Methods for identifying epitopes

Abstract

A method for identifying epitopes on a protein that can be bound by an antibody is provided. [Solution] Disclosed is a method for identifying epitopes on a protein that can be bound by an antibody, comprising the steps of performing limited proteolysis of the protein and identifying sites on the protein that are cleaved by the protease used. Also disclosed are epitopes identified by the method of the invention and antibodies that bind to the epitopes.

Description

[Technical Field]

[0001] The present invention relates to several novel methods for selecting epitopes of target proteins, which are used in antibody (e.g., functional antibodies) production, but is not limited to these. Thus, the present invention relates in some aspects to methods for producing antibodies. Such methods typically involve the identification of antigenic epitopes and the production of antibodies against those antigenic epitopes. The present invention also relates to antigenic epitopes and antibodies that bind to such antigenic epitopes. [Background technology]

[0002] The antibody therapy market is experiencing rapid growth, largely due to the clinical success of several monoclonal antibody (mAb) therapies, such as Humira, Avastin, Herceptin, and promising new cholesterol-lowering mAb treatments targeting PCSK9, including alirocumab and evolocumab. However, all antibodies currently on the market and those in advanced clinical development generally target extracellular targets, and these antibodies are generally discovered and developed using screening platforms that focus on affinity or binding strength. Developing intracellular antibodies and antibodies against "hard targets"—that is, targets against which conventional antibody discovery methods have been unsuccessful—remains a challenging task, requiring advances in new technologies for discovering and developing effective antibodies. Intracellular antibodies also require new means for the antibody to be internalized into the cells of the appropriate target organ. Furthermore, current antibody discovery and development platforms typically lack the ability to correlate functional, pharmacological, and mechanism of action, which can predict how a particular antibody will function in a given biological system, such as a disease.

[0003] Today, strategies for developing and discovering successful antibody therapies are not limited to full-size monoclonal antibodies. Advances in protein engineering have led to the production of a wide variety of modified antibody fragments over the past two decades. Examples include Fab fragments, ScFv fragments, diabodies, tetrabodies, antibody fragments functionalized with protein complexes, and bispecific fragments that bind to two antigens. These new constructs provide a fairly large toolbox when attempting to develop antibodies and antibody-derived biologics with high specificity and affinity, deep tissue penetration, high stability, and low toxicity. However, one of the major obstacles associated with antibody therapy remains: antibody therapy is generally limited to extracellular targets. Antibodies are too large and polar to pass through the cell membrane. In addition, antibodies are generally unstable in the reducing environment of the cytoplasm. Several techniques have been developed to access intracellular targets, including the transport of antibodies across the cell membrane using various transport media, such as transfection reagents and protein transduction domains (PTDs), as well as the direct expression of antibodies into target cells, so-called intracellular antibodies. Electroporation techniques have also been used for antibodies, though not as broadly as for small molecules and genetic material. Intracellular antibodies can be constructed to target various cellular compartments by fusing the gene sequence of the intracellular antibody with an intracellular transport signal. Nevertheless, since the genetic material encoding the intracellular antibody is still required for delivery to target cells, the need for efficient delivery vectors is a crucial step in intracellular antibody therapy.

[0004] The production of monoclonal antibodies using hybridoma technology was first developed in 1975. Simply put, this involves injecting a target antigen into a mammal to induce an immune response. Splenocytes are then extracted from the animal's spleen and fused with immortalized myeloma cells. The cells are then diluted to single cells and separated into multi-well plates. Since each cell forms an independent colony, the antibody produced in a single well is monoclonal. The next step is to screen all the different wells for the best candidate antibody that binds to the antigen.

[0005] A major advantage of small antibody fragments compared to full-size antibodies is that they can be produced in various expression systems, such as E. coli, yeast, and mammalian cells, and are no longer limited to production using hybridoma technology. This enables low-cost, large-scale production and increases the potential for genetically modifying antibody properties. Antibody fragments can be displayed on the surface of filamentous phages, a process known as phage display. This display can be used to create large antibody libraries screened against desired antigens. The screening procedure evaluates antibody candidates that bind to the antigen. Since non-specific binding occurs in the first cycle, this is often repeated for several cycles. The conditions between screening cycles can be varied to find the candidate best suited to a particular environment; for example, more stable antibodies can be selected by using harsher conditions. Another way to select antibodies with very high affinity is to screen using very low concentrations of the antigen, leaving only antibodies that can bind under such conditions. Several companies have developed their own screening technologies and often possess large antibody libraries. See, for example, Regeneron (regeneron.com) or Alligatorbioscience (alligatorscience.se). [Overview of the project]

[0006] In one aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Subjecting the protein to limited proteolysis by contacting it with at least one protease, thereby forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and identifying the antigenic epitope of the protein by producing an antigenic epitope based on the surface-exposed peptide, and (ii) comprising producing an antibody against the antigenic epitope.

[0007] In another aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying an antigenic epitope by identifying a surface-exposed epitope in the at least one surface-exposed peptide located in a region of the protein that results in a lack of or significant change in the biological function of the protein when the peptide is cleaved or removed from the protein during the limited proteolysis or restriction proteolysis; or selecting at least one target region in the protein based on known data of bioinformatics and / or the biological function of the protein, and identifying an antigenic epitope by identifying a surface-exposed epitope among the at least one surface-exposed peptide located in the at least one target region; and (iii) comprising producing an antibody against the antigenic epitope.

[0008] In another aspect, the present invention provides a method for identifying an antigenic epitope, the method being: (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying an antigenic epitope by identifying a surface-exposed epitope in the region of the protein where the peptide is cleaved or removed from the protein during the limited proteolysis, resulting in a lack of or significant change in the biological function of the protein, or The method includes selecting at least one target region within the protein based on known bioinformatics and / or biological function data of the protein, and identifying an antigenic epitope by identifying a surface-exposed epitope with the at least one surface-exposed peptide within the at least one target region.

[0009] This invention relates to a method for detecting and identifying the amino acid sequence of a protein, wherein the amino acid sequence is well-exposed and functionally relevant, and at least those amino acid sequences are well-exposed. Therefore, these amino acid sequences, which we call "hot spots," can be used as antigenic epitopes to guide antibody targeting, discovery, and development. Furthermore, these amino acid sequences can be ranked based on their appearance after proteolytic digestion and on functional relevance already known from bioinformatics data or functional / pharmacological studies. Thus, from a list of several amino acid sequences resulting from proteolytic digestion, the optimal amino acid sequence (based on functional and structural arguments) can be selected for antigenic epitope discovery and development. Proteolytic digestion is performed under limited conditions; that is, the activity of the protease or some proteases is very low, with only one or a few surface-exposed peptides being cleaved from the target protein at once. In other words, the protease is used as a draggability probe for antibodies that bind to the target protein.

[0010] In one embodiment, the antibody is pharmacologically active. In another embodiment, the antibody is pharmacologically active and developed for therapeutic use. More specifically, such methods include proteomics tools that reveal hotspot epitopes of target proteins.

