Epitope Sequence and Modification Fingerprints of Anti-Aβ Antibodies

Reviewer #1 (Public review):

The manuscript by Ivan et al aimed to identify epitopes on the Abeta peptide for a large set of anti-Abeta antibodies, including clinically relevant antibodies. The experimental work was well done and required a major experimental effort, including peptide mutational scanning, affinity determinations, molecular dynamics simulations, IP-MS, WB, and IHC. Therefore, it is of clear interest to the field. The first part of the work is mainly based on an assay in which peptides (15-18-mers) based on the human Abeta sequence, including some containing known PTMs, are immobilized, thus preventing aggregation. Although some results are in agreement with previous experimental structural data (e.g. for 3D6), and some responses to disease-associated mutations were different when compared to wild-type sequences (e.g. in the case of Aducanumab) - which may have implications for personalized treatment - I have concerns about the lack of consideration of the contribution of conformation (as in small oligomers and large aggregates) in antibody recognition patterns. The second part of the study used full-length Abeta in monomeric or aggregated forms to further investigate the differential epitope interaction between Aducanumab, donanemab, and lecanemab (Figures 5-7). Interestingly, these results confirmed the expected preference of these antibodies for aggregated Abeta, thus reinforcing my concerns about the conclusions drawn from the results obtained using shorter and immobilized forms of Abeta. Overall, I understand that the work is of interest to the field and should be published without the need for additional experimental data. However, I recommend a thorough revision of the structure of the manuscript in order to make it more focused on the results with the highest impact (second part).

Reviewer #2 (Public review):

This paper investigates binding epitopes of different anti-Abeta antibodies. Background information on the clinical outcome of some of the antibodies in the paper, which might be important for readers to know, is lacking. There are no references to clinical outcomes from antibodies that have been in clinical trials. This paper would be much more complete if the status of the antibodies were included. The binding characteristics of aducanumab, donanemab, and lecanemab should be compared with data from clinical phase 3 studies.

Aducanumab was identified at Neurimmune in Switzerland and licensed to Biogen and Eisai. Aducanumab was retracted from the market due to a very high frequency of the side-effect amyloid-related imaging abnormalities-edema (ARIA-E). Gantenerumab was developed by Roche and had two failed phase 3 studies, mainly due to a high frequency of ARIA-E and low efficacy of Abeta clearance. Lecanemab was identified at Uppsala University, humanized by BioArctic, and licensed to Eisai, who performed the clinical studies. Eisai and Biogen are now marketing lecanemab as Leqembi on the world market. Donanemab was developed by Ely Lilly and is sold in the US as Kisunla.

Limitations:

(1) Conclusions are based on Abeta antigens that may not be the primary targets for some conformational antibodies like aducanumab and lecanemab. There is an absence of binding data for soluble aggregated species.

(2) Quality controls and characterization of different Abeta species are missing. The authors need to verify if monomers remain monomeric in the blocking studies for Figures 5 and 6.

(3) The authors should discuss the limitations of studying synthetic Abeta species and how aggregation might hide or reveal different epitopes.

(4) The authors should elaborate on the differences between synthetic Abeta and patient-derived Abeta. There is a potential for different epitopes to be available.