title: Molecular epitope and affinity determination of protein-protein interactions by a combination of biosensor and mass spectrometric methods
Creators: name: Wiegand, Pascal
Publisher: name: UNSPECIFIED
Year: 2024

description: The focus of the present work is the characterization of binding sites (epitopes) of antibodies and aptamers against the four model proteins interleukin-8 (IL8, CXCL8), cathepsin D (CTSD), α-glucosidase A (GAA) and survival motor neuron protein (SMN). Common to all subprojects was the isolation of potential epitope peptides from enzymatic digestion of the respective protein using affinity chromatography with immobilized antibody or aptamer. This experimental setup is called epitope extraction,¹ and eluated fractions were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-tof MS)² to identify the epitope peptide sequence based on the peptide masses. For further validation of the identified peptides, their affinity to the antibodies was determined by surface plasmon resonance (SPR) biosensor analysis.³ Epitope extraction with Sepharose-bound antibody was also compared with a variant in which the antibody was immobilized directly on an SPR chip.⁴ Epitope characterization is essential for the understanding of antibody function and for the development of various bioassays. The model proteins are all associated with different diseases, either as target proteins in the treatment of diseases or as potential biomarkers. IL8⁵ is associated with chronic inflammatory diseases such as rheumatoid arthritis (RA), SMN⁶ with muscular atrophy (SMA), and CTSD⁷ and GAA⁸ with various lysosomal storage diseases (LSD). The elucidation of the epitopes of antibodies relevant to these proteins can help us understand their biological functions. For example, the antibody against IL8 inhibits its receptor binding and corresponding functions but can also be used for its detection. Epitope peptides can also be used to quantify a protein that serves as a biomarker for a specific disease, such as the SMN protein, a biomarker for spinal muscular atrophy. In CTSD, the binding sites of an antibody should be compared with those of an aptamer, and the epitope peptides of GAA should help to remove antibodies, which are formed against the drug in enzyme replacement therapy (ERT) from blood of patients using apheresis. In the epitope extraction experiments for the complex of IL8 with the monoclonal antibody mAB-I2519, epitope peptides could be identified particularly well when the corresponding antibodies were bound to gold chips via protein G (PG). The investigation of IL8 and its peptides was complicated by their relatively strong binding to various surfaces, such as sepharose 4B. When peptides from a tryptic digest were used, a discontinuous epitope for mAB-I2519 consisting of IL8[12-20] and IL8[55-60] was obtained. The affinities of the antibody-antigen complex were determined by using the SPR-based biosensor. The immobilization of the antibody via protein G yielded stable and highly active surfaces. For the interaction of IL8 with mAB-I2519, a KD of 7.4 nM was determined; for the peptide IL8[12-20], a KD of 75.1 μM and for IL8[55-60] of 0.98 mM. Both peptides bind the antibody paratope with low affinity, which is why both must be presented in a specific conformation in order to bind the antibody with a correspondingly high affinity. IL8[12-20] is part of the receptor binding site, and IL8[55-60] is responsible for binding to glycans on cell surfaces, which is important for IL8 gradient formation required for leukocyte recruitment. Therefore, mAB-I2519 simultaneously inhibits the binding of IL8 to its receptor and cell surfaces, thus preventing its biological effect. The SMN protein and its monoclonal antibody mAB-7B10 were analyzed similarly to IL8 and its antibody mAB-I2519. For the complex of SMN and mAB-7B10, a known epitope on the recombinant SMN (rSMN) protein was extracted in the form of the tryptic peptide rSMN[37-57]. In this context, epitope extraction was also successfully performed using an SPR-based biosensor. The affinity of mAB-7B10 to rSMN is 0.25 - 0.69 nM; for the synthetic epitope peptide rSMN[37-57], it is 3.2 nM. Due to the well-preserved affinity of the epitope peptide, it was successfully used in a proof-of-concept for a diagnostic assay to quantify the SMN protein from biological samples by epitope extraction and MALDI-tof MS. Appropriate calibration curves in conjunction with a first successful extraction of the epitope peptide from whole blood lysate were obtained. In addition, polyclonal antibodies with an epitope in the C-terminal region of SMN were tested with mAB-7B10 in a sandwich SPR assay, which could be used as an alternative strategy to quantify the SMN protein. The signal amplification by the second antibody could increase the assay’s sensitivity. The examination of the CTSD-antibody and -aptamer complexes, respectively, and the GAA antibody complex did not lead to unambiguous identification of epitopes or binding sites. This was mainly due to the large amount of unspecific sepharose-binding peptides originating from the respective protein digestion with trypsin and chymotrypsin. Therefore, alternative methods would have to be used for epitope identification of these proteins. In order to recognize protein epitopes that are difficult to access via epitope extraction, at an early stage, specific scouting experiments for unspecific column-binding peptides from digestions with different proteases must be carried out in the future. This would allow the determination of the difficulty level of an epitope to be determined using the epitope extraction method. For the GAA antibody, it was known that the epitope should be located in the 70 kDa fragment of the protein complex, which is why at least the epitope could be narrowed down to a region around GAA[317-324], GAA[304-349], and GAA[411-458].
In addition to the experimental lab work, in-silico predictors for B-cell epitope prediction were used to evaluate the identified peptides a part of a potential epitope. The predictions of the software tools DiscoTope 2.0,⁹ Seppa 3.0,¹⁰ BepiPred 2.0,¹¹ and BCEPS¹² included the epitopes for SMN and IL8. For CTSD and GAA, many of the identified peptides were identified by the epitope predictors, but due to the large proportion of non-specific binders, no unique epitope peptide could be assigned. However, for the investigated proteins, the epitope predictions showed that other proteases, such as Glu-C, Lys-C, or Arg-C, would have been advantageous for the formation of epitope-forming peptides, as they do not cleave at potentially critical positions for antibody binding. Therefore, in-silico analysis can help to design and improve the experimental setup of a particular epitope identification. Finally, the enzymatic digestion of proteins under high-pressure was tested with the four model proteins, as the digestion is essential for epitope extraction. High-pressure digestion was carried out under repeated cycles of high and low pressure and is referred to as "pressure cycling technology" (PCT).¹³ The tests were performed with trypsin, and the peptides were analyzed by MS. Performance parameters such as sequence coverage, digestion speed, enzyme specificity, and peptide peak patterns were compared with a standard procedure under atmospheric pressure. The parameters showed similar values for digestion under atmospheric and high pressure. There is evidence that much lower amounts of the enzyme could be used for high-pressure digestion, with a few hours or less sufficient for complete specific digestion, but this will need further investigation. At last, these experiments confirmed that trypsin does not change its specificity and activity under high pressure.