A multivalent biparatopic EGFR-targeting nanobody drug conjugate displays potent anticancer activity in solid tumor models

The epidermal growth factor receptor (EGFR), one member of the ErbB family of receptor tyrosine kinases, has been implicated in various epithelial malignancies. As a clinically validated target, both small-molecule tyrosine kinase inhibitors and antibody-based drugs have been approved by regulatory agencies. Anti-EGFR antibody-drug conjugates (ADCs) could kill target tumor cells irrespective of EGFR signaling. However, cancer patients treated with EGFR-targeted antibody therapy eventually develop acquired epitope substitutions such as S492R, G465R, G465E, and I491M, which abolish cetuximab or panitumumab binding and further gives rise to resistance to anti-EGFR ADC therapy. 1 Our team has constructed a tetravalent biparatopic anti-EGFR nanobody-drug, consisting of two tandemly fused anti-EGFR nanobodies (7D12 and 9G8) targeting two distinct non-overlapping epitopes (9G8-7D12-Fc, abbreviated as 97m). Coupling anti-mitotic agent monomethyl auristatin E (MMAE) to the conservative engineered surface cysteine S7C on 7D12 part of 97m resulting its conjugate, abbreviated as S7 ADC (Fig. 1a and Supplementary Fig. S1a–c ). The tetravalent biparatopic ADC demonstrated high conjugation efficiency, small binding interfaces to overcome cetuximab-resistant mutations, enhanced complement-dependent cytotoxicity (CDC), and highly potent antitumor activity. The introduction of E430G in the Fc domain would further boost the complement-mediated immune response of S7 ADC, synergizing with the cytotoxicity of drug payload during cancer treatment. Fig. 1 Multivalent anti-EGFR nanobody drug conjugate. a Schematic diagram of 97m and Hydrophobic interaction chromatography (HIC) analysis of S7 ADC. b Flow cytometry analysis of concentration-dependent binding of cetuximab, 97m and S7 ADC with EGFR-positive A431 cells. c Binding ability of biparatopic nanobody 97m and cetuximab with EGFR-S492R NIH-3T3 cells. d The inhibition effect of 97m and cetuximab on A431 tumor cell proliferation. e CDC activity of biparatopic nanobody and its conjugates on EGFR-wt, EGFR-G465R, and EGFR-S492R NIH-3T3 cells. f Internalization of cetuximab, 97m and S7 ADC in A431 cells. g Quantification of internalized and degraded EGFR through flow cytometry. h Intracellular trafficking and lysosomal localization of cetuximab, 97m and S7 ADC in A431 cells at 37 °C for 1.5 h antibodies (green), lysosomes (red), nucleus (blue). i In vitro cytotoxicity of S7 ADC and S7/E430G ADC on A431, MDA-MB-468, and BxPC-3 tumor cells. j In vivo antitumor activities of multi-dose 97m and S7 ADC in A431 xenograft models. k In vivo antitumor activities of single-dose 97m and S7 ADC in A431 xenograft models. l CDC activity of 97m and its conjugates on MDA-MB-468 and BxPC-3 cells. m Flow cytometry analysis of C1q deposition on A431 cells in the presence of S7 ADC, S7/E430G ADC, or cetuximab. n In vivo antitumor activities of single-dose S7 ADC and S7/E430G ADC in BxPC-3 xenograft models. o In vivo antitumor activities of single-dose S7 ADC and S7/E430G ADC in MDA-MB-468 xenograft models Full size image