Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic payloads with the goal of killing those cancer cells with high receptor levels. Delivery can be accomplished by attaching a toxic payload to either a receptor-binding antibody or a receptor-binding ligand. Generally, the cell-binding domain of the toxin is replaced with a ligand or antibody that dictates a new binding specificity. The advantage of this “immunotoxin” approach lies in the potency of these chimeric molecules for killing cancer cells. However, receptor expression on normal tissue represents a significant obstacle to therapeutic intervention.

Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop. Methods: The 286–303 peptide was coupled chemically to keyhole limpet hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V regions were cloned and sequenced. Receptor binding was characterized using enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines. Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be cytotoxic to MDA-MB-468, A431 and F98_npEGFRvIII expressing cells. Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC₅₀ values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC₅₀ values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p<0.0001) or 40H3-MMAE (p<0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p<0.0001) and 26% with 40H3-MMAE (p<0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs. Citation Format: Sonia Jain, Eric Chun Ho, Kendra A. Porath, Antonella Antignani, David J. FitzGerald, Jann N. Sarkaria. Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 556.