Targeting of T lymphocytes to melanoma cells through chimeric anti-GD3 immunoglobulin T-cell receptors

Abstract

Immunoglobulin T-cell receptors (IgTCRs) combine the specificity of antibodies with the potency of cellular killing by grafting antibody recognition domains onto TCR signaling chains. IgTCR-modified T cells are thus redirected to kill tumor cells based on their expression of intact antigen on cell surfaces, bypassing the normal mechanism of activation through TCR-peptide-major histocompatibility complex (MHC) recognition. Melanoma is one of the most immunoresponsive of human cancers and has served as a prototype for the development of a number of immunotherapies. The target antigen for this study is the ganglioside GD3, which is highly expressed on metastatic melanoma with only minor immunologic cross-reaction with normal tissues. To determine an optimal configuration for therapy, four combinations of IgTCRs were prepared and studied: sFv-epsilon, sFv-zeta, Fab-epsilon, Fab-zeta. These were expressed on the surface of human T cells by retroviral transduction. IgTCR successfully redirected T-cell effectors in an MHC-unrestricted manner, in this case against a non-T-dependent antigen, with specific binding, activation, and cytotoxicity against GD3+ melanoma cells. Soluble GD3 in concentrations up to 100 microg/ml did not interfere with recognition and binding of membrane-bound antigen. Based on the outcomes of these structural and functional tests, the sFv-zeta construct was selected for clinical development. These results demonstrate key features that emphasize the potential of anti-GD3 IgTCR-modified autologous T cells for melanoma therapies.

Figure 1

Schematic representation of antibody (Ig), TCR complex, and IgTCR structures. The TCR complex is comprised of six proteins. The structure of the αβ receptor chains is analogous to the Fab portion of an antibody with variable (V) and constant (C) domains. TCR chains γ, δ and ε also have immunoglobulin-like domains. sFv-ζ, sFv-ε, Fab-ζ, and Fab-ε are shown within the complete IgTCR complex. Boxes in the cytoplasmic domains represent signaling motifs of all chains except α and β, which lack them.

Figure 2

Surface expression of IgTCR on transiently transduced 293. Cells were assayed by flow cytometry with staining anti-Id antibody against MB3.6 (solid line) or with negative control antibody (dotted line).

Figure 3

Surface expression of MB3.6 IgTCR on stably transduced Jurkat cells. Cells were stained as in Figure 2.

Figure 4

Recognition-mediated adhesion of IgTCR+ T cells. Modified or nonmodified Jurkat T cells were incubated on plates coated with either anti-Id or nonspecific antibody (UPC). Anti-GD₃ IgTCR modified T cells bind immobilized anti-Id tightly, inducing cells to flatten on the plate surface (a). On plates coated with UPC, cells did not adhere, and kept their normal round morphology (b). Similarly, no binding occurred with nonmodified Jurkat cells on anti-Id-coated plates (c).

Figure 5

Cell conjugation of IgTCR+ T cells with GD₃+ melanoma cells. Noncytotoxic Jurkat T cell effectors were used to avoid M21 target cell lysis during the binding assay. After co-incubation of nonadherent T-cell effectors and adherent tumor cell targets on plates, unbound T cells were removed by washing. Nonmodified T cells were nearly completely washed away, showing only the tumor cells left in plate (a). However, IgTCR+ modified T cells were tightly bound to M21 (b). In panel (c), fluorescence staining was employed. A single giant melanoma tumor cell target (orange; apparently in mitosis) is bound by 19 effector T cells (green).

Figure 6

IL2 secretion of IgTCR transduced cells after stimulation with immobilized proteins and melanoma cell lines. Unmodified and sFv-ζ modified Jurkat T cells were coincubated with various stimuli, and the supernatants were harvested and tested for IL2 concentration. Stimulators were: UPC, nonspecific antibody; OKT3, anti-CD3 antibody; anti-Id, anti-idiotypic antibody to MB3.6; M21, GD₃+ melanoma cell line; M24, GD₃- cell line. IL2 levels were standardized to OKT3-stimulated secretion for each group (= 100) to control for variation in cell numbers and cell condition, with a range of 400–1200 IU/ml in different assays. The use of no stimulator or bovine serum albumin (BSA) gave no IL2 secretion (not shown) that was equivalent to UPC. The SE was ±20% in quadruplicate assays.

Figure 7

IgTCR expression on primary T lymphocytes. T lymphocytes were purified from PBMCs, transduced with the sFv-ζ IgTCR encoding retroviral vector, and analyzed for surface expression with anti-Id (IgTCR+) and negative control antibody, as indicated.

Figure 8

Cytotoxicity of IgTCR+ CTLs against GD3+ melanoma cells. GD₃+ M21 tumor targets were seeded at 2x10⁵ cells per well (120 mm²) in six-well tissue culture plates, and then anti-GD3 IgTCR+ CD8+ CTLs were added at different E:T ratios, ranging from 0.2:1 to 2:1. 0:1 indicates growth of tumor cells without added effector CTLs. Similar results were obtained from three independent experiments.