[0011] In aspects of the present invention, proteins are digested, broken down, and / or truncated through protease activity, and all well-exposed amino acid sequences are used for antigenic epitope production. Antibodies developed based on these antigenic epitopes are then tested for efficacy, efficacy, pharmacological profiling, and other tests, as is common practice in antibody discovery in the pharmaceutical industry.

[0012] In aspects of the present invention, proteins are digested, broken down, and / or truncated through protease activity, and concurrently examined by functional assays of the digested, broken down, and / or truncated proteins to identify functionally important regions of the proteins. The term "related protein" here sometimes refers to the "target protein."

[0013] In one embodiment, the digestion, degradation, and / or truncation of the target protein occur in parallel and simultaneously to reveal functionally important regions of the target protein that lead to epitope selection for antibody production.

[0014] In one embodiment, digestion, degradation, and / or truncation of proteins and native target proteins, along with functional assays, combined with other bioinformatics facts and other known facts regarding protein function, reveal functionally important regions of target proteins to guide epitope selection for antibody production.

[0015] In one embodiment, a single protease may be used to digest, degrade, and / or truncate a target protein. In another embodiment, multiple proteases may be used to digest, degrade, and / or truncate a target protein one by one sequentially or in parallel and simultaneously. Examples of such proteases include, but are not limited to, Arg-C proteinase, Asp-N endopeptidase, clostrypain, glutamyl endopeptidase, Lys-C, Lys-N, trypsin, chymotrypsin, proteinase K, and thermolysin. Regions readily digested by several proteases should be exposed regions of the protein, while regions digested by only a single protease are likely located in more hidden regions. Alternatively, proteases may possess unique cleavage specificity and / or physicochemical properties and / or structural features that allow them to identify surface-exposed peptides of a target protein that other proteases cannot recognize. Therefore, the use of multiple proteases is preferable, as each different protease can generate complementary or unique information regarding the suitability of surface-exposed peptides as antigenic epitopes.

[0016] The embodiments enable novel methodologies / technologies for the rapid and precise development of pharmacologically active antibodies that can be used in pharmacological studies, for example, these antibodies can be used as tools for detecting biocompounds in cell-based methods or in vitro assays. More importantly, these antibodies can be used to treat diseases in humans and animals. The embodiments can be applied to soluble or membrane-bound, extracellular or intracellular proteins. Furthermore, the embodiments can be used to generate a new fundamental understanding of protein function.

[0017] Furthermore, the present invention provides antibodies produced by the method of the present invention.

[0018] Furthermore, the present invention provides antigenic epitopes identified by the method of the present invention.

[0019] The present invention also provides an antibody against the antigenic epitope of the present invention.

[0020] Other features and advantages of the present invention will be apparent from the following detailed description.

Brief Description of the Drawings

[0021] Embodiments, as well as further objects and advantages, will be better understood by reference to the following description taken in conjunction with the accompanying drawings. [Figure 1] Peptides detected from TRPV1 after limited proteolysis at room temperature using 5 μg / ml trypsin, n = 6. A: Position of the detected peptides shown in the 3D model of TRPV1. Peptides were detected after 0.5 minutes (magenta), 5 minutes (orange), and 15 minutes (blue). B: Position of the detected peptides shown in the schematic of TRPV1. Peptides were detected after 0.5 minutes (magenta), 5 minutes (orange), and 15 minutes (blue). C: Bar plot of the peptides digested from and detected from TRPV1 after limited proteolysis using 5 μg / ml trypsin, showing at which time points those peptides were identified. [Figure 2] Changes in current responses after digestion of peptides from TRPV1 and removal of them after 5-minute exposure to 5 μg / ml, 20 μg / ml, or 40 μg / ml trypsin (Tr). A - C: Positions of the digested peptides of TRPV1. Peptides digested in the flow cell (cyan) and peptides digested in the flow cell and then completely digested overnight (yellow) are shown. D: Representative trace of an inside - out recording of TRPV1 when activated with 1 μM capsaicin (Cap), then subjected to either buffer or trypsin for 5 minutes, and further activated with capsaicin. From top to bottom: Exposed to 5 μg / ml, 20 μg / ml, and 40 μg / ml trypsin for 5 minutes, respectively. For the purpose of presentation in the figure, the trace has been digitally filtered at 100 Hz. [Figure 3]Electrophysiological patch-clamp recordings of TRPV1 function showing the current trace time integral for the second activation with capsaicin, calculated as a percentage of the integral for the first activation with capsaicin, after treatment with either buffer n=11 or antibody n=6. Data are presented as mean ± SEM. [Figure 4] Visualized positions of the OTV1 antigenic determinants (red) and peptides aa96-117 in the surface model of hTRPV1. A: Side view of TRPV1 with each monomer alternately colored blue and purple. B: Top view of TRPV1 with each monomer alternately colored blue and purple. [Figure 5] Visualized OTV2 antigenic determinants (red) and the positions of peptide aa785-799 in the surface model of hTRPV1. A: Side view of TRPV1 with two monomers omitted for clarity. B: Bottom view of TRPV1 with each monomer alternately colored blue and purple. [Figure 6] Localization of OTV1 (left) and OTV2 (right) in fixed cells with and without TRPV1 expression (A). OTV1 and OTV2 were visualized using a goat anti-rabbit Alexa488 secondary antibody. Intensity values ​​along the line segments (black) crossing the cells are given below each image. Different laser settings were used for OTV1 and OTV2, and comparisons between these antibodies should not be made. [Figure 7]Electrophysiological patch-clamp recordings of TRPV1 function after antibody treatment. A: Current trace time integral for secondary activation with capsaicin, calculated as a percentage of the integral for primary activation with capsaicin, after treatment with either buffer (n=11) or OTV1 (n=6). B: Current trace time integral for secondary activation with capsaicin in the presence of calmodulin (CaM) and OTV2, calculated as a percentage of the integral for primary activation with capsaicin, after treatment with either calmodulin alone (n=11) or calmodulin and OTV2. Treatment with OTV2 is divided into measurements within 15 minutes of tip sonication (n=4) and measurements within 30 minutes of tip sonication (n=7). Data are presented as mean ± SEM. [Figure 8] A: TRPV1-mediated YO-PRO uptake after electroporation with OTV1 in calcium-free phosphate-buffered saline. Top panel: Fluorescence intensity of OTV1 (n=11) and control (n=11). Bottom panel: Corresponding fluorescence intensity percentages for OTV1 and control. B: TRPV1-mediated YO-PRO uptake after electroporation with OTV2 in the presence of 50 μM Ca2+. Top panel: Fluorescence intensity of OTV2 (n=9) and control (n=7). Bottom panel: Corresponding fluorescence intensity percentages for OTV2 (green) and control (red). Data are presented as mean ± SEM values. [Figure 9] Fluorescence was used to verify antibody internalization by electroporation. Cells were electroporated, fixed, permeabilized, and incubated with goat anti-rabbit Alexa 488 secondary antibody. Fluorescence intensity was measured by confocal microscopy. Intensities were compared between electroporated and unelectroporated cells, and between cells treated only with the secondary antibody, using either OTV1 or OTV2. Different laser settings were used for OTV1 and OTV2, and intensity values ​​should not be directly compared. Data are presented as mean ± SEM values. [Figure 10]Peptides detected in TRPV1 after limited proteolysis using trypsin. The positions of the detected peptides are shown on a 3D model of TRPV1. Experimental details are given in Example 3. Peptides digested first are shown in black. Peptides digested later are shown in gray. [Figure 11] Peptides detected in TRPV1 after limited proteolysis using Asp-N. The positions of the detected peptides are shown on a 3D model of TRPV1. Experimental details are given in Example 3. Peptides digested first are shown in black. Peptides digested later are shown in gray. [Figure 12] Peptides detected in TRPV1 after limited proteolysis using chymotrypsin. The positions of the detected peptides are shown on a 3D model of TRPV1. Details of the experiment are given in Example 3. Peptides digested first are shown in black. Peptides digested later are shown in gray. [Figure 13] Peptides detected in TRPV1 after limited proteolysis using pepsin. The positions of the detected peptides are shown on a 3D model of TRPV1. Details of the experiment are given in Example 3. Peptides digested first are shown in black. Peptides digested later are shown in gray. [Figure 14] Peptides detected in TRPV1 after limited proteolysis using proteinase K. The positions of the detected peptides are shown on a 3D model of TRPV1. Experimental details are given in Example 3. Peptides digested first are shown in black. Peptides digested later are shown in gray. [Figure 15] This figure shows the similar overall dimensions of the protease and antibody Fab regions. Top panel: Part of the HMM5 Fab crystal structure (PDB: 5I8O), with the paratope-containing region highlighted in red R. Bottom panel: Crystal structure of bovine trypsin (PDB: 1MCT), with the region containing the active pocket highlighted in red R. Bar = 10 angstroms. [Figure 16]This figure exemplifies a workflow for identifying accessible / cleavage sites of proteases. However, it does not include in silico modeling, Fab-protease homology binding, and epitope release based on microfluidic multiprotease digestion / MS-MS detection. (a) In silico digestion, combined with protein homology modeling and Fab-protease docking, predicts protease cleavage sites on the native protein structure. (b) Proteoliposomes containing native proteins are immobilized and digested with a set of proteases using a microfluidic platform, and the resulting peptides are identified by LC-MS / MS. This allows mapping of accessible cleavage sites of proteases. Protease 1 peptide is marked in red (R), protease 2 peptide in blue (B), and protease 3 peptide in green (G). (c) Experimentally determined cleavage sites are compared with predicted sites in silico to determine unpredicted and missed cleavages. (d) To investigate missed cleavage sites, construct antibodies against 7-8 amino acid sequences containing the missed cleavage site using a frame shift approach to include a suitable region (e.g., -20 to +20 amino acids around the cleavage site). Screen the antibodies to find the best binding agent. (e) Use the best binding antibody to explore native and partially digested proteins for structural information. For example, if the antibody binds to a missed cleavage site on a native protein, this may indicate that the protease binds but does not cleave for some reason. Antibodies can also be used to explore peptide cleavage, truncation, or loss of local structure in a domain, for example, caused by protease action. Furthermore, they are also valid for detecting structural rearrangements caused by ligand- or protein-protein interactions. [Figure 17]This figure shows an exemplary multi-protease digestion platform for identifying epitope candidates. (a) Proteoliposomes containing the target are extracted from cells and subjected to limited proteolysis by several proteases in parallel. Various reaction parameters are used (duration, enzyme concentration). Digested peptides from each reaction are eluted and identified using LC-MS / MS. (b) Surface accessibility is ranked according to reaction parameters, e.g., rate, with peptides digested using the slowest kinetics located in areas exposed to the surface and consequently receiving a high rank. (c) Peptides are further ranked by functional relevance using bioinformatics and experimental data, depending on which type of effect is desired (agonism, antagonistism, or simple binding). Epitopes suitable for antibody development have good accessibility and relevant function. (d) Epitopes are optimized and validated in silico, for example, by docking simulations to the epitope using Fab fragments. Finally, epitopes are used as immunogens for animal antibody production. [Modes for carrying out the invention]

