An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells

Abstract

MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours.

Figure 1

Expression cassettes coding for the recombinant anti-MUC1 fusion proteins C595scFv-Fc and C595scFv-Fc-IL2. The expression cassettes were generated as described in Materials and Methods. Lk: leader sequence of the Ig κ chain; C595scFv: anti-MUC1 scFv; Fc: IgG1 CH2CH3. The expression cassettes are driven by the RSV LTR.

Figure 2

Detection of the fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 in the supernatants of transfected 293 T cells. Serial dilutions of culture supernatants from 293 T cells transfected with pRSV-C595scFv-Fc (▪), pRSV-C595scFv-Fc-IL2 (•) and, as control, from mock-transfected cells (*), respectively, were incubated in microtitre plates coated with an anti-human IgG antibody. Bound fusion proteins were detected by a biotin-labelled anti-human IgG (A) or anti-human IL2 (B) antibody.

Figure 3

The fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 are expressed as homodimers. Cell culture supernatants from 293 T cells transfected with pRSV-C595scFv-Fc DNA (lane 1) or with pRSV-C595scFv-Fc-IL2 DNA (lane 2) as well as recombinant human IL2 (1000 U, lane 3) were electrophoretically separated under nonreducing conditions, blotted onto nitrocellulose membrane and probed with an anti-human IgG antibody (A) and an anti-human IL2 antibody (B). The calculated molecular weight of the monomeric form of the fusion protein C595scFv-Fc is 60 kDa, of the C595scFv-Fc-IL2 protein is 75 kDa.

Figure 4

Antigen binding profile of the recombinant fusion proteins C595scFv-Fc and C595scFv-Fc-IL2. Microtitre plates were coated with a set of MUC1-related (glyco-) peptides (10 μg ml⁻¹) corresponding to mono- and oligorepeats and containing the RPAP motif (see Table 1), partially deglycosylated MUC1 antigen (100 μg ml⁻¹), and, as control, BSM (100 μg ml⁻¹), an anti-human IgG antibody (1 μg ml⁻¹) and an anti-mouse IgG antibody (1 μg ml⁻¹), respectively. The coated wells were incubated with cell culture supernatants containing the fusion proteins C595scFv-Fc (A) and C595scFv-Fc-IL2 (B), respectively, and as control with the mAb C595 (C) and a mouse IgG1 control antibody (D) (both 0.25 μg ml⁻¹). Bound fusion proteins and antibodies were detected by a biotin-labelled anti-human IgG mAb (A and B) or a biotin labelled anti-mouse IgG mAb (C and D). The assay was performed in triplicate; data are presented as the mean±s.e.m.

Figure 5

Binding-inhibition assay. Microtitre plates coated with the peptide antigen TR5 (2.5 μg ml⁻¹) were incubated with dilutions of C595scFv-Fc (•), C595scFv-Fc-IL2 (▪), and C595 mAb (▴), respectively, that result in half-maximal binding. The binding to immobilized TR5 antigen was specifically competed by addition of increasing amounts of soluble TR5 antigen. Bound fusion proteins were detected by the biotin-labelled anti-human IgG antibody; bound C595 mAb was detected by the biotinylated anti-mouse IgG antibody. The percentage of inhibition was determined as described in ‘Material and Methods’.

Figure 6

Fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 bind specifically to MUC1 positive tumour cells and TR5 peptide-coated beads. Cell culture supernatants from transfected 293 T cells containing the C595scFv-Fc and C595scFv-Fc-IL2 fusion protein (thick lines), respectively, and, as control, supernatant from 293 T cells transfected with an irrelevant DNA (thin lines), were incubated (A) with T-47D cells (MUC1+/CD25−), 293 T cells (MUC1−/CD25−), L540 cells (MUC1−/CD25+), TR5 peptide-coated beads or, as control, with human serum albumin-coated beads, and (B) with quiescent lymphocytes from the peripheral blood (PBL) or with PBL activated by preincubation with the anti-CD3 mAb OKT3 plus IL2. Bound fusion proteins were detected by an FITC-labelled anti-human IgG antibody. Expression of the IL2 receptor CD25 was monitored by incubation with a FITC-conjugated anti-CD25 mAb (anti-CD25).

Figure 7

Activated lymphocytes proliferate in the presence of soluble C595scFv-Fc-IL2 fusion protein. Lymphocytes from the peripheral blood (2.5 × 10⁵ cells per well) were activated by incubation with the anti-CD3 mAb OKT3 plus IL2 and cultured in the presence of cell culture supernatants containing the fusion proteins C595scFv-Fc-IL2 (•), C595scFv-Fc (▪), and as control IL2 (▴) (2 000 U ml⁻¹), respectively. After 72 h, lymphocyte proliferation was quantified by means of the ‘XTT cell proliferation kit’ (Roche Diagnostics). The assays were performed in triplicate; data are presented as mean±s.e.m.

Figure 8

C595scFv-Fc-IL2 fusion protein bound to MUC1 antigen stimulates proliferation of activated lymphocytes. Micotitre immunoplates were coated with increasing amounts of deglycosylated MUC1 (A), TR5 peptide (B), and asialo-BSM (C), respectively, and subsequently incubated with cell culture supernatants containing the fusion proteins C595scFv-Fc (▪) and C595scFv-Fc-IL2 (•), respectively. After extensive washing, activated lymphocytes (2.5 × 10⁵ per well) were incubated in coated plates for 72 h. In a second set of experiments, beads coated with TR5 peptide antigen (D) and, as control, coated with human serum albumin (E) were incubated with culture supernatant containing the fusion protein C595scFv-Fc or C595scFv-Fc-IL2, respectively, washed extensively and incubated in serial dilutions (15 × 10⁵–0.6 × 10⁵ beads per well) with activated lymphocytes (2.5 × 10⁵ per well). After 72 h, lymphocyte proliferation was quantified by means of the ‘XTT cell proliferation kit’. The assays were performed in triplicate; data are presented as the mean±s.e.m.

Figure 9

The fusion protein C595scFv-Fc-IL2 activates resting NK cells to tumour cell lysis (A). MCF7 cells (MUC1⁺) (5 × 10⁴ cells per well) were cocultured with resting NK cells (1x10⁵ cells per well) in the presence of C595scFv-Fc-IL2 fusion protein or IL2 (400 U ml⁻¹). As control, MCF7 cells were cocultured with NK cells in cell culture medium without additives. (B) MCF7 and T-47D tumour cells (MUC1⁺) were preincubated with cell culture supernatants containing C595scFv-Fc, C595scFv-Fc-IL2, or IL2 (400 U ml⁻¹), respectively. After four rounds of washings, cells were seeded into round-bottom microtiter plates (5 × 10⁴ cells per well) and cocultured with resting NK cells (1 × 10⁵ cells per well). The viability of MCF7 and T-47D cells was determined by an XTT-based viability assay as described in Material and Methods. The assays were performed in triplicate; data are presented as the mean±s.e.m. Statistical significance was determined utilising Student's t-test.