Zylberman, V. | bioRxiv Science

bioRxiv2025

Development of novel high-affinity nanobodies against EGFR for cancer therapy

The epidermal growth factor receptor (EGFR) is a member of a family of transmembrane tyrosine kinase receptors that plays a pivotal role in regulating diverse cellular processes such as cell proliferation, survival, migration, and differentiation. Aberrant activation of EGFR signaling has been implicated in various pathological conditions, particularly cancer, making it an attractive target for therapeutic intervention. While several anti-EGFR monoclonal antibodies have been developed and demonstrated their clinical value for the treatment of various solid tumors, smaller antibody fragments such as nanobodies (Nb) offer distinct advantages over conventional antibodies, including reduced immunogenicity and enhanced tumor penetration. In this paper, we report the isolation and characterization of two novel high-affinity Nb targeting EGFR. These Nb were identified and characterized using ELISA, flow cytometry, microscopy, and SPR. Furthermore, these Nb and bivalent Nb engineered from them were tested for their effects on cancer cell proliferation. We demonstrate that the novel Nb exhibit high affinity and potent anti-tumor activity in vitro in their bivalent form, positioning them as promising candidates for cancer treatment.

molecular biology↗

bioRxiv2024

Cryo-EM structures of engineered Shiga toxin-based immunogens capable of eliciting neutralizing antibodies with therapeutic potential against Hemolytic Uremic Syndrome

Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is a serious disease that causes renal failure predominantly in children. Despite its significant impact, there are currently no licensed vaccines or effective therapies available. The B subunits of Shiga toxins 1 and 2 (Stx1B and Stx2B) are suitable targets for developing neutralizing antibodies, but their pentameric assembly is unstable when isolated from the whole toxin. Taking advantage of the oligomeric symmetry shared between Stx1B and Stx2B with the lumazine synthase from Brucella spp. (BLS), we have previously engineered the chimeric toxoids BLS-Stx1B and BLS-Stx2B as immunogens to generate therapeutic equine polyclonal antibodies. The resulting product (INM004) has successfully passed phases 1 and 2 clinical trials, and a phase 3 has been launched in Argentina and seven European countries. In this work, we present the cryo-EM structures of BLS-Stx1B and BLS-Stx2B, which confirm that these engineered immunogens effectively stabilize the StxB pentamers. Moreover, our results reveal that both chimeric constructs present high flexibility at their extremes, corresponding to motions of the StxBs with respect to the BLS core. Additionally, we present structural evidence of the interaction between the chimeras and polyclonal Fab (pFab) fragments derived from INM004, demonstrating that the elicited neutralizing antibodies block most of the interaction surface of the toxins with their cellular receptors. These findings further validate this promising antibody-based therapy for mitigating STEC-HUS and demonstrate that the BLS-Stx1B and BLS-Stx2B chimeras are potential candidates for developing a human vaccine.

immunology↗