[0022] The aforementioned and other embodiments of the invention are described in more detail in relation to the descriptions and methodologies provided herein. It should be understood that the invention can be embodied in various forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so as to ensure that this disclosure is thorough and complete and fully conveys the scope of the invention to those skilled in the art.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. In the specification, singular forms also include plural forms unless the context clearly indicates otherwise. Methods and materials similar to or equivalent to those described herein may be used in the execution or testing of the invention, but preferred methods and materials are listed below. Publications, patent applications, patents, and other references referenced herein are incorporated by reference. References cited herein are not considered prior art to the claimed invention. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are for illustrative purposes only and are not intended to limit the invention.

[0024] The terminology used herein to describe the present invention is for the purpose of describing specific embodiments and is not intended to limit the invention. When used to describe embodiments of the invention, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context clearly indicates otherwise. Furthermore, “and / or” herein refers to and includes any possible combination of one or more related enumerations. In addition, when referring to measurable values ​​such as the amount of a compound, dosage, time, or temperature, the term “about” herein means to include variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. When a range (e.g., a range of x to y) is used, the measurable value means any range within that, such as about x to about y or about x1 to about y1. Furthermore, as used herein, the terms “comprises” and / or “comprising” specify the presence of a given feature, integer, step, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Unless otherwise defined, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0025] In one aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Subjecting the protein to limited proteolysis by contacting it with at least one protease, thereby forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and identifying the antigenic epitope of the protein by producing an antigenic epitope based on the surface-exposed peptide, and (ii) comprising producing an antibody against the antigenic epitope.

[0026] In another aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying an antigenic epitope by identifying a surface-exposed epitope in the region of the protein where the peptide is cleaved or removed from the protein during the limited proteolysis, resulting in a lack of or significant change in the biological function of the protein, or Identifying antigenic epitopes by selecting at least one target region within the protein based on known bioinformatics and / or biological function data of the protein, and identifying surface-exposed epitopes with at least one surface-exposed peptide within the at least one target region, and (iii) comprising producing an antibody against the antigenic epitope.

[0027] Alternatively, the present invention provides a method for producing antibodies against a protein, and the method is (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying antigenic epitopes by identifying cleaved surface-exposed peptides having amino acid sequences that are functionally important to the protein or are predicted to be so, and producing antigenic epitopes based on such surface-exposed peptides, and (iii) comprising producing an antibody against the antigenic epitope.

[0028] In another aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, thereby identifying the surface-exposed peptide of the protein by forming at least one digested, degraded, or truncated form of the protein and at least one peptide cleaved from the protein by the action of the protease, and (ii) constructing a linear or three-dimensional antigenic epitope based on at least one surface-exposed peptide, (iii) comprising producing an antibody against the antigenic epitope.

