Footnotes:

(a) names of the anti-amyloid beta antibodies. (b) Source of the antibodies used in this study, either commercial or academic. (c) Clonality of the antibodies: “mono” indicates monoclonal, while “poly” stands for polyclonal antibodies. (d) Species of origin for the IgGs: mouse, rat, human, or rabbit. (e) Immunogens, as reported in the literature, that were used to generate the corresponding antibodies. (f) If available, the anticipated PTM specificity is indicated: “p” represents phosphoserine (at positions Ser8 or Ser26), and “pE3” refers to an N-terminal pyroglutamate (at position 3). (g) epitope identified in this study on microarrays.

Microarray workflow and Aβ-Ab epitope pre-screening.

(a) Schematic of the microarray workflow. The library comprised the amyloid precursor protein (APP) residues 649-722 (APP770 numbering), corresponding to Aβ residues -23 to 51, as a library of 15-mer peptides overlapping by 14AAs, copied in dozens, and probed for Aβ-Abs binding followed by analysis in MARTin. (b). The epitope screening binding data are summarized as a heatmap. The antibody-binding peptides were presented in a semi-quantitative manner. Blue and red shades indicate the relative binding strength of the related arrayed peptides to the left and cognate Aβ-Ab on top. The blue – red scale is visualizing the normalized binding intensity for each antibody individually. The applied antibody concentrations ranged between 0.5-1 µg/mL. Note: Among the 20 tested antibodies, 1E4E11, 5H11C10, 2-48, Aβ-pE3, and Donanemabbs have known PTM-selectivity, which could not be fully addressed with the initial microarray consisting of unmodified Aβ fragments only.

Titration series of N-terminal specific Anti-Aβs

(a) Titration of monoclonal Aβ-Ab 3D6, 82E1 and 7H3D6 at concentrations ranging from 2 to 0.00041 µg/mL to study the selectivity for free-Asp(1) over Asp(1) within an N-terminal elongated peptide. The signals were normalized internally and presented as a heat map with blue-to-red shades ranging from 0 to 1 (b) monoclonal Aβ-Ab 11H3D6 and 80C2 exhibited high selectivity towards free-Asp(1) across the chosen concentration range of 2 µg/mL down to 0.0041 µg/mL.

Deep Mutational Scans Define Aβ-Ab Core Sequences

(a) Schematic summary of the three deep positional scans in the N-terminal region. Seven reported APP mutations are indicated as yellow boxes. From each 18-mer epitope, positional substitutions were generated (342 variants for each epitope) and tested semi-quantitatively in microarray format. The wild-type sequence was sequentially scanned from N to C terminal by turning each position into every proteinogenic amino acid. Consequently, the contribution of each residue is depicted in blue-white-red shades, with white corresponding to no variation from wt [1] and blue-to-red representing loss or gain of binding strength. (b) Fingerprint analysis for the first peptide group covering the sequence KTEEISEVKMDAEFRHDS. The Aβ-Abs tested here are 101-1-1 and 14-2-4 (c) Fingerprint analysis for the second peptide group covering the sequence VKMDAEFRHDSGYEVHHQ. The Aβ-Abs tested here are IC16 and 1E8 (d) Fingerprint analysis for the third peptide group covering the sequence DAEFRHDSGYEVHHQKLV. The Aβ-Abs tested here are Aducanumab, Donanemab and Lecanemab biosimilars. In addition, commercially available Aβ-Abs such as 3D6, 82E1, 11H3, 80C2, 7H3D6 and 1E4E11 were included. Note: The antibodies, 1E4E11 and Donanemabbs have known reported PTM-preference which will be addressed in the next paragraphs.

