Dual- and triple-targeting of the HER-family members using combinations of mono- and bispecific antibodies

Abstract

Epidermal growth factor receptor (EGFR)-targeted cancer treatments with antibodies like Cetuximab are successfully used in the clinic for about 20 years. However, intrinsic, as well as newly developed resistance mechanisms to EGFR-targeted therapies, are the main reason for their failure. Activation of human epidermal growth factor receptor 3 (HER3)-signaling upon EGFR-targeted therapies is frequently observed and has motivated the development of combination therapies that simultaneously block EGFR and HER3. In this study, bispecific and bivalent, or tetravalent, respectively, single-chain diabody (scDb) and scDb-Fc molecules were developed comprising the antigen-binding sites of a humanized version of Cetuximab (hu225) as well as a recently developed anti-HER3 antibody (3-43). In total, eight molecules (two scDb and six scDb-Fc) with varying linkers were engineered. The scDb hu225x3 43 Fc showed the most favorable properties regarding production yield, purity, homogeneity and linker setup. Binding of the scDb-Fc to recombinant receptors, as well as to HER-family receptor expressing cell lines revealed retained binding properties, compared to parental antibodies. Furthermore, the scDb hu225x3 43 Fc showed strong and long-lasting inhibition of downstream signaling by EGF, HRG or combination of both ligands. Proliferation studies on head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), and colorectal cancer (CRC) cell lines revealed either similar, or stronger inhibition, compared to parental antibodies as single or combination treatment, which translated into to long-lasting growth suppression in a s.c. xenograft tumor model. Treatment with the bispecific antibody inhibited in vitro HRG-stimulated oncosphere formation of two TNBC cell lines. In an orthotopic MDA-MB-468 tumor model, superior antitumor effects were observed compared to those obtained by the parental antibodies alone or in combination. Furthermore, this was associated with a reduced number of cells with stem-like properties demonstrating that the bispecific antibody not only efficiently blocks TNBC proliferation but also the survival and expansion of the cancer stem cell population. The high degree of plasticity and compensatory signaling within the HER-family not only leads to compensatory crosstalk by HER3 but also HER2 giving the rational to combine the EGFR- and HER3-targeting scDb-Fc with a HER2-targeting antibody like Trastuzumab. The triple-targeting approach with the scDb-Fc and Trastuzumab was superior in inhibition of HRG-stimulated proliferation of the CRC cell line LIM1215 compared to the combination of IgG hu225, Trastuzumab and IgG 3 43. This was also observed in primary and secondary CRC oncosphere formation assays. Finally, in CRC patient derived organoids (PDOs) grown in HRG-supplemented medium the triple-targeting of EGFR, HER2 and HER3 provided broader efficacy than dual- or mono-targeting of receptors of the HER family. In contrast to Afatinib (anti-EGFR, -HER2, -HER4), the triple-targeted antibody approach showed efficient inhibition in all tested PDOs. Thus, the bispecific scDb-Fc alone or in combination with Trastuzumab represents a superior strategy to deal with primary and acquired resistances compared to targeting a single receptor with different antibodies or any combination of antibodies targeting two receptors of the HER-family.