategy to provide potent antitumor activity in vivo.

Figure 1. Generation and specific immune recognition by FRα CAR-transduced human T cells in vitro

A. Schematic representation of MOv19 based Chimeric Antigen Receptor (CAR) constructs containing the CD3ζ cytosolic domain alone (MOv19-ζ) or in combination with CD137 costimulatory module (MOv19-BBζ). FRα-specific CAR with a truncated CD3ζ domain (MOv19-Δζ) and anti-CD19-BBζ CAR are shown. MOv19, anti-FRα scFv; VL, variable L chain; L, Linker; VH, variable H chain; TM, transmembrane region. B. MOv19 CAR expression (solid black line) on human CD3-gated cells after transduction with lentivirus compared to parallel untransduced T cells (filled gray histograms). Percent transduction is indicated. C. Surface FRα expression (solid black line) by various human ovarian cancer cell lines by flow cytometry; isotype antibody control (filled gray histograms). D. Antigen-specific IFN-γ secretion by MOv19-ζ and MOv19-BBζ CAR-transduced T cells but not MOv19-Δζ anti-CD19-BBζ T cells, following overnight incubation with FRα+ cancer cell lines. Mean IFN-γ concentration ± SEM (pg/ml) from triplicate cultures is shown. E. Antigen-specific killing of FRα+ tumor cells by FRα CAR+ CD8+ T cells in 18hr bioluminescence assay at the indicated effector to target (E/T) ratio. Untransduced T cells (UNT) or gfp transduced human CD8+ T cells served as controls.

Figure 2. Human MOv19-BBζ CAR T cells eradicate large pre-established tumors in vivo: effect of CD137 costimulatory signaling domains and route of administration

A. NSG mice bearing established s.c. tumor were treated with i.t. injections of 8 × 10⁶ CAR+ T cells on day 0 and 5 and imaged every two weeks. Tumor growth was assessed by caliper measurement (V = 1/2(length × width²). B. SKOV3 fLuc+ bioluminescence signal was decreased in MOv19-BBζ CAR treated mice compared with the MOv19-ζ and the control treatment groups 2 weeks and 4 weeks after last T cell dose. C. SKOV3 fLuc-bearing NSG mice were treated with 8 × 10⁶ MOv19-BBζ T cells via i.t., i.p., or i.v. routes. Tumor growth was assessed by caliper measurement. D. CD137 signaling enhances the survival of human CD4+ and CD8+ T cells in vivo on day 73 (4 weeks following last T cell dose) in the peripheral blood. CD4 and CD8 T cells were quantitated from blood using the TruCount method. Mean cell concentration (cells/ul) ± SD for all evaluable mice in each treatment group is shown.

Figure 3. Tumor eradication by CAR T cells is antigen specific

A. NSG mice with subcutaneous SKOV3 fLuc+ tumor were treated with 8 × 10⁶ T cells (40% transduction efficiency) expressing MOv19-BBζ, anti-CD19-BBζ or gfp via i.t. infusion on days 0 and 5 and measured for tumor volume by calipers every 2-3 days. B. Peripheral blood was collected 3 weeks following last T cell infusion and quantified for the absolute number of human CD4+ and CD8+ T cells/ul of blood. Mean cell count ± SD is shown. C. FRα- and CD19-specific CAR expression on human CD3+ T cells from peripheral blood of treated mice measured by flow cytometry using goat anti-mouse IgG F(ab’)₂. Mean CAR+ expression frequency ± SD per group is shown. D. Absolute CAR+ T-cell count was calculated as number of human CD3+ T cells/ul of blood times percent CAR+. Mean count ± SD was determined.

Figure 4. CAR T cell localization to tumor in vivo is antigen-specific

NSG mice with subcutaneous SKOV3 fLuc+ tumors were treated with intravenous injections of 8 × 10⁶ T cells expressing MOv19-BBζ (upper), anti-CD19-BBζ (middle) or gfp (lower) on day 0 and 5. SKOV3 tumors grown for ~40 additional days were collected from euthanized mice and stained for human CD3 expression (brown). Representative sections are shown at 100 × magnifications.

Figure 5. Mov19-BBζ T cells inhibit tumor growth and ascites formation in SKOV3 murine model of peritoneal carcinomatosis

A. NSG mice received intraperitoneal injection of 5 × 10⁶ SKOV3 fLuc+ tumor cells and were randomized into four groups before beginning therapy with 9×10⁶ T cells expressing MOv19-BBζ or anti-CD19-BBζ via i.p. or i.v. infusion on day 30 and 35 after tumor inoculation. B. Representative NSG mice treated with MOv19-BBζ T cells (left) via i.v. (upper) or i.p (lower) infusion; Mice treated with anti-CD19-BBζ T cells (right) developed ascites as evidenced by a distended abdomen (middle). Postmortem visualization of the peritoneum shows nodular tumor masses (arrows; far right). C. Kaplan-Meier survival curve of tumor-bearing NSG mice treated with either MOv19-BBζ or anti-CD19-BBζ T cells via i.v. or i.p. injection.

Figure 6. Adoptive transfer of FRα-specific T cells induces regression of ovarian cancer lung metastasis

A. NSG mice with 3 day established SKOV3 fLuc+ tumor in the lungs received tail-vein injections of 6 × 10⁶ T cells expressing either MOv19-BBζ or anti-CD19-BBζ on day 3 and day 8 and were monitored by bioluminescence imaging. B. Photon emission from fLuc+ tumor cells was quantified and the mean ± SD bioluminescence signal determined.