Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Abstract

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K = 220 pM) and HER3-expressing tumor cells with EC values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.

Targeting scFv-Fc-scTRAIL fusion proteins to tumor cells

“…In these formats, a single-chain version of TRAIL that consists of amino acids 118 to 281 with a single glycine residue as linker to connect the protomers [ 21 ] was fused to the C-terminus of a human γ1 Fc region, while a TAA-targeting single-chain variable fragment was optionally located N-terminally (Figure 1A, 1B ). Five different antibody moieties directed against four distinct tumor-associated antigens were employed, including the EGFR-targeting antibody hu225 derived from antibody C225 used in cetuximab [ 29 ] and humanized by CDR grafting [ 30 ], the trastuzumab-derived 4D5 directed against HER2 [ 31 ], the HER3-targeting antibodies 3M6 (a modified version of MM-121, Ab#6 [ 32 ] with a mutation of C89 of the V L according to the Kabat numbering scheme to serine) and 3-43 [ 33 ], as well as the humanized version 323/A3hu3 [ 34 ] of the anti-EpCAM antibody 323/A3 [ 35 , 36 ]. All molecules further comprised a FLAG-tag at the N-terminus allowing purification of the proteins from the supernatant of stably transfected HEK293T cells by FLAG affinity chromatography.…”

“…Modular vectors for sequential insertion of the Fc region, scTRAIL, and the antibody moiety were created based on pSecTagAL1, a modified version of pSecTagA (Invitrogen, Thermo Fisher Scientific, V90020) comprising an AgeI restriction site at the 3’-end of the Igκ chain leader sequence. In order to eliminate potential protease cleavage sites, K447 of the human IgG1 Fc (EU numbering scheme) was mutated to Q. Single-chain variable fragments hu225 [ 30 ], 4D5 [ 31 ], 3M6 (derived from MM-121, Ab #6 [ 32 ] comprising a cysteine to serine mutation at position 89 (Kabat numbering scheme) in the variable domain of the light chain), 3–43 [ 33 ], and 323/A3hu3 [ 34 ] directed against EGFR, HER2, HER3, and EpCAM were used. A single-chain version of TRAIL (scTRAIL) containing amino acid residues 118–281 of human TRAIL and a linker composed of a single glycine residue was used to create the various fusion proteins [ 21 ].…”

Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules

“…The variable domains of a humanized version of anti-EGFR antibody cetuximab (hu225) 28 and anti-HER3 antibody IgG 3-43 29 were used to generate tetravalent bispecific Db-Ig molecules. In total, four different tetravalent bispecific molecules were produced using different dimerization domains (DD): C H 1/C L , EHD2, MHD2, and hetEHD2 (Figure 2(a)).…”

“…Similar to the ELISA binding studies, concentration-dependent binding was detected for all analyzed antibodies. As FaDu cells express very high amount of EGFR (~140,000 receptors per cell) and low amounts of HER3 (~3,000 receptors per cell) on their surface, 29 cell binding was obviously dominated by EGFR, which resulted in binding of IgG hu225 and of all bispecific antibodies with similar EC 50 values in the range between 129 and 238 pM ( Table 1).…”

Designing Human Antibodies by Phage Display

“…These libraries were derived from 44 donors and contain around 5 billion independent clones. From these libraries, more than 1,000 antibodies for international proteome binder consortia [43][44][45], diagnostic antibodies [46,47] as well as candidates for therapeutic applications [48][49][50] were selected. The next generation of human naïve antibody gene libraries (HAL9/10) was derived from 98 donors with a broad spectrum of ethnic origins and contains a diversity of more than 10 billion independent clones [32].…”