Patent Grant
11982679
This application is the 35 U.S.C. § 371 national stage application of PCT application No. PCT/IB2017/057422, filed Nov. 27, 2017, where the PCT claims the priority to and benefit of Italian Patent Application No. 102016000120204, filed Nov. 28, 2016 both of which are herein incorporated by reference in their entireties.
This application contains a sequence listing filed in ST.25 format entitled “221007-1660 Sequence Listing” created on Jan. 25, 2023. The content of the sequence listing is incorporated herein in its entirety.
The present invention relates to a method for obtaining protein mimotopes occurring in at least two distinct 3D conformations. In a further embodiment, a method for detecting antibodies with diagnostic relevance using said mimotopes is claimed. In a preferred embodiment, said methods are applied to Alzheimer's disease.
According to the estimates reported by the World Alzheimer Report in 2015, there are 46.8 million people in the world living with a form of dementia. The most common of these forms, with an incidence between 50 and 70% of all forms of dementia, is Alzheimer's disease (AD). AD evolves until compromising, in an affected patient, the ability to perform daily actions.
The analysis and monitoring of two proteins, amyloid P protein 1-42 (Aβ42) and Tau protein, are to date fundamental instruments for a correct and early diagnostic classification of AD. However, these analyses require diagnostic techniques of the invasive type. Alternative approaches have been developed, with uncertain results.
The detection of AD-specific biomarkers present in the serum is the approach of choice. It is known that autoantibodies against Aβ42 are naturally present in human blood, in free or antigen-complexed form, both in AD patients and in healthy subjects (Szabo et al 2008, Gustaw et al 2008). The levels thereof change with the progression of the disease, but the data reported in the literature relating to antibody values present in the serum are conflicting. A series of works describe decreased serum levels of free β-autoantibodies, not attached to the antigen, decreased in AD patients with respect to the controls (Song et al 2007, Moir et al 2005, Weksler et al 2002, Du et al 2001, Britschgi et al 2009, Brettschneider et al 2005); on the other hand, another series of works reports increased values (Nath et al 2003, Mruthinti et al 2004), or unchanged values (Hyman et al 2001, Baril et al 2004). The studies mentioned above are mainly focused on autoantibodies against Aβ42 monomer.
Therefore, it is assumed that autoantibodies against the oligomer or aggregated Aβ42 were the most clinically relevant target (Hock et al 2003). Titration methods of autoantibodies in serum have been proposed by acid dissociation of β-immune complexes, highlighting higher Aβ-specific antibody levels in patients affected by AD with respect to healthy controls (Gustaw et al 2008, Gustaw-Rothenberg et al 2010). However, using a similar procedure, Klaver et al (2011) have not found any significant difference between the two groups, AD and control.
The data inhomogeneity obtained to date does not make a diagnostic approach based on the evaluation of the autoantibodies against the Aβ42 protein feasible.
A non-invasive and accurate diagnostic method is strongly felt, which allows the diagnosis of AD, as well as of other pathologies characterized by the presence of proteins taking on more than one 3D conformation, even in early stages of the pathology. A method is equally strongly felt, which allows, having identified the protein responsible for the disease and the 3D conformations which it takes on, to efficiently develop antibodies capable of recognizing it.
It is the object of the present invention to identify methods and instruments for developing diagnostic and/or therapeutic methods with the desired features.
The variability of the data found in the vast literature available about autoantibody levels against Aβ42 has led to assuming that different and distinct Aβ42 variants or multimers, involved in AD, may be recognized as conformational antigens. This could lead to the generation of different antibody populations, not equally recognizable with the different approaches followed in the different studies. The presence of different autoantibodies would, therefore, be considered responsible for the variability of the observed data.
The authors of the present invention surprisingly developed a completely new approach allowing the identification of mimotopes conformationally homologous to Aβ42. Said mimotopes, or sequences capable of mimicking conformational epitopes of a protein, have been successfully used as a probe for identifying specific AD antibodies in diagnostic systems.
