Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression

Abstract

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40(+) tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40(+) tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19(+) systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy.

Figure 1

Constitutive expression of CD40L by human T cells. (a) Schematic of retroviral construct encoding human CD40L vector; LTR, long terminal repeat; SD, SA, splice donor and acceptor; Ψ, packaging element. (b) Flow cytometry of CD4⁺ and CD8⁺ CD40L-modified T cells following retroviral gene transfer; x-axis, APC-conjugated antihuman CD40L (CD154). Results shown are representative of at least three independent experiments. (c) Enhanced proliferation of CD40L-modified T cells compared to mock-transduced T cells. (d) Enhanced secretion of soluble CD40L (sCD40L), IFN-γ, and GM-CSF of CD40L-modified T cells compared to mock-transduced T cells. Results for proliferation and cytokine secretion are combined from three independent experiments. (*Denotes statistical significance where P < 0.001 using Mann–Whitney test). GM-CSF, granulocyte–monocyte colony-stimulating factor; IFN-γ, interferon-γ.

Figure 2

Augmented immunogenicity of CD40⁺ tumor cells by CD40L-modified T cells. (a) Flow cytometry showing upregulation of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), HLA molecules (HLA Class I and HLA-DR), and the Fas-death receptor (CD95) on DOHH2 tumor cell line following coculture with CD40L-modified T cells (solid line) compared to culture with mock-transduced T cells from the same donor (gray line). (b) CD40⁻ tumor (NALM6 shown) demonstrating no phenotypic changes following coculture with CD40L-modified T cells. All results are representative of at least three separate experiments. MFI, mean fluorescence intensity.

Figure 3

Augmented immunogenicity of CLL cells by autologous CD40L-modified T cells. (a) Flow cytometry of patient-derived CD40L-modified T cells following retroviral gene transfer with CD40L containing retroviral vector; x-axis, APC-conjugated antihuman CD40L (CD154). (b) Flow cytometry showing upregulation of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), HLA molecules (HLA Class I and HLA-DR), and the Fas-death receptor (CD95) on CLL cells after coculturing with autologous CD40L-modified T cells (solid line) compared to cocultures with mock-transduced T cells from the same donor (gray line). All results are representative of at least three experiments using three separate donors. CLL, chronic lymphocytic leukemia; MFI, mean fluorescence intensity.

Figure 4

Secretion of IL-12 and maturation of monocyte-derived dendritic cells (moDCs) by CD40L-modified T cells. (a) Cytokine analysis of culture media for cocultures (24 hours) between moDCs and CD40L-modified T cells from three separate donors demonstrating elevated IL-12p70 secretion. (b) Flow cytometry of moDCs demonstrating maturation following coculture with CD40L-modified T cells (solid line). All results are representative of at least three separate experiments. IL-12, interleukin-12.

Figure 5

Efficient transduction of human T cells with a CAR/CD40L vector demonstrates enhanced cytotoxicity. (a) Schematic of retroviral construct containing 1928z-IRES-CD40L and Pz1-IRES-CD40L genes; LTR, long terminal repeat; SD, SA, splice donor and acceptor; Ψ, packaging element; CD8 indicates CD8 leader sequence; scFv, single chain variable fragment; V_H and V_L, variable heavy and light chains; TM, transmembrane domain. (b) FACS analysis of human T cells transduced to express 19-28z/CD40L vector (prestimulation) with subsequent enhanced expression of CAR/CD40L following coculture on AAPCs (NIH 3T3 fibroblasts expressing CD19 and CD80; 1928z/CD40LT cells shown) used for in vivo experiments. x-axis, PE-conjugated 1928z CAR-specific antibody (19e3); y-axis, APC-conjugated antihuman CD40L (CD154). Results are representative of at least three independent experiments. (c) As determined by standard ⁵¹Cr release assay 19-28z/40L T cells have significant increased ability to lyse DOHH2 tumor cells compared to 19-28z T cells. Results of cytotoxicity assay are combined from three independent experiments and effectors have not undergone prestimulation on AAPCs. (*Denotes statistical significance where P < 0.05 using Mann–Whitney test). AAPC, artificial antigen-presenting cell; FACS, fluorescence-activated cell sorting.

Figure 6

Tumor eradication and long-term survival following 1928z/CD40L T-cell infusion. Survival curve of SCID-Beige mice inoculated with DOHH2 tumor cells by intravenous (i.v.) injection 2 days before a single i.v. dose of CAR-modified T cells. Enhanced long-term survival was demonstrated in mice treated with 1928z/CD40L T cells (n = 22) as compared to a panel of control T cells (1928z group n = 13; Pz1 and Pz1/40L group n = 9). Results are combined from two independent experiments. (*Denotes statistical significance between the 1928z/40L group versus 1928z group where P < 0.005). CAR, chimeric antigen receptor.