Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains

Abstract

Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-X(L), and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rgamma(-/-) mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.

Fig. 1.

Generation and cytolytic activity of antimesothelin lentiviral vector-engineered T cells. (A) Schematic representation of the mesothelin-binding chimeric receptors. A binding-control chimeric receptor with a truncated TCRζ domain and a specificity control receptor with an anti-CD19 scFv were also constructed. (B) Expression of the SS1 scFv fusion proteins was examined on human primary CD4 T cells. Transduction efficiencies are indicated with mean fluorescence intensity of the transduced populations in parentheses. (C) Cell surface expression of mesothelin on K562, K562.meso, OvCa61.4, OvCa68.4, and M108 was determined by flow cytometry (Top). Cells were incubated with either the mouse anti-human mesothelin antibody CAK1 (solid black line) or an isotype control (dotted line) followed by staining with a PE-conjugated goat anti-mouse Ig. The cytolytic activity of the chimeric receptors on primary human CD8+ T cells targeting cell lines expressing mesothelin was determined using a 4 h ⁵¹Cr release assay (Bottom). Results are expressed as a mean and SD of triplicate wells from 1 of at least 3 separate experiments.

Fig. 2.

Mesothelin retargeted T cells eradicate large pre-established tumors in vivo: effect of costimulatory signaling domains and route of administration. (A) Human primary M108 tumors were established in the flanks of NOG mice. After 6 weeks, when the tumors reached a volume of ≈500 mm³, the mice were treated with 2 intratumoral injections of 15 × 10⁶ T cells (≈70%–80% chimeric receptor-positive) on days 46 and 53 (arrows). Results are expressed as a mean tumor volume (mm³ ± SEM) with n = 8 for all groups, and are representative of 2 experiments. The plots for BBz, CD28z, and CD28BBz are significantly different from the control treatment groups (P < 0.0001 by a Wald test that all groups gave equal log tumor volume curves). (B) The tumor-growth curves of the individual mice in each group are shown. (C) M108-bearing NOG mice were treated with T lymphocytes expressing the 28BBz chimeric receptors (against mesothelin or CD19) via intratumoral (IT), i.p. (IP), and i.v. (IV) routes and the effect on tumor growth was assessed. After tumors reached a mean volume of ≈500 mm³, 2 injections of 10 × 10⁶ T cells (>90% CIR-positive) on days 43 and 49 (arrows) were performed. The results are expressed as a mean tumor volume (mm³ ± SEM) with n = 8 for the saline and intratumoral injection groups, and n = 7 for the i.v. and i.p. groups.

Fig. 3.

CD28 and 4–1BB signals enhance the persistence of human T lymphocytes after treatment of M108 tumor. (A) Peripheral blood from M108-bearing NOG mice treated with intratumoral injections of SS1 chimeric receptor-transduced T cells was obtained on day 73 and quantified for the presence of CD4 and CD8 T cells by a FACS Trucount assay. Analysis of variance of the CD4 and CD8 data indicates significant differences among the treatment arms (P < 0.0001). Results are expressed as a mean absolute count per μL of peripheral blood ± SD with n = 8 for all groups. (B) Persistence of T cells in vivo was assessed after i.v. (IV), intratumoral (IT), or i.p. (IP) route of administration. Mice from the experiment in Fig. 2C were bled and analyzed for peripheral T cell persistence on day 65 by a FACS Trucount assay. Absolute T-cell count per μL of peripheral blood is shown for individual mice in each group with corresponding tumor volumes directly below at day 65. The overall F test comparing the mean T-cell counts across groups was significant (P < 0.0001). In Tukey-adjusted pairwise comparisons of the means (at P = 0.05), i.v. and intratumoral SS1 administration gave a significantly higher T-cell count than i.p. SS1 administration.