The approach herein described for the first time has an application thereof for identifying mimotopes of any protein which is in more than one conformation. The patent may be extended to all the pathologies caused by the alteration of proteins which from the standard conformation generate misfoldings, thus becoming pathological. The altered protein forms could lead to the exposure of new epitopes, potentially involved in the antibody response. Included in these pathologies are: prion proteins involved in Creutzfeldt-Jakob disease; synuclein which, transformed into α-synuclein, leads to the formation of fibrillar forms in Parkinson's disease; huntingtin, involved in Huntington's disease; phosphorylated Tau, involved in neurodegenerative diseases; amyloidosis, characterized by the deposition at the extracellular level of protein material with a β-sheet structure, which is the reason why the use of the term β-fibrillosis is preferred for the disease, β-fibrillosis involving different proteins, including 2β-microglobulin, transthyretin, lysozyme, fibrinogen; poly(A)-binding proteins, involved in oculopharyngeal muscular dystrophy; proteins of the Serpin superfamily (serine protease inhibitor). Therefore, the present invention relates to a method for obtaining mimotopes for proteins of interest. By way of example, said method is successfully applied for obtaining mimotopes of proteins selected from the group comprising: Aβ42, antibodies responsible for autoimmune diseases, prion proteins, synuclein, huntingtin, phosphorylated Tau, 2β-microglobulin, transthyretin, lysozyme, fibrinogen, poly(A)-binding proteins, proteins of the Serpin superfamily (serine protease inhibitor).
The present invention further relates to a diagnostic method comprising the use of phage clones expressing protein mimotopes of diagnostic relevance for the evaluation of the presence of specific antibodies for said protein in the serum.
The present invention also relates to the use of a target against which to generate antibodies capable of recognizing and binding proteins of therapeutic interest. In a preferred embodiment, said protein of therapeutic interest is Aβ42 and said target is the Chaperon protein caf1M of Yersinia pestis.
Phage clones are also described which comprise the sequences SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 5 and the use thereof in AD diagnosis and in generating therapeutic antibodies capable of recognizing and binding Aβ42.
Also claimed are the sequences SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 5 and the use thereof in AD diagnosis and/or in generating therapeutic antibodies capable of recognizing and binding Aβ42. In a preferred embodiment, said peptide sequences are synthesis sequences.
The present invention relates to a method for obtaining mimotopes of proteins of interest characterized by occurring in at least two distinct 3D conformations, wherein said method comprises:
In a preferred embodiment, said procedure of selecting the phage library comprises three selection cycles. In the first cycle, the phage population capable of binding the IgG-test protein is isolated. The eluted phages in the first step are used in the second selection cycle for screening against the monoclonal bait. Only the phages which cross react with the IgG-test protein and with the bait antibody have the possibility of being selected. Lastly, a third selection cycle is carried out, directed again against the test IgG-protein, so as to select the phage clones having more affinity for the latter.
Preferably, said test IgG-protein and said bait antibody are immobilized on beads, for example on Dynabeads Protein G. Typically, the selected phage clones are amplified and the DNA thereof sequenced. From the obtained genomic sequences, encoded amino acid sequences are thus deduced, by means of bioinformatics programs, for example using ExPASy.
Phage clones expressing protein mimotopes with diagnostic relevance are applied in diagnostic methods, wherein said method comprises:
Typically, an ELISA assay is used and the reading carried out is of the colorimetric type. The accomplished reaction is indicative of the presence of the researched antibody with diagnostic relevance in the test serum.
In a further embodiment, it is herein claimed the use of selected mimotopes as described above for generating antibodies capable of recognizing and binding proteins of therapeutic interest. By way of a non-limiting example, said protein of therapeutic interest is Aβ42 and said target is the Chaperon protein caf1M of Yersinia pestis. As an alternative, said method provides the use not of the entire protein but of the fragment thereof for which the greatest conformational homology with the protein of interest has been shown. Said fragment has a diagnostic or therapeutic value, as described above, where said protein fragments are used for generating antibodies which recognize and bind proteins of interest.
In a particularly preferred embodiment, the ELISA assays are performed on chips, wherein the selected phage clone is immobilized on a chip then exposed to the serum of interest. Typically, said chips allow a reading of the optical, electrochemical or capacitive type.
