A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

Abstract

Human epithelial growth factor receptor-2 (HER2) plays an oncogenic role in numerous tumors, including breast, gastric, and various other solid tumors. While anti-HER2 therapies are approved for the treatment of HER2-positive tumors, a necessity persists for creating novel HER2-targeted agents to resolve therapeutic resistance. Utilizing a synthetic nanobody library and affinity maturation, our study identified four anti-HER2 nanobodies that exhibited high affinity and specificity. These nanobodies recognized three distinct epitopes of HER2-ECD. Additionally, we constructed VHH-Fc and discovered that they facilitated superior internalization and showed moderate growth inhibition. Compared to the combination of trastuzumab and pertuzumab, the VHH-Fc combos or their combination with trastuzumab demonstrated greater or comparable antitumor activity in both ligand-independent and ligand-driven tumors. Most remarkably, A9B5-Fc, which targeted domain I of HER2-ECD, displayed significantly enhanced trastuzumab-synergistic antitumor efficacy compared to pertuzumab under trastuzumab-resistant conditions. Our findings offer anti-HER2 nanobodies with high affinity and non-overlapping epitope recognition. The novel nanobody-based HER2-targeted antibody, A9B5-Fc, binding to HER2-ECD I, mediates promising receptor internalization. It possesses the potential to serve as a potent synergistic partner with trastuzumab, contributing to overcoming acquired resistance.

Keywords: Fc fusion; HER2; ligand-dependent heterodimerization; nanobody; synergistic efficacy; trastuzumab-resistance.

Figure 1

Screening and characterization of HER2-specific nanobodies. (A) The flowchart summarized the overall process of nanobodies screening from the AUAM synthetic nanobody library (ASyNAL). (B) HER2-specific nanobodies with His-tag were purified from BL21 (DE3) and showed high purity in SDS-PAGE. (C) The cell-surface binding activity of nanobodies was quantified by flow cytometry in SKBR3 tumor cells. (D) Nanobodies showed target specificity and species cross-reactivity. ELISA assay was performed to analyze the binding of nanobodies against human EGFR, human HER2, human HER3, human HER4, mouse Her2, and monkey Her2. In c and d, a non-specific IgG antibody was used as a negative control.

Figure 2

Affinity maturation of HER2-targeted nanobodies by phage display and identification of VHH-Fc. (A) The flowchart summarized the overall process of affinity maturation. (B) The sequence alignments of CDR regions were represented by Weblogo (http://weblogo.berkeley.edu/logo.cgi). All CDR regions of A2H4 and H2C11 were designed to generate random mutations. As for A9H5 and G1H2, their CDR2 region remained unchanged. (C) VHH-Fc fusions were purified from the 293T system and resolved by SDS-PAGE. (D) VHH-Fc derived from HER2-nanobodies with high affinity retained target specificity and species cross-reactivity. VHH-Fc showed a potent binding activity to human HER2 and monkey Her2 recombinant proteins. (E) VHH-Fc exhibited greater saturation activity in tumor cells compared to related parental nanobodies. (F) Confocal images of NCI-N87 cells identified the binding of VHH-Fc to the cell surface. Cells were treated with 50 nM antibodies and were stained by DyLight^® 488 for detection of the HER2-antibody complex. Alexa-Fluor 647-labeled secondary antibody and DAPI were used to visualize lysosomes and nuclei, respectively. Scale bars = 10 μm.

Figure 3

Epitope analysis of HER2-targeted nanobodies. (A) Schematic diagram of the construction of chimeric HER2-ECD proteins with domain substitution. HER2-mD1, HER2-mD2, HER2-mD3, and HER2-mD4 corresponded to the ECD I (T23-R217), ECD II (T218-C342), ECD III (Y344-A510), and ECD IV (C511-T652) of HER2 replaced by that of murine homolog, respectively. All chimeric and wild-type HER2 proteins were expressed in the 293T system. (B) ELISA analysis of the key domains of interactions between HER2 and nanobodies. The EC₅₀ value of nanobodies against different recombinant HER2 proteins was calculated and displayed by a heat map. (C) Competitive ELISA confirmed the binding domains of nanobodies. A2G5 and A9B5 competed with each other to bind HER2. H2F5 partially competed with pertuzumab, and G1E4 competed with trastuzumab.

Figure 4

HER2-targeted VHH-Fc promotes internalization and mediates growth inhibition in SKBR3 and NCI-N87 tumor cells. (A) HER2 internalization induced by VHH-Fc in comparison with trastuzumab and pertuzumab. SKBR3 cells were incubated with antibodies for 0.5 or 4 hours. The flow cytometry was used to determine the mean percent internalization. Data were mean ± SD (n=3) and two-way ANOVA with Tukey’s multiple comparisons test was performed. The p values were provided in Supplementary Table 1 . (B) VHH-Fc mediated tumor growth inhibition in NCI-N87 cells. The percentage of cell viability was measured by CCK-8 assay. (Up) The representative curve means the concentration-dependent inhibition induced by VHH-Fc. (Down) A2G5-Fc, A9B5-Fc, and H2F5-Fc conferred moderate cell proliferation blocking, but G1E4-Fc showed significant agonistic effect. Data were mean ± SD (n=3) and one-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 2 . *p < 0.0332, ***p < 0.0002, ****p < 0.0001.

Figure 5

VHH-Fc exhibits potent synergistic efficacy with antibodies recognizing non-overlapping epitopes irrespective of the presence of ligands. (A) VHH-Fc mixtures mediated the inhibition of ligand-dependent growth in NCI-N87 cells. A heat map was used to represent the inhibitory rate (mean, n=3, two-way ANOVA with Dunnett’s multiple comparisons test). The p values were provided in Supplementary Table 3 . (B) VHH-Fc showed greater synergistic efficacy with trastuzumab than a combination of trastuzumab and pertuzumab. A2G5-Fc and A9B5-Fc, derived from nanobodies binding to HER2 ECD I, both induced a significantly greater combined effect with trastuzumab than pertuzumab. Data were mean ± SD (n=6) and one-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 4 . (C-E) A9B5-Fc mediated superior synergistic efficacy with trastuzumab to block ligand-independent and ligand-driven tumor cell growth. Data were mean ± SD (n=3) and two-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 5 . *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001.

Figure 6

Mechanisms of action of HER2-targeted VHH-Fc. HER2-specific nanobodies were obtained from a well-established synthetic nanobody library (ASyNAL). Affinity maturation based on soft randomization led to a pronounced increase in nanobodies binding to HER2 receptors of tumor cells. These nanobodies could recognize at least three different epitopes, encompassing ECD I, ECD II, and ECD IV. We further constructed the VHH-Fc fusions, which theoretically could prolong the plasma half-life of VHH, improve therapeutic benefit by bivalent binding, and induce Fc-effector functions (ADCC and CDC). The HER2 receptor internalization induced by VHH-Fc was notably observed in HER2-positive tumor cells, indicative of VHH-Fc potentially mediating receptor clustering on the cell surface. A9B5-Fc, targeting ECD I, exhibited potent combined efficacy with trastuzumab in ligand-independent and EGF/HRG-stimulating tumor cell growth. We posited that a combination of A9F5-Fc and trastuzumab effectively disturbed HER2 heterodimerization and homodimerization with EGFR family members, resulting in a blockade of ligand-independent and ligand-driven resistance. In summary, these mechanisms contribute to the growth suppression of trastuzumab-resistant tumor cells by the novel nanobody-based VHH-Fc.