[0029] In another aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, thereby identifying the surface-exposed peptide of the protein by forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and (ii) Identifying surface-exposed peptides that are cleaved or removed from a protein during limited or restrictive proteolysis, resulting in a deficiency or significant alteration in the biological function of the protein, or selecting at least one identified (i) surface-exposed peptide based on its association with known bioinformatics and / or biological function data of the protein, and (iii) constructing a linear or three-dimensional antigenic epitope based on at least one surface-exposed peptide, (iv) The method includes producing an antibody against the antigenic epitope.

[0030] In another aspect, the present invention provides a method for producing antibodies against a protein, the method being: (i) Subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, thereby identifying the antigenic epitope of the protein by forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and (ii) comprising producing an antibody against the antigenic epitope.

[0031] The antibody production method according to the present invention may, in other embodiments, be seen as an alternative method for producing antibodies that specifically bind to proteins. Furthermore, the exemplary and preferred embodiments of the antibody production method described herein can be applied, with modifications as necessary, to methods for producing antibodies that specifically bind to proteins.

[0032] In another aspect, the present invention provides a method for identifying an antigenic epitope, the method being: (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying an antigenic epitope by identifying a surface-exposed epitope in the region of the protein where the peptide is cleaved or removed from the protein during the limited proteolysis, resulting in a lack of or significant change in the biological function of the protein, or The method includes selecting at least one target region within the protein based on known bioinformatics and / or biological function data of the protein, and identifying an antigenic epitope by identifying a surface-exposed epitope with the at least one surface-exposed peptide within the at least one target region. Optionally, the method of the present invention further comprises the step of producing an antibody against the antigenic epitope.

[0033] Alternatively, the present invention provides a method for identifying antigenic epitopes, the method being: (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying antigenic epitopes by identifying cleaved surface-exposed peptides having amino acid sequences that are functionally important to the protein or are predicted to be so, and producing antigenic epitopes based on such surface-exposed peptides. Optionally, the method of the present invention further comprises the step of producing an antibody against the antigenic epitope.

[0034] Detailed insights into functionally active epitopes exposed on the surface of proteins can aid in the development of effective antibodies and reduce the need for complex screening procedures by decreasing the amount of antibody candidate material. A possible method for evaluating protein surface topology is to limit protease activity by performing limited and controlled proteolysis, digesting only the most flexible and surface-exposed portions of the protein. The idea is to reduce the kinetics of protease activity so that peptides are cleaved one at a time or at most a few at a time. The cleaved peptides can then be ranked based on the order in which they appear after being subjected to the protease. Peptides cleaved first are well exposed by the protein and easily accessible by the protease. We give these peptides a high rank, and we hypothesize that peptides easily cleaved by the protease are also easily recognized by antibodies. We give peptides that are cleaved later a low rank, and all peptides in between are given scores from high to low based on their appearance time after being subjected to the protease. Therefore, since this method is based on amino acid sequences, and the sequences are known to us, we can specifically determine where the antibody binds to the target protein. In the second step, since we know the specific amino acid sequence targeted in the protein, we can investigate the functional significance of the amino acid sequence from previously reported data, other known bioinformatics data, or pharmacological studies of the truncated protein. If the amino acid sequence matches, relates to, or overlaps with known amino acid sequences that have functional importance, such as binding sites, regulatory sites, structurally important sites, or channel regions, the peptide is given a high score and is judged to be a good candidate for antigenic epitope and subsequent antibody development. Specifically, this can be achieved, for example, by controlling the activity of the protease using low temperature, low concentration, and / or short digestion time. When limited proteolysis occurs in proteins with known structures, it is understood that three main structure-determining factors influence where proteolytic activity occurs.These include flexibility, surface exposure, and the number of local interactions. Flexibility and the ability of the protein to locally unfold are required for the peptide chain to enter the active site within the protease. Surface exposure is more likely to provide cleavage sites for proteolysis due to the fact that regions on the surface unfold more easily and experience less steric hindrance. The amount of local interactions in terms of hydrogen bonding and disulfide crosslinking is also important. Fewer local interactions are favorable for proteolysis. All three of these structure-determining factors are usually related to each other within a protein. Therefore, limited proteolysis primarily involves cleaving surface-exposed regions where the protein chain can locally unfold. Limited proteolysis has been used as a method to determine the surface-exposed regions of proteins whose detailed structure is unknown.

[0035] Lipid-based protein immobilization (LPI) techniques enable flexible chemistry on membrane proteins. By obtaining proteoliposomes from cells and immobilizing them in a flow cell, a stationary phase of membrane proteins is created, which can be subjected to a series of solutions multiple times and, for example, various types of chemical modifications by enzymes. Sequential trypsin digestion protocols have been developed for proteomics characterization, in which peptides resulting from the stepwise enzymatic digestion of proteoliposomes are analyzed by liquid chromatography and tandem mass spectrometry (LC-MS / MS) [1-3].

[0036] In some embodiments of the method of the present invention, the protein is a protein (e.g., a membrane protein) present in proteoliposomes (e.g., cells, e.g., human cell-derived proteoliposomes) (e.g., in the lipid bilayer of the proteoliposome). Therefore, in some embodiments, limited proteolysis is carried out in proteoliposomes. Proteoliposomes are lipid vesicles containing proteins. Proteoliposomes can be reconstituted from purified membrane proteins and lipids, or obtained directly from cell membranes (e.g., through vesicle formation) or through cell lysis. Preferably, proteoliposomes are obtained (or prepared) from the cell membrane of lysed cells. Proteoliposomes can be obtained from any target cell type. A convenient cell type is Chinese hamster ovary (CHO) cells.

[0037] Methods for preparing proteoliposomes are known in the art, and any of them may be used (for example, the method described in Jansson et al. Anal. Chem., 2012, 84:5582-5588). Exemplary and preferred methods for preparing proteoliposomes are described herein in the examples. Typically, proteoliposomes with a diameter of about 50 nm to about 150 nm are preferred.

[0038] Using proteoliposomes (prepared) derived from the cell membrane of lysed cells is preferable because, for example, using the methods mentioned in the examples, the proteoliposomes prepared in this manner may be present in the intracellular portion (or domain) of the outer membrane protein of the proteoliposome, thus making available for proteolytic cleavage (and consequently antigenic epitope identification) of portions of the protein that would otherwise be inaccessible to proteases.

[0039] In one aspect, we have developed targeted antibody technologies by utilizing the LPI microfluidics platform [1, 4] to produce potential epitope candidates. This is a mechanism-based methodology rather than a screening-based one. Briefly, LPI technology allows flexible chemistry, such as limited proteolysis, to be performed on membrane proteins. A stationary phase of membrane proteins is created by obtaining proteoliposomes from cells and immobilizing them in a flow cell. A sequential digestion protocol has been developed for proteomics characterization, in which peptides resulting from stepwise enzymatic digestion of proteoliposomes are analyzed by LC-MS / MS. Such peptides are produced from kinetically controlled digestion in the LPI flow cell, revealing regions within the target protein that are exposed and accessible, regions with potential for readily utilized for antibody binding. Furthermore, these potential epitopes are correlated against known functional data to identify epitopes that will result in antibodies with superior binding properties and biological efficiency. Finally, it should be mentioned that selected epitopes / peptides can be used to immunize host animals to produce antibodies. Other methods and techniques for performing limited proteolytic digestion are known in the art and can be used, for example, for soluble proteins.