The following examples aim at describing particularly preferred embodiments of the invention, not to be understood as limiting the scope of the present invention.
Through the use of bioinformatics platforms, proteins have been researched which optionally had conformations homologous to Aβ42. Through the use of the bioinformatics tool UniProt the sequence of the APP (Amyloid Precursor Protein, UniProt ID:P05067) protein was studied which is a transmembrane macroprotein of 770 amino acids (aa) with the extracellular N-terminus (amino-terminus) and the cytoplasmic C-terminus (carboxyl-terminus), encoded by a gene located on chromosome 21. The APP is known to be cleaved in a series of functional fragments by proteases, the secretases.
The mechanism which leads to the formation of Aβ42 by APP provides a β-secretase (BACE: beta APP cleavage enzyme) intervention which cleaves on the extracellular domain, at amino acids 671-672. With the instruments Protein Data Bank (PDB) and 3D Molecular Viewer, the 3D conformations of Aβ42 in the different conformations thereof, single and fibrillar, have been identified and reprocessed. The returned structures are 10 possible conformation models which the single considered protein may assume in the aqueous medium and 10 possible conformation models for the same fibrillar protein. In
Having the models obtained as described above, conformational homology analyses were conducted, using the bioinformatics instrument PDBeFold (EMBL-EBI). The analysis carried out is based on identifying residues occupying an equivalent geometric shape in space. The bioinformatics platform allowed a comparison between the 3D structures in the FDB database with the “bait” sequence selected by us (query sequence). The conformation alignment between the 3D structures of Aβ42 in the different conformations thereof was performed by setting, one at a time, all the filters proposed by the system, that is: Q-score (Cα-alignment); P-score (which takes into consideration RMSD, the number of aligned residues, any gaps, the number corresponding to secondary structure elements); Z-score (which takes into account the Gaussiane statistics); % of sequence identity.
With this approach, the system returned a series of proteins. Those having only one homology section either for single Aβ42 or for fibrillar Aβ42 were discarded, and on the other hand those proteins having a homology for both forms were selected. A protein being homologous to more Aβ42 conformations was thus identified, the Chaperon protein caf1M (PDB ID=1p5u) of Yersinia pestis. To confirm the conformational homology, the same approach followed using the 3D structure of Aβ42 in PDB as bait was carried out, using the 3D structure of the Chaperon protein caf1M as bait. The returned proteins as homology output were analyzed and Aβ42 resulted therefrom.
The conformational homology was also verified with another bioinformatics instrument in PDB, instrument allowing to identify a conformational homology on the secondary structure between the proteins of interest. In this bioinformatics platform, the homology with Chaperon protein Caf1M of the different Aβ42 forms is present in the points highlighted in
In
With Aβ42 in fibrillar form,
The conformational homology found between the different forms taken by Aβ42 and the Chaperon protein caf1M confirms the non-probability that these are randomly similar. In fact the homology found in multiple elements of the same working matrix, in different points of a same protein increases the probability that these are actually shared, and which therefore the homology thereof is no coincidence (Bastas G et al. 2008). Having found a sufficient degree of conformational homology between Aβ42 in single form and Aβ42 in fibrillar form on the Chaperon protein caf1M implies that the latter protein includes more misfolding forms, or more conformations, of the Aβ42 protein.
Having identified the protein(s) with a high degree of conformational homology, specifically the Chaperon protein caf1M, monoclonal antibodies were used directed against the same for selecting, by cross selections against IgG in sera of patients affected by AD, different phage libraries constructed on the pVIII of the filamentous bacteriophage M13, through the Phage Display technique.
For the screening of any conformational mimotopes, homologous to caf1M of Yersinia pestis, the monoclonal antibody YPF19 (AbDSerotec® A Bio-Rad Company) anti-F1 of Y. pestis was used, reactive only against the EV76 Y. pestis strain, as it recognizes discontinuous epitopes on the Chaperon protein caf1M. Furthermore, the antibody used is capable of recognizing different Yersinia pestis fragments equal and/or homologous to the Chaperon protein caf1M region comprised between the aa of positions 1-160, region which is that of interest for the conformational homology with the different forms of Aβ42.