N-terminally elongated Deep Mutational Scans of Aducanumabbs and 6E10 with core motifs summary

(a) Fingerprint analysis for the first peptide group covering the sequence KTEEISEVKMDAEFRHDS. The Aβ-Ab tested here is Aducanumabbs (b) Fingerprint analysis for the second peptide group covering the sequence VKMDAEFRHDSGYEVHHQ. The Aβ-Ab tested here is Aducanumabbs (c) Fingerprint analysis of 6E10 on the sequence KTEEISEVKMDAEFRHDS. (d) Summary of the antibody core motifs, “X” within the sequence stands for non-conserved residue. When an amino acid variation led to a decrease below 50% wt it was marked as dominant negative. If more than 10 amino acid substitutions were dominant negative, the entire position was designated as part of the core motif (represented in single-letter coding). (e) Aβ-Ab species preferences. In red, amino acid positions which are different in rat and mouse are highlighted. These specific positions were probed. Antibodies showing altered binding strength after replacing R5 or Y10 in the human Aβ sequence by G5 or F10 (rat/mouse Aβ sequence) are highlighted in blue. Human: Homo Sapiens; Dog: Canis Lupus Familiaris; Pig: Sus Scrofa; Rabbit: Oryctolagus Cuniculus; Sheep: Ovis Aries; Rat: Rattus Norvegicus; Mouse: Mus Musculus.

Aβ1-15 carrying the English mutation (H6R) shows reduced affinity for Aducanumabbs

(a) Dot Blot from the purified 6xHis peptides DAEFRHDSGYEVHHQ and DAEFRRDSGYEVHHQ was printed using Slide Spotter robot. IgG signals were detected using Goat anti-human DL650, in square brackets the µg/mL of Aducanumabbs (b) The Biolayer interferometry responses of Aducanumabbs (10nM) over the NTA-loaded DAEFRHDSGYEVHHQ and DAEFRRDSGYEVHHQ peptides are shown.

Multiplex Analysis of Aβ-Abs with preference for post-transcriptionally Modified Abeta variants

The signals were normalized internally and presented as a heat map ranging from 0 to 1. Blue and red shades indicate the binding strength over the related arrayed post-transcriptionally modified peptide shown on top and cognate anti-Aβ antibody to the left. The antibodies were tested under optimal dilutions: 6E10, IC16, pEAB, 1E411, 101-1-1, 5H11C10, 2-48 at 0.25µg/mL; Lecanemabbs, Donanemabbs at 0,0012µg/mL; Aducanumabbs at 0.02µg/mL; 3D6, 11H3, 82E1 at 0.037 µg/mL; 7H3D6 at 0.012µg/mL. The dataset is split over different sections (a-f) to allow proper visualization. Each antibody was screened in parallel against all modifications. (a) The control peptide Aβ(-1-14) was used alongside Asp(1) isomers and stereoisomers. (b) The region overlapping with unmodified Serine 8. (c) the region overlapping with modified phospho Serine 8 (represented as ’2’), (d) the control unmodified 3-17 was used alongside pE3. (e) The region overlapping with unmodified Serine 26 (f) region overlapping with modified phospho Serine 26 (represented as ’2’).

IP-MS signature of different biosimilar Aβ-Abs.

A mixture of synthetic Aβ peptides was immunoprecipitated with the following Aβ-Abs (from top to bottom): 4G8 (pan-antibody), Donanemabbs (Don-bs), Lecanemabbs (Lec-bs), Aducanumabbs (Adu-bs), and 1E4E11 (phospho-selective control). Mass spectra were generated by MALDI-TOF-MS in reflector mode from TOPAC matrix and are representative for two independent experiments. We noted that the signal pattern slightly varied between experiments, which we mainly attributed to some immunoprecipitation of co-aggregated Aβ species, an effect that has to be considered under the experimental conditions applied.

Antibody 1E4E11 shows high preference for phosphorylated pSer8 Aβ1-15 in µSPOT and BLI assays.