[0040] In some embodiments of the present invention, the protein (e.g., membrane protein) is immobilized (e.g., on a solid support) before limited proteolysis or restriction proteolysis to create a protein stationary phase. Thus, in some embodiments, the protein is bound to a surface.

[0041] In some embodiments, the protein (e.g., a membrane protein) is present in a proteoliposome (e.g., a cell-derived proteoliposome), which is immobilized (e.g., on a solid support) before limited proteolysis or restrictive proteolysis to create a stationary phase for the protein.

[0042] In some embodiments of the method of the present invention, the protein is a membrane protein present in cell-derived proteoliposomes, and the proteoliposomes are immobilized in a flow cell to create a stationary phase of membrane proteins. Suitable flow cells are known in the art, such as the flow cell described by Jansson et al. (Anal. Chem., 2012, 84:5582-5588).

[0043] In some embodiments, the protein (e.g., a membrane protein) is present in (or on) a proteoliposome (e.g., a cell-derived proteoliposome), and the proteoliposome is in a suspension (e.g., suspended in a solution).

[0044] In some embodiments, the protein is bound to a surface or is present in (or on) protein-containing lipid vesicles in a suspension (e.g., suspended in a solution).

[0045] In some embodiments, the protein is part of or present on any suitable entity, for example, as part of a lipid bilayer or membrane, or on a scaffold or particle, so that its functional or native conformation is preserved.

[0046] In some embodiments, the protein is bound to a surface or is present in (or on) particles such as nanoparticles or other colloidal particles that are in a suspension (e.g., suspended in a solution).

[0047] In some embodiments, the protein is present on (or on) other chemical entities such as scaffolds or caged compounds that are bound to the surface or in suspension (e.g., suspended in solution).

[0048] In some embodiments, the protein is bound to the surface or present in (or on) intact cells (living cells, e.g., human cells) in a suspension (e.g., suspended in a solution).

[0049] In relation to proteins within proteoliposomes, proteins containing vesicles or intact cells include proteins that extend outside (and are consequently exposed to the outside) the proteins containing the proteoliposomes, lipid vesicles, or cells.

[0050] In some embodiments, the protein is in a solution. The solution may be a solution of purified protein, or it may contain a mixture of proteins.

[0051] In some embodiments, cells (e.g., CHO cells) overexpress proteins via an expression system that is controllable, for example, tetracycline-controlled. In some embodiments, proteoliposomes derived from such cells are used.

[0052] We investigated peptides produced from limited proteolysis of the transient receptor potential vanilloid 1 (TRPV1) ion channel with the aim of identifying potential epitopes for developing biologically active antibodies capable of altering the function of this ion channel. TRPV1 was subjected to limited proteolysis with two different proteases, and the digested peptides were correlated with functional data. Using this information, we developed two polyclonal antibodies, OTV1 and OTV2, that act intracellularly on the human TRPV1 (hTRPV1) ion channel. Both antibodies are pharmacologically active, and their target epitope regions were selected based on their digestibility (or surface exposure (peptides highly ranked after limited proteolysis)) and functional importance. When stimulated with the agonist capsaicin, OTV1 exhibits a strong inhibitory effect on the protein. OTV2 is the calmodulin / Ca of TRPV1 process, which is induced by calcium influx through TRPV1. 2+It interferes with dependent desensitization. The efficacy of OTV1 and OTV2 was investigated using both inside-out patch clamp assays, which expose the intracellular side of TRPV1 to antibody solution, and TRPV1-mediated fluorescence uptake assays after the antibody has been electroporated into living cells.

[0053] For rapid solution exchange suitable for patch-clamp experiments, a method combining an LPI flow cell and an open-volume microfluidic flow cell has been reported for some time. The advantage of this method is that it allows for the inversion of the cell membrane, enabling direct examination of the intracellular domain of ion channels. This method allows for the acquisition of relevant structural and functional data using limited and controlled proteolysis. TRPV1 is a cation channel expressed in nociceptive primary sensory neurons. While detailed crystal structures of the full-length protein have not been obtained, the N-terminal ankyrin repeat domain (ARD) has been successfully crystallized in rat TRPV1. Peptides digested in a short time during limited proteolysis of TRPV1 have been compared to known functionally active regions. One-third of the detected peptides contained residues suggested to be functionally important.

[0054] Screening of TRPV1 surface topology, as described in the survey of the field, was performed by immobilizing TRPV1-containing proteoliposomes in a flow cell and then exposing them to limited trypsin proteolysis [1, 4]. Trypsin activity was controlled by using various digestion times at room temperature. Cumulative incubation times and sequential protocols were used, and digested peptides were detected by LC-MS / MS. An increase in peptide number was detected over time, revealing accessible and easily digestible protein regions and rigid regions, as shown in Figure 1. After limited proteolysis of TRPV1 in an LPI flow cell, several regions observed by LC-MS / MS as cleaved peptides were associated with known interaction sites for calmodulin, ATP, and PIP2.

[0055] Furthermore, we investigated the functionality of TRPV1 after removing various structural segments by trypsin digestion [4]. The activity of the TRPV1 ion channel was investigated by inside-out patch-clamp recording and flow cell digestion, followed by proteomic analysis to assess the structural effects of chemical truncation. We used an inside-out patch-clamp recording configuration, which allowed us to expose the intracellular portion of TRPV1 to trypsin and measure the decrease in current response with increasing trypsin concentration (Figure 2).

[0056] We demonstrate that the ion channel TRPV1 can be exposed to limited and controlled trypsin proteolysis in two different microfluidic flow cells under identical experimental conditions. In one example, patch-clamp recordings were performed for pharmacological studies, and information on channel function dynamics was obtained using an open-volume microfluidic instrument. This design allowed patch-clamp pipettes and cell patches to gain access to the perfusion channel. In another example, the ion channel peptide was digested without sample dilution using an equivalent closed-volume flow cell. The cleaved peptide was identified by LC-MS / MS. Data from the two experiments were then compared to assess the structure-function relationship. Using this methodological approach, we identified a highly flexible region of TRPV1 and a critical region that influences its functional channel properties when activated by its agonist, capsaicin.

[0057] Furthermore, this type of methodology can be used for other proteins (i.e., proteins other than TRPV1).