As a source of antibodies directed against possible conformational epitope of human Aβ42, a pool of 5 human sera from AD patients (IgG-AD) (average age of 77.4 years, average MMSE value=15.2) was selected to be used.
Material preparation protocol for the selection: Phage libraries. Phage M13 libraries were used, kindly donated by Prof. Franco Felici, expressing random peptides, exposed on the pVIII protein, based on the phagemid vector pC89 (Felici et al., 1991) on which random oligonucleotide sequences were inserted in the region 5′ of the VIII gene present in the vector, under the control of the LacZ promoter. The digestion with the restriction enzymes EcoRI and BamHI linearized the vector and allowed the insertion of the oligonucleotides with random sequences which, flanked by the same restriction sites, allow the recircularization of the vector through ligase reaction.
For the selection, two types of peptide libraries were used, expressing twelve amino acids in the pVIII amino terminus region: pVIII-12aa and pVIII-12aa-cys, wherein the latter has a cysteine-cysteine constriction expressed in the peptide, so as to stabilize the structure thereof. The amplitude of the library is comprised between 10 and 100 million independent clones.
Monoclonal antibody YPF19 anti-F1 of Yersinia pestis: AbDSerotec® A Bio-Rad Company IgG1-9820-5007.
Human AD antibodies: pool of 5 human sera from AD patients (average age of 77.4 years, average MMSE value=15.2) (IgG-AD). For the immobilization of the antibodies, Dynabeads® Protein G (Thermo Fischer scientific) super magnetic beads of 2.8 μm were used. The saline solutions used to form the reaction buffers were purchased at Sigma Aldrich.
Coating Procedure:
Coating of the Dynabeads® Protein G with the monoclonal antibody YPF19:
The procedure for coating the Dynabeads® Protein G described above was carried out except for step 3), changed as follows:
100 μl of each library (1×1012 viral particles) were added to 50 μl of Dynabeads® Protein G and resuspended in 190 μl of TBS-Tween 0.1%. After 30′ incubation, separation with a magnetic device for 1-2′; the supernatant is recovered and used to carry out again the two preceding steps twice before the use of the library for the selection.
New Selection Procedure Methodology
In order to isolate phages expressing common sequences recognized both by the monoclonal YPF19 and by the human sera pool IgG-AD, a selection procedure referred to as “double-blinding” was developed.
In brief, 3 selection cycles were carried out for each library, referred to as biopanning cycles, using the same execution protocol. This cross-selection procedure allows, in the first round, to select the phage population capable of binding the IgG-AD immobilized on the beads. The eluted phages in this step are used in the second selection round, for screening against the monoclonal YFP19. In this case, only the phages which cross react also with the monoclonal YPF19 will have the possibility of binding the target, so as to limit the number of reactive phages to those common between the two antibody classes IgG-AD and mAb-YPF19. Another biopanning cycle is then carried out, with the eluted phages from the second selection round, again against the IgG-AD, in order to select the phage pools having more affinity for the latter.
First Selection Round
Materials: IPTG stock (11.9 mg/ml) Molecular Biology Certified; Ampicillina stock solution (100 mg/ml) Sigma Aldrich; Kanamycin stock solution (10 mg/ml) Sigma Aldrich; X-Gal stock Molecular Biology Certified.
The saline solutions used to prepare the buffers and all the reagents used for the procedure were purchased at Sigma Aldrich. TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl); PEG 20% NaCl 2.5 M. Amplification of the eluates coming from the first, second and third selection rounds:
I Step (Plate Amplification)
Precipitation of the amplified product coming from the eluate of the three selection rounds:
The titration is carried out to determine the number of infecting phage particles per μl. Two different phage titration protocols were carried out.
First Protocol
According to the protocol, physical titration by a UV/Vis spectrophotometer reading.
