Therapeutic potential of a tumor-specific, MHC-unrestricted T-cell receptor expressed on effector cells of the innate and the adaptive immune system through bone marrow transduction and immune reconstitution

Abstract

T-cell receptor (TCR) with unique major histocompatibility complex (MHC)-unrestricted antigen-binding properties was isolated from a human T-cell clone specific for the tumor antigen MUC1. This TCR binds its epitope on the MUC1 protein without the requirement of processing and presentation. A single-chain Valpha/Vbeta/Cbeta (scTCR) was fused to a CD3 zeta (zeta) chain to allow expression on the surface of cells of the innate (granulocytes, macrophages, natural killer [NK] cells) as well as the adaptive (T and B cells) immune system. To test the ability of the cells of the innate immune system to reject a tumor when provided with a tumor antigen-specific TCR, we reconstituted severe combined immunodeficiency (SCID) mice with bone marrow cells transduced with a retroviral vector encoding this receptor and challenged them with a MUC1-positive human tumor. These mice controlled the growth of the tumor significantly better than the control mice. We performed a similar experiment in immunocompetent mice transgenic for human MUC1. Expression of the TCR on large percentages of cells did not result in infiltration or destruction of tissues expressing MUC1. Reconstituted mice controlled the outgrowth of a MUC1-transfected but not the parental control tumor. scTCR expression appears lifelong, suggesting a successful transduction of the self-renewing stem cells.

Figure 1.

Comparison of the CDR2 and CDR3 regions of the MUC1-specific antibody SM3 and the MA TCR and modeling their interactions with MUC1. (A) Amino acid sequence alignment of SM3 and MA TCR. Numbering corresponds to MA TCR sequence. Residues in bold are hypothesized to be involved in the binding. (B-C) SM3 and (D-E) MA TCR computer-based models of the interactions with the MUC1 epitope. The colors indicate the following: red, CDR1; green, CDR2; blue, CDR3; yellow, MUC1; white, the SM3 light chain and MA TCR α chain; gray, the SM3 heavy chain and MA TCR β chain. Contact residues are shown as stick diagrams with nitrogens in blue and oxygens in red, in close-up comparison of SM3 (C) and MA TCR (E) computer-based models of their interactions with MUC1 and labeled by chain, residue, and number in chain.

Figure 2.

Cells transfected with scTCR recognize MUC1-positive tumors and synthetic MUC1 antigen. (A) Mammalian expression vector encoding MA scTCR gene. The scTCR was cloned into the pEF6 vector. BGH indicates bovine growth hormone; EF-α, elongation factor α. (B) Cell surface expression of the scTCR in RBL cells or (C) BWZ cells stably transfected with the scTCR-pEF6 vector. Cells were stained with anti-TCR βF1 (open histogram) or with isotype control (filled histogram) antibody. (D) Degranulation of RBL cells or RBL-scTCR cells following stimulation with platebound βF1 antibody or MUC1 140-mer peptide. Specific degranulation is presented as percent of maximum degranulation induced by TCR cross-linking with βF1 antibody. (E) IL-2 enzyme-linked immunosorbent assay (ELISA) for BWZ or BWZ-scTCR following stimulation with platebound anti-TCR βF1 antibody, ionomycin plus PMA (I/P), DM6 (MUC1-negative tumor), HPAF, or T3M4 (MUC1-positive tumors). IL-2 in culture supernatant was measured by ELISA, and values were plotted on the y-axis as picograms per milliliter. Cells were stimulated as indicated.

Figure 3.

Transduction of the long-term reconstituting hematopoietic stem cell population (c-Kit⁺ Sca-1⁺ Lin^- Thy1.1^-) with scTCR-EGFP MFG retroviral vector. (A) Schematic diagram of the scTCR-EGFP MFG retroviral vector. (B) BM cells transduced with the scTCR-EGFP MFG retroviral vector were stained on day 7 in culture for hematopoietic stem cell surface markers (c-Kit and Sca-1) and for lineage markers (Lin). Cells that expressed high levels of Sca-1 and c-Kit (C) and that lacked expression of Lin (D), R2, and R3 were gated on and were plotted against EGFP (E). FSC indicates forward scatter. (F) Mock-transduced BM cells. All cells in culture were Thy1.1^- (not shown). Percentages are percentages of EGFP⁺ cells.

Figure 4.

Detection of scTCR-expressing cells at various times after reconstitution with transduced BM cells. Mice were bled at indicated time points, and leukocytes were stained for the appropriate cell surface markers plotted on the y-axis: (A) GR-1 for granulocytes, (B) Mac-3 or F4/80 for monocytes/macrophages, (C) DX5 for NK cells, (D) CD3 for T cells, and (E) B220 for B cells. EGFP expression is plotted on the x-axis. Percentages of EGFP-positive cells in each lineage are indicated.

Figure 5.

SCID mice reconstituted with transduced BM cells can control the growth of the MUC1-positive tumor xenograft. (A) Control (▴) or scTCR-reconstituted (○) mice were injected subcutaneously with 2 × 10⁶ HPAF (MUC1-positive) tumor cells. Tumor size is shown on the y-axis while days after tumor challenge is plotted on the x-axis. P values were calculated by running t test using Microsoft Excel software. Data are presented as mean ± SE. (B) H&E staining of HPAF tumor sections from control mice (left) or from scTCR-reconstituted mice (right). (C) Staining of tumor sections from scTCR-reconstituted mice for myeloperoxidase (neutrophil marker), F4/80 (monocyte/macrophage marker), or granzyme B (NK cell marker). Images were taken under × 20 magnification. Images in lower right squares were taken under × 100 magnification.

Figure 6.

Expression of scTCR on immune cells has no deleterious effects on MUC1-positive normal tissues. (A) C57BL/6 (wild-type) and MUC1 Tg mice were reconstituted with BM cells transduced with the scTCR-EGFP MFG retroviral vector. Untreated mice served as controls. Spleen, lung, and pancreas were harvested 6 weeks after reconstitution and microscopically examined for infiltration with EGFP-positive cells (A) or stained with H&E (B) and examined for tissue destruction.

Figure 7.

scTCR-reconstituted MUC1 Tg mice rejected MUC1-positive tumor challenge. MUC1 Tg mice were reconstituted with BM cells transduced with scTCR (▴) or with control supernatant (▪) and challenged 6 weeks later with MUC1^- tumor (RMA) or with RMA cells transfected with MUC1 (RMA-MUC1). Data are presented as mean ± SE. The number of mice at each time point ranged from 5 to 10.