[0058] The amino acid sequence of hTRPV1 is shown below (SEQ ID NO: 1). MKKWSSTDLGAAADPLQKDTCPDPLDGDPNSRPPPAKPQLSTAKSRTRLFGKGDSEEAFPVDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVAA STEKTLRLYDRRSIFEAVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLLKAMLNLHDGQNTTIPLLLEIARQTDSLKELVNASYTDSYYKGQTALHIA IERRNMALVTLLVENGADVQAAAHGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQTADISARDSVGNTVLHALVEVADNTADNTKFVTSMYNEIL MLGAKLHPTLKLEELTNKKGMTPLALAAGTGKIGVLAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVLEVIAYSSSETPNRHDMLLVEPL NRLLQDKWDRFVKRIFYFNFLVYCLYMIIFTMAAYYRPVDGLPPFKMEKTGDYFRVTGEILSVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEMLFFLQSLFML ATVVLYFSHLKEYVASMVFSLALGWTNMLYYTRGFQQMGIYAVMIEKMILRDLCRFMFVYIVFLFGFSTAVVTLIEDGKNDSLPSESTSHRWRGPACRPPDSSYN SLYSTCLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQESKNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPD GKDDYRWCFRVDEVNWTTWNTNVGIINEDPGNCEGVKRTLSFSLRSSRVSGRHWKNFALVPLLREASARDRQSAQPEEVYLRQFSGSLKPEDAEVFKSPAASGEK

[0059] Therefore, the present invention enables functional studies of specific epitopes or evaluation of presumed binding sites of novel antibodies with respect to target membrane proteins present in their natural lipid environment.

[0060] In this invention, antigenic epitopes are typically based on surface-exposed peptides cleaved from proteins during limited or restrictive proteolysis. Alternatively, surface-exposed peptides are typically used to produce antigenic epitopes.

[0061] In this regard, antigenic epitopes may include the amino acid sequence of the surface-exposed peptide or sequences substantially homologous thereto. Antigenic epitopes may consist of the amino acid sequence of the surface-exposed peptide or sequences substantially homologous thereto. Antigenic epitopes may overlap with the amino acid sequence of the surface-exposed peptide or sequences substantially homologous thereto.

[0062] Examples of amino acid sequences "substantially homologous" to a surface-exposed peptide include sequences having or containing one, two, or three amino acid substitutions (preferably one or two, more preferably one) compared to the amino acid sequence of a given surface-exposed peptide.

[0063] Examples of amino acid sequences "substantially homologous" to surface-exposed peptides include sequences containing (or consisting of) at least five or at least six consecutive amino acids of the surface-exposed peptide (or containing or consisting of at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least fifteen, at least twenty, or at least twenty-five consecutive amino acids of the surface-exposed peptide). Six amino acids is a typical length for a peptide / protein sequence that is recognized or bound by an antibody.

[0064] Examples of amino acid sequences that are "substantially homologous" to a surface-exposed peptide include sequences having or containing sequences with at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence homology to a given surface-exposed peptide sequence. Sequence homology of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% is preferred.

[0065] The antigenic epitope may include (or consist of) an elongated form of the surface-exposed peptide, or an elongated form of an amino acid sequence substantially homologous to the surface-exposed peptide. For example, one or more additional amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty amino acids) may be present at one or both ends of the surface-exposed peptide sequence (or a sequence substantially homologous thereto). In some embodiments, up to two, up to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, up to fifteen, or up to twenty amino acids may be present at one or both ends of the surface-exposed peptide sequence (or a sequence substantially homologous thereto).

[0066] The antigenic epitope may include (or consist of) a truncated form of the surface-exposed peptide, or a truncated form of an amino acid sequence substantially homologous to the surface-exposed peptide. For example, one or more amino acids (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) may not be present at one or both ends of the surface-exposed peptide sequence (or a sequence substantially homologous thereto). In some embodiments, up to two, up to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, up to fifteen, or up to twenty amino acids may not be present at one or both ends of the surface-exposed peptide sequence (or a sequence substantially homologous thereto).

[0067] When surface-exposed peptides are arranged in close proximity to each other spatially, the antigenic epitope may be, for example, a cyclic peptide substantially homologous to one or more surface-exposed peptides.

[0068] The antigenic epitope may have a length of at least 5, or at least 6, or at least 7 amino acids, for example, a length of 6-10, 6-12, 6-15, 6-20, 6-25, 6-30, 6-40, 6-50, 6-60, or 6-75 amino acids. The antigenic epitope may have a length of, for example, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids. The antigenic epitope may have a length of, for example, 5-30, 6-30, 7-30, 5-25, 6-25, or 7-25 amino acids. Antigenic epitopes can be, for example, 5-7, 5-8, or 5-9 (e.g., 7-9 amino acids) in length. To avoid misunderstanding, longer proteins or polypeptides, for example, those with a length exceeding 100 amino acids, are not considered epitopes in this invention.

[0069] Homology (e.g., sequence homology) can be evaluated by any simple method. However, to determine the degree of homology (e.g., identity) between sequences, a computer program that creates alignments of multiple sequences is useful, such as the Clustal W algorithm (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can use the BLOSUM62 score matrix (Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA, 89:10915-10919, 1992) along with a gap start penalty of 10 and a gap extension penalty of 0.1, resulting in a top-level match between the two sequences, where at least 50% of the full length of one of the sequences is included in the alignment. Other methods that can be used to align sequences include the Needleman and Wunsch alignment method (Needleman and Wunsch, J.Mol.Biol., 48:443, 1970), modified by Smith and Waterman (Smith and Waterman, Adv.Appl.Math., 2:482, 1981), which yields a top-level match between two sequences and determines the number of identical amino acids between them. Other methods for calculating the percentage of identity between two amino acid sequences are generally recognized in the art, such as those described by Carillo and Lipton (Carillo and Lipton, SIAM J.Applied Math., 48:1073, 1988) and those described in Computational Molecular Biology, Lesk, ed. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects.

[0070] Generally, computer programs are employed for such calculations. Programs for comparing and aligning pairs of sequences, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc.Natl.Acad.Sci.USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990), and gap BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984), are also useful for this purpose. Furthermore, the Dali server at the European Institute for Bioinformatics provides structure-based alignment of protein sequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J.Mol.Biol.,233:123-38, 1993; Holm, Nucleic Acid Res.,26:316-9, 1998).

[0071] The antigenic epitope according to the present invention may be a linear epitope or a three-dimensional epitope.

[0072] In some embodiments, the antigenic epitope according to the present invention may be a cyclized epitope.

[0073] A common method used to prepare linear antigenic epitopes for immunization is Fmoc SPPS (solid-phase peptide synthesis). In SPPS, small porous beads on which peptide chains can be constructed using a wash-coupling-wash cycle are treated with a functional linker. The synthesized peptides are then cleaved from the beads using chemical cleavage. For the synthesis of cyclic peptides, common methods utilize cyclization by forming disulfide crosslinks (crosslinks formed by two cysteine ​​bonds) or by forming "head-to-tail" crosslinks consisting of typical peptide bonds. Cyclic peptides can be formed on solid supports. Generally, antibodies against structural epitopes are produced using the entire protein or a large portion of the protein.

[0074] Restrictive proteolysis involves the proteolytic digestion of a protein that does not proceed to completion. Therefore, through restricted proteolysis, a given protein may only be partially digested (or partially broken down or partially truncated). Restrictive proteolysis may be considered as partial proteolysis. Under restricted proteolysis, if a given protein has a number of potential cleavage sites (i.e., sites recognizable by a given protease for cleavage) for a given number of proteases, that protease may only cleave at some of those cleavage sites.

[0075] Furthermore, restricted proteolysis includes proteolysis performed under restrictive conditions in which the kinetics of protease activity are reduced to the extent that peptides are cleaved from the protein one at a time or up to several at a time. In some embodiments, the kinetic activity of the at least one protease is reduced to such an extent that the surface-exposed peptides are cleaved one at a time or up to several at a time, for example, up to eight at a time (one, two, three, four, five, six, seven, or eight) (for example, as otherwise described herein, for example, up to eight peptides or up to eight unique peptides in the sample), or up to seven at a time (one, two, three, four, five, six, or seven) (for example, as otherwise described herein, for example, up to seven peptides or up to seven unique peptides in the sample), or up to five at a time (one, two, three, four, or five) (for example, as otherwise described herein, for example, up to five peptides or up to five unique peptides in the sample). In some such embodiments, the proteolysis reaction may proceed to completion so that the protein is exhausted of peptides that can be cleaved by the given protease.

[0076] As described elsewhere in this specification, limited proteolysis typically results in only the most flexible and / or surface-exposed portions of the protein being cleaved by the protease.

[0077] In some embodiments of the present invention, the at least one protease is used under conditions such that up to eight surface-exposed peptides (e.g., one, two, three, four, five, six, seven, or eight surface-exposed peptides) (e.g., up to eight peptides in a sample or up to eight unique peptides, as described elsewhere herein) are cleaved from the protein by the action of the protease.

[0078] In a preferred embodiment, the at least one protease is used under conditions such that up to seven surface-exposed peptides (e.g., one, two, three, four, five, six, or seven surface-exposed peptides) or up to five surface-exposed peptides (e.g., one, two, three, four, or five surface-exposed peptides) (for example, as otherwise described herein, up to seven or up to five peptides or up to seven or up to five unique peptides in the sample) are cleaved from the protein by the action of the protease.

[0079] The limited proteolysis or restriction proteolysis according to the present invention can typically be achieved, for example, by reducing the kinetics of protease activity to such an extent that peptides are cleaved from the protein one at a time or up to several at a time. In some embodiments, the kinetic activity of at least one protease is reduced to such an extent that the surface-exposed peptides are cleaved one at a time or up to several at a time, for example, up to eight at a time (one, two, three, four, five, six, seven, or eight) as described above, or up to seven at a time (one, two, three, four, five, six, or seven), or up to five at a time (one, two, three, four, or five).

[0080] Any preferred conditions may be used for limited or restriction proteolysis to result in only the most flexible and / or surface-exposed portion of the protein being cleaved by the protease, for example, up to 8 surface-exposed peptides, or up to 7 surface-exposed peptides, or up to 5 surface-exposed peptides being cleaved by the protease. The conditions leading to limited or restriction proteolysis can be determined by varying the temperature of the digestion reaction and / or the concentration of the protease and / or the time of the digestion reaction and / or the buffer conditions. The number of peptides cleaved from the peptide under specific conditions can be determined by those skilled in the art (e.g., based on mass spectrometry or protein chemistry or biochemistry). Furthermore, preferred methods for establishing appropriate conditions for limited or restriction proteolysis are described elsewhere in this specification. Appropriate limited or restriction proteolysis conditions can be established for various proteins or various proteases or specific combinations of proteins and proteases used. Particularly preferred conditions for limited or restriction proteolysis are described in the examples herein. Typically, the conditions used for limited or restriction proteolysis do not alter (or significantly alter) the natural composition (natural form) of the protein. The protein's cofactors may be present during limited or restriction proteolysis, but are not necessarily required.

[0081] The conditions suitable for limited proteolysis or restricted proteolysis may vary depending on the protease and / or protein, but generally, they are conditions below the optimal level for the protease, for example, conditions that significantly slow down or reduce the kinetics of the protease activity.

[0082] Conditions that impart (or result in) low protease proteolytic activity (e.g., lower than or significantly lower than optimal proteolytic activity) are commonly used. Such conditions include, but are not limited to, the use of low concentrations of protease and / or operating temperatures below the optimal level for the protease and / or buffers that are not standard for the protease or are below the optimal level and / or short contact (incubation) times between the protease and the protein.

[0083] In some embodiments, limited proteolysis (e.g., using trypsin, or using proteases with an optimal working temperature of 37°C or higher) was performed at room temperature (e.g., about 20°C or 17-23°C).

[0084] In some embodiments, limited or restriction proteolysis is performed at a temperature at least 2°C, at least 5°C, at least 10°C, or at least 20°C higher or lower, or significantly higher or lower (preferably lower) than the optimal working temperature of the protease used. In some embodiments, limited or restriction proteolysis is performed at a temperature at least 2°C to 5°C, 2°C to 10°C, 2°C to 20°C, 2°C to 30°C, 5°C to 10°C, 5°C to 20°C, 5°C to 30°C, 10°C to 20°C, 10°C to 30°C, or 20°C to 30°C higher or lower (preferably lower) than the optimal working temperature of the protease used.

[0085] In some embodiments, protease concentrations (e.g., trypsin) up to 5 μg / ml are used for limited or restriction proteolysis. In some embodiments, protease concentrations of up to 0.5 μg / ml, up to 1 μg / ml, up to 2 μg / ml, up to 10 μg / ml, or up to 20 μg / ml are used for limited or restriction proteolysis. In some embodiments, the limited proteolysis reaction can proceed for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, or up to 5 hours, with shorter incubation times generally preferred. For example, in some embodiments, the limited proteolysis reaction can proceed for 5 minutes, 10 minutes, 15 minutes, or up to 30 minutes. In some such embodiments, limited proteolysis is carried out at room temperature. Therefore, in some embodiments, limited proteolysis is carried out at room temperature for up to about 5 minutes (e.g., about 5 minutes) at a concentration of up to 5 μg / ml protease (e.g., about 5 μg / ml protease).

[0086] In some embodiments, the proteolytic digestion reaction can be stopped using formic acid or aqueous ammonia. For example, trypsin, Asp-N, proteinase K, and chymotrypsin can be stopped using formic acid. Pepsin can be stopped using aqueous ammonia.

[0087] In some embodiments of the present invention, cleaved surface-exposed peptides are ranked based on the order in which they appear after contact with at least one protease, with surface-exposed peptides that are cleaved first (or early) and detected at the first (or early) sampling point being given a high rank, and surface-exposed peptides that are cleaved later and detected at later sampling points being given a low rank. Highly ranked peptides that detach from the target protein in a short time and also have functional importance can typically be used for epitope development, immunization, and subsequent antibody production.

[0088] In some embodiments of the present invention, surface-exposed peptides that are cleaved under conditions of low (not harsh) proteolytic activity as described herein (e.g., low (lower) protease concentration, low (lower) incubation temperature, and / or short (shorter) incubation time, generally easily digestible peptides) are given a high rank, and surface-exposed peptides that are cleaved under conditions of high (more harsh) proteolytic activity as described herein (e.g., high (higher) protease concentration, high (higher) incubation temperature, and / or long (longer) incubation time, generally not easily digestible peptides) are given a low rank.

[0089] In some embodiments, multiple samples of the proteolytic digestion material (or eluent from the proteolytic digestion reaction) may be taken during a limited proteolytic or restrictive proteolytic reaction (e.g., sequentially), and / or multiple samples (e.g., multiple limited proteolytic or restrictive proteolytic reactions) may proceed (or be processed) separately (e.g., simultaneously in parallel).

[0090] In some embodiments, multiple samples of proteolytic digestion material (or eluent from a proteolytic digestion reaction) may be taken (or obtained) at time intervals (e.g., 1-minute, 2.5-minute, or 5-minute intervals) during limited or restrictive proteolysis of the protein. In some such embodiments, the samples may vary based on the time (or duration) of contact (or incubation) with the protease, while the protease and / or (typically "and") protease concentration (and / or other conditions resulting in proteolysis as described elsewhere herein) may be constant for each sample (or within each sample). In some such embodiments, the samples may be obtained sequentially (continuous digestion).

[0091] In some embodiments, multiple samples (e.g., multiple limited or restrictive digestion reactions) are carried out (or processed) separately, and each sample has different proteolytic conditions or proteolytic activities for limited or restrictive proteolytic degradation of proteins, for example, different proteases and / or different protease concentrations and / or different temperatures and / or different incubation times may be used for different samples, as discussed elsewhere herein. In some such embodiments, the time (or duration) of contact (or incubation) with the protease is typically (and preferably) constant for each sample (or within each sample). In some such embodiments, the samples may be processed (or processed) in parallel and simultaneously.

[0092] In some embodiments of the method of the present invention, the number of surface-exposed peptides cleaved from the protein by the action of the protease is adjusted over time at a constant protease concentration, and several samples are collected over time; or the number of surface-exposed peptides cleaved from the protein by the action of the protease is adjusted over time by the protease concentration, and several samples can be collected (or processed) at several different concentrations of the protease; or the number of surface-exposed peptides cleaved from the protein by the action of the protease is adjusted by both time and the protease concentration.

[0093] Each sample (or preferred sample) preferably contains one or more peptides cleaved from the protein (e.g., up to eight peptides or up to eight unique peptides). Therefore, one or more peptides cleaved from the protein (e.g., up to eight peptides or up to eight unique peptides) may be detected in each sample. Unique peptides are those that are not present in the previous sample or are not present in the sample under weaker (or less severe) proteolytic conditions (e.g., different from peptides present in the previous sample or present in the sample under weaker proteolytic conditions). Thus, a sample containing up to eight unique peptides may contain more than eight distinct peptides, but one or more of these peptides may be detectable in the sample under the previous sample or under weaker proteolytic conditions (therefore, one or more of these peptides may be non-unique peptides).

[0094] Ideally and preferably, each sample will contain only a single cleaved peptide. For example, a single cleaved peptide may be detected in the first sample (or sampling point). A single cleaved peptide may be detected in one or more subsequent samples (or sampling points). In other examples, multiple cleaved peptides (e.g., up to 8 peptides or up to 8 unique peptides) may be detected in the first and / or subsequent samples (sampling points). Conditions that produce one or more cleaved peptides per sample (e.g., up to 8 peptides or up to 8 unique peptides per sample) can be established by using short sampling intervals, various protease concentrations, various buffer compositions, various temperatures, various salt concentrations, or protease inhibitors (or combinations thereof). Cleaved peptides may be ranked based on the sample (sampling point) in which they appear. For example, under conditions that result in the detection of only one peptide per sampling point, the peptide in the first sample taken may be given the highest rank, the peptide in the second sample taken may be given rank 2, and so on. It is possible to rank individual peptides using conditions in which only a single cleaved peptide is detected at each sampling point. It is also possible to rank groups of peptides using conditions in which only multiple cleaved peptides are detected at each sampling point.

[0095] In some embodiments, higher-ranked surface-exposed peptides (cleaved peptides) are preferred. In some embodiments, the surface-exposed peptide according to the present invention (e.g., a high-ranked peptide) is a cleaved peptide detected (or present) in the first sample taken. In some embodiments, the surface-exposed peptide according to the present invention (e.g., a high-ranked peptide) is a cleaved peptide present in a sample containing, in terms of the order in which it appears in a sample taken during limited or restrictive proteolysis of a protein, one of the top eight ranked peptides (e.g., the top seven or top five ranked peptides) (e.g., the top eight, top seven, or top five unique peptides). Such peptides may be detected (or present) in the first sample taken, or they may be present in one or more subsequently taken samples.

[0096] Peptides that are cleaved from the protein first (or early) (e.g., those in the sample taken first (at the first sampling point) as described above, or those ranked as the top eight peptides (e.g., the top eight unique peptides) based on the order in which they appear during limited proteolysis or restriction proteolysis as described above)) are typically well exposed (e.g., exposed to the surface) and therefore readily accessible by proteases. Such first (or early) digested peptides are given a high rank (e.g., the first peptide to appear is given rank 1, the second peptide to appear is given rank 2, and so on). Peptides digested later (e.g., at a sampling point later than the early peptides) are typically not well exposed and therefore not readily accessible by proteases. Such later digested peptides are given a lower rank. In this invention, peptides with a high rank are typically preferred.

[0097] In some embodiments, cleaved peptides having the amino acid sequence most exposed on the protein surface (surface-exposed peptides) are preferred for antigenic epitope development.

[0098] In some embodiments, peptides (cleaved peptides) may be ranked based on their functional importance or predicted functional importance to the protein. Typically, peptides having amino acid sequences that are functionally important or predicted to be functionally important to the protein are given a higher rank than those that are not functionally important or predicted to be functionally important. In some embodiments, higher-ranked peptides are preferred.

[0099] In some embodiments, peptides having an amino acid sequence that is functionally important to the protein or is predicted to be functionally important (e.g., having a high rank in terms of functional importance), and in addition having a high rank based on surface exposure (e.g., the peptide from the first sample taken (first sampling point) as described above, or peptides ranked among the top 8 peptides based on the order in which they appear during limited proteolysis or restriction proteolysis as described above (e.g., unique peptides ranked among the top 8)), are preferred for antigenic epitope development (or, in other words, preferred peptides that form the basis of antigenic epitopes).

[0100] In some embodiments, peptides that have an amino acid sequence that is functionally important to the protein or is predicted to be functionally important (e.g., having a high rank in terms of functional importance), and that also have a high rank based on surface exposure (e.g., not being the first sample (first sampling point) peptide as described above, nor being one of the top eight peptides ranked based on the order in which they appear during limited or restrictive proteolysis as described above (e.g., a unique peptide ranked among the top eight)), can be used for antigenic epitope development.

[0101] In some embodiments, peptides having amino acid sequences that are functionally insignificant to the protein or are predicted to be functionally insignificant (e.g., having a low rank in terms of functional importance) but have a high rank based on surface exposure (e.g., peptides from the first sample taken (first sampling point) as described above, or peptides ranked as the top eight peptides based on the order in which they appear during limited or restrictive proteolysis as described above (e.g., unique peptides ranked as the top eight