Table 1 shows the values obtained from the different selection rounds, indicating the input of the quantity of engineered bacteriophages placed at the start of the bioplanning process (IN), the quantity recovered at the end of the process (OUT), and finally the ratio between them (YIELD) to indicate the number of bacteriophages that have instead remained adhered to the selection target:
Selection of positive clones for both antibodies of interest The phage clones coming from the third round were used in a double immunoscreening assay which allows to identify, in a phage population, the positive clones against the antibodies of interest.
To check the reactivity against the monoclonal YPF19:
Check reactivity against the AD sera pool:
The colonies, which were reactive in both filters, were individually amplified.
The amplified of single clones are precipitated as follows: 1) Aliquot the filtrate in 2 250 ml sterile test tubes with 125 ml each;
An ELISA assay was carried out adsorbing the selected single clones on ELISA well plates (Multisorp Nunc) to confirm the binding specificity with the monoclonal antibody YPF19 and with the IgG-AD of the AD sera pool used for the selection.
Check the monoclonal antibody YPF19 specificity:
To check the specificity of response with the AD sera pool:
The selected clones which simultaneously show greater reactivity against the two antibody categories, or IgG present in the serum of AD subjects and specific monoclonal antibodies for the identified proteins having a high conformational homology degree for Aβ42, were sequenced and therefore the compatibility with the predicted 3D conformational homology regions was evaluated.
From the analyzed clones, the clone referred to as 12III1 was selected, capable of discriminating a pool of IgG-AD through the ELISA test.
The DNA thereof was amplified for PCR, sequenced and the amino acid sequences encoded from the same were deducted from the obtained genome sequences.
The clone 12III1 expresses the sequence RWPPHFEWHFDD (SEQ ID No.1). The testing was extended against samples of AD patient sera and of controls, showing the capability of discriminating between the two assayed population types, with significance greater than 99% (pValue=0.0044). Furthermore, through the ELISA test with Urea, a high binding avidity was observed by the clone 12III1 against the antibodies IgG-AD.
For the amplification of the DNA, protocols known to those skilled in the art were carried out, herein summarized:
For the subsequent sequencing, the DNA was purified with the extraction kit Nucleo Spin® (Macherey-Nagel).
Besides the already mentioned clone 12III1, the following further reactive clones were also identified:
The clones showing a greater reactivity were analyzed in the amino sequences thereof. The peptides were then compared for sequence and frequency homology of the single amino acids, among those of the same group.
Through the CLUSTAL X sequence alignment program, using the BLOSUM matrix (Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. NucleicAcids Res. 22:4673-4680), the clone sequences were aligned along the portions of interest conformational on the chaperon protein caf1M (that is the fragments 20-29 and 148-161) and with the Aβ42; the results are shown in
The clones isolated from the two libraries have a low homology degree both with the portion of the Caf1 involved in the recognition and with the Aβ42, as expected since they are not linear epitopes. With the CLUSTAL X program, tracts which are homologous to the two Aβ42 regions, as well as to the chaperon protein caf1M fragments, were identified.
The clone 12III1, coming from the library 12-mer, has a homology along the initial region of the Aβ42,
Furthermore, the alignment was carried out on the Aβ42 of the clone sequences which have besides the insert also the amino acids that are exposed along the pVIII, which may give a contribution to the recognition participating to the correct folding of the peptide. The results are shown in
The clone 12III1 (panel A) aligns on the region (1-16) of the Aβ42, region implicated in the recognizing by the immunoglobulins and in the formation of amyloid protofilaments. The clone 12cIII1 (panel B) aligns along the region (25-35), known as the shortest Aβ42 fragment which exhibits the beta-sheet structure (Hughes et al 2000). The clone 12cIII4 (panel C) aligns with the region (16-22) which represents the hydrophobic core of the Aβ42 mainly subject to aggregation, known as the autorecognition site (Neddemiep et al 2011).
It is possible to note how also in the presence of the remaining part of pVIII, the peptide maintains, in any case, the same homology region with the Aβ42, if not for some position changes of a few amino acids. Only the clone 12cIII4 is more affected by the presence of amino acids along the pVIII.