(a) presents the titration of monoclonal Aβ-Ab 1E4E11 at concentrations ranging from 0.25 to 0.001 µg/mL to achieve high selectivity for Aβ1-15 phosphorylated at Ser(8). The modified pSer is represented as ’2’. The signals were globally normalized against DAEFRHDpSGYEVHHQ and presented as a heat map with blue-to-red shades ranging from 0 to 1. (b) The Biolayer interferometry dose-response curve of mAb 1E4E11 binding to the NTA-loaded DAEFRHDSGYEVHHQ-6xHis-amide peptide is shown. (c) The Biolayer interferometry dose-response curve of mAb 1E4E11 binding to the NTA-loaded DAEFRHDpSGYEVHHQ-6xHis-amide peptide is shown. (d) BLI steady-state analysis revealed KDs of 4.1nM and 1.3µM for Aβ1-15 with phosphorylated and unphosphorylated Ser (8), respectively (1:1 model).

Phosphorylation aids Aducanumabbs binding to synthetic monomeric Aβ1-15.

(a) shows the titration of Aducanumabbs at different concentrations to achieve high selectivity for phosphorylated pSer8-Aβ1-15 over unphosphorylated Aβ1-15. The signals were internally normalized and fitted as a sigmoidal curve (Boltzmann). The phospho signal for pSer8-Aβ1-15 is shown in red (r2 0.98) and the signal for unphosphorylated Aβ1-15 is shown in blue (r2 0.96). (b) BLI steady-state analysis revealed KDs of 1.4nM and 8.7nM for phosphorylated and unphosphorylated Aβ1-15, respectively (1:1 model) (c) The Biolayer interferometry dose-response curve of Aducanumab binding to the NTA-loaded DAEFRHDSGYEVHHQ-6xHis-amide peptide is shown. (d) The Biolayer interferometry dose-response curve of Aducanumab binding to the NTA-loaded DAEFRHDpSGYEVHHQ-6xHis-amide peptide is shown.

MD simulation of Aducanumab with pSer(8)-Aβ2-8

(a) sticks and cartoon depiction of the MD simulations between Aducanumab and AEFRHDS (b) electrostatic density for the non phosphorylated sequence interaction (c-d) RMSD analysis of the backbone atoms of the unphosphorylated candidate (average conformation over 600ns) (e) sticks and cartoon depiction of the MD simulations between Aducanumab and AEFRHDpS (f) electrostatic density for the pSer8 sequence interaction (g-h) RMSD analysis of the backbone atoms of the PTM candidate (average conformation over 600ns)

pSer8 but not Ser8-Aβ recapitulate the X-Ray resolved binding mode.

Comparison of MD simulations with the structural data. The MD simulation was conducted with the resolved epitope peptide EFRHDS, but no additional ions predict a largely altered binding mode characterized by over 10 Å movements of R317 and R318. In contrast, the crystal structure (PDB 6CO3) harbors a sulfate ion that neutralizes the positive charge introduced by R317 and R318. Strikingly the MD of the pSer(8) modified peptide recapitulates the resolved X-ray binding mode to monomeric Aβ. Aducanumab targets aggregated Aβ, while still engaging with low micromolar affinity a monomeric Aβ.

Aducanumabbs shows strong preference for highly aggregated fibrillar Aβ1-42.

(A) HFIP-treated Aβ1-42 monomer (m), oligomer (o) and fibril preparations (f) (1.44 µg each) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Aducanumabbs (5 min exposure, F 0.84, image display: High: 65535; Low: 0; Gamma: 0.85). Following image recording the blot membrane was reprobed without prior stripping (no removal of primary and secondary antibodies) with mAb4G8 (1 min exposure, image display: High: 54971; Low: 0; Gamma: 0.99). That way the 4G8 signals were essentially added on top of the initial Aducanumabbs signals and background artifacts. The positions of pre-stained protein marker bands on the blot membrane are shown on the left-hand side. (B) Parallel sections from an AD patient were stained with Aducanumabbs and Aducanumabbs that had been pre-adsorbed with either Aβ1-42 monomers (Aβ1-42m) or Aβ1-42 fibrils (Aβ1-42f). Scale bar: 200 µm.

Lecanemabbs recognizes an epitope located in the N-terminal region.

