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 expession 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.

Detection of Circulating Anti-Mucin 1 (MUC1) Antibodies in Breast Tumor Patients by Indirect Enzyme-Linked Immunosorbent Assay Using a Recombinant MUC1 Protein Containing Six Tandem Repeats and Expressed in Escherichia coli

“…Studies have demonstrated that circulating anti-MUC1 antibodies may be used as a favorable prognostic factor for patients with early breast cancer and pancreatic cancer (7,25). In addition, previous studies have shown that the antibodies might contribute to limit tumor outgrowth and dissemination by antibody-dependent cellular cytotoxicity (1,8,28). It is believed that free anti-MUC1 antibodies can bind MUC1 and form MUC1 circulating immune complexes (MUC1-CIC) in blood circulation (3); however, patients with stage IV of breast cancer present low MUC1-CIC, although more common anti-MUC1 antibodies and MUC1 exist in their sera (4,26).…”

Antibody–cytokine fusion proteins: applications in cancer therapy

“…This C595scFv was genetically fused to the amino-terminus of the hinge-Fc region of human IgG1 (scFv-Fc), while IL-2 was fused to the carboxy-terminus of this Fc, generating the mouse/human C595 scFv-Fc-(IL-2) ( Figure 2A (III)). This fusion protein retained the ability to bind MUC-1-positive T-47D (human mammary carcinoma) cells and also retains IL-2 bioactivity as evidenced by the induction of proliferation of activated CD25 + lymphocytes and the activation of NK cells resulting in the destruction of MUC-1-positive tumor cells in vitro [59].…”

“…Fusion of IL-2 to an antibody specific for a TAA has conferred the ability to concentrate this cytokine in the tumor microenvironment, decreasing its systemic side effects. Biodistribution analysis in different murine models has shown that the tumorspecific antibody-cytokine fusion proteins preferentially accumulate in malignant tissues [57][58][59]. Table 1 summarizes the antibody-(IL-2) fusion proteins described in this review.…”

“…The C595 mouse monoclonal antibody that recognizes the peptide RPAP in the sequence of the human MUC-1 protein core was used to generate a scFv that retained binding specificity [59]. This C595scFv was genetically fused to the amino-terminus of the hinge-Fc region of human IgG1 (scFv-Fc), while IL-2 was fused to the carboxy-terminus of this Fc, generating the mouse/human C595 scFv-Fc-(IL-2) ( Figure 2A (III)).…”

Simultaneous targeting of IL2 and IL12 to Hodgkin's lymphoma cells enhances activation of resting NK cells and tumor cell lysis

“…17 The secreted fusion proteins were detected by ELISA as described below and purified from cell culture supernatants by affinity chromatography as described. 18 Binding of the fusion protein to CD30 was determined by ELISA utilizing CD30-Fc fusion protein that was coated (1 mg/ml) onto 96-well microtiter plates (Nunc, Wiesbaden, Germany). Bound fusion proteins were detected by biotinylated anti-human IgG (0.05 mg/ml; Southern Biotechnology, Birmingham, AL), antihuman IL2 (0.5 mg/ml) or anti-mouse IL12 antibodies and visualized by peroxidase-coupled streptavidin (1:10,000) and ABTS 1 (Roche Diagnostics).…”