The three phage clones selected in the above-described examples were simultaneously tested against a sera pool of 5 healthy subjects, (MMSE>26, no pathology of the nervous system), homogeneous in age, average age=77.2 years and with the AD sera pool used for the screening, average age=77.4 years. Therefore an ELISA test was developed, adsorbing on the bottom of the wells logarithmic scale dilutions of the phage clone 12III1 (1010, 1011, 1012, 1013 PFU/ml) and concentrations of sera equal to 1:10, 1:50 and 1:100. The procedure was standardized using the phage concentration at 1×1012 PFU/ml (OD269˜1), and the serum dilution at 1:50. Below is the description of the optimized procedure of the test.
The values obtained by the test are shown in table 2, wherein the average value of the obtained results from the three repetitions of each sample was inserted; the significance was evaluated with the Student test.
The obtained results show that only the clone 12III1 discriminates with significance the two subject groups. Therefore the preferred embodiment of the present invention is the phage clone 12III1 and the use thereof as an antigenic mimotope to determine the antibody levels IgG-AD.
In order to validate the system, 20 patients affected with AD with a MMSE index comprised between 20.8-6.8 and 18 healthy controls were recruited and the samples were tested according to the ELISA method as follows:
The obtained results are shown in
The difference between the averages of the healthy control group and those relating to the AD patients, is significant at 99% (pValue=0.0044).
It was then tested whether the antibodies in the sera of the patients with AD which recognize the peptide sequence of 12III1 would show a low or high avidity, through the ELISA test with the use of the Urea 4M. The test was conducted on 20 samples, 10 AD sera and 10 control sera, according to the following protocol:
The avidity (Avi %) is evaluated determining the ratio between the obtained result without the use of Urea and that obtained after treatment with Urea.
The avidity test showed that the treatment with Urea does not significantly alter the bond with the target (average avidity % of the considered samples=81%, comprised between 121.53 and 61.33). For such a reason it is required that the antibodies bound by the phage clone 12III1 are free antibodies at a high avidity.
Optical Detection
It was obtained using a device consisting of a silicon chip (substrate) and a polycarbonate plastic support which creates a reaction chamber of a volume of about 400 μl. In particular, said substrate has a dimension of about 1.2*1.7 cm and is coated with a layer of aluminum, typically with a thickness of 1 μm on which a layer of SiO2 (TEOS 8000 A) lies. With the aim of obtaining a stable bond between said support and the bacteriophage, a functionalization is carried out which provides the use of a coupling agent such as for example 1-Ethyl-3-(3-dymethylaminopropyl) carbodiimide (EDC).
The wells are coated with 20 μl of MES buffer (ethanesulfonic acid 2-(N-morpholine)) and left to decant in stasis for 10′.
From the moment of use, a solution of 10 mg EDC/250 μl of MES is prepared and the same is dispensed in each well wherein 10 μl of phage suspension of the clone 12III1 (measuring 1.4×1013 PFU/ml) is then added, to have a total final concentration of 1×1011 phages in the reaction.
The chip is incubated for 2 h at room temperature in a humid room in stasis. After eliminating the supernatant and washing the well with 20 μl of PBS for 5′, the blocking solution (PBS+MILK 6%+Tween 20 0.05%) is added and the chip is incubated in a humid room for 2 h in stasis at room temperature. Finally, the supernatant is eliminated and a further wash is carried out as described above. An ELISA test is then carried out on the silicon surface, operating as follows:
The conducted experiment had the following results (the numbers refer to the spectrophotometer reading):
As an alternative to the optical detection, electrochemical detection may be used. In this case, a silicon chip is used on the surface of which electrodes to form an electrochemical cell were integrated, according to the diagram shown in
In this case, the ELISA test takes place with the use of an electrochemical probe, followed by a reading of the electrochemical type.
In a third alternative embodiment, the detection is of the capacitative type and does not require markers. In this embodiment, the device in silicon has a dielectric material on the surface on which the phage is immobilized. Following the ELISA assay, the electrical capacity of said device immersed in water is measured.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102016000120204 | Nov 2016 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/057422 | 11/27/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/096512 | 5/31/2018 | WO | A |
| Number | Date | Country | |
|---|---|---|---|
| 20190383834 A1 | Dec 2019 | US |