(A) shows an example of CIEF-immunoassay results for Lecanemabbs displayed as lane view (Western blot simulation). The indicated Aβ peptides were separated by isoelectric focusing in microcapillaries on a Peggy Sue device, immobilized photochemically to the inner capillary wall and subjected to immunological detection. Under the tested conditions, Lecanemabbs detected Aβ1-40 (pI 5.31), Aβ2-40 (pI 5.98), Aβ3-40 (pI 5.97), Aβ-3-40 (pI 6.04), Aβ1-42 (pI approx. 5.3) and pSer8Aβ1-40 (amide) (pI 5.13-5.14). (B) Synthetic Aβ2-40 (361 ng), and monomerized (m), oligomeric (o) and fibrillar (f) preparations of Aβ1-42 (361 ng of each) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Lecanemabbs (0.5 µg/mL). Exposure: 2 min 7.5 sec; F 0.84; image display: high: 47545; low: 0; gamma: 1.0. After blot development, the blot membrane was reprobed with mAb 4G8 without prior stripping. That way the 4G8 signals were essentially added on top of the initial Lecanemabbs signals and background artifacts. Exposure: 1 min F 0.84; image display: high: 46277; low: 0; gamma: 1.0. (C) The indicated Aβ peptides (50 ng of each) were separated by Bicine Tris (peptide) SDS-PAGE, blotted on PVDF, and probed with Lecanemabbs. Exposure: 5 min F 0.84, image display: high: 65535; low: 0; gamma: 0.85. (D) To confirm loading and successful blotting of all Aβ peptides, the blot membrane was re-probed with mAb 4G8 without prior stripping (see above). Exposure: 5 min F 0.84; image display: high: 65535; low: 0; gamma: 1.0. (E) Parallel sections from an AD patient were stained with Lecanemabbs and Lecanemabbs that had been pre-adsorbed with either Aβ1-42 monomers (Aβ1-42m), Aβ1-42 oligomers (Aβ1-42o) or Aβ1-42 fibrils (Aβ1-42f). Scale bar: 200 µm.

Donanemabbs shows high preference for pyroglutamate bearing pEAβ3-40.

The upper panel shows lane views (Western blots simulation) of a CIEF-immunoassay for assessing the detection of different Aβ variants by (A) mAb 4G8 (positive control antibody) and (B) Donanemabbs. The indicated Aβ variants were separated by isoelectric focusing in microcapillaries on a Peggy-Sue device, immobilized photochemically to the inner capillary wall and subjected to immunological detection by (A) mAb 4G8 and (B) Donanemabbs. (C) Synthetic Aβ peptides (50 ng per lane) were separated by Bicine Tris (peptide) SDS-PAGE, blotted on PVDF and probed with Donanemabbs (0.5 µg/mL) (upper image). Exposure: 5 min F 0.84; image display: high: 59406; low: 0; Gamma: 0.65. To confirm that all Aβ variants were loaded and blotted, the blot membrane was reprobed with mAb 4G8 without prior stripping. That way, the 4G8 signals were essentially added on top of the initial Donanemabbs signals and background artifacts (lower image). Exposure: 5 min F 0.84; image display: high: 65535; low: 0; gamma: 0.77. (D) AβpE3-40 and Aβ1-42 monomer (m), oligomer (o) and fibril preparations (f) were separated by 4-12% Bistris SDS-PAGE, blotted onto nitrocellulose and probed with Donanemabbs (image display: High: 64091; Low: 0; Gamma: 0.75), followed by reprobing without prior stripping with mAb4G8 (image display: High: 46984; Low: 0; Gamma: 1.0). The positions of prestained protein marker bands on the blot membrane are shown on the left-hand side. (E) A pre-adsorption experiment employing synthetic Aβ peptides in human AD brain tissue showed that the immunoreactivity of Donanemabbs was not suppressed by pre-adsorption of the antibody with excess amounts of synthetic Aβ1-40 and Aβ3-40 peptides. In contrast, a pre-incubation with AβpE3-40 effectively attenuated the antibody signal in extracellular Aβ deposits as well as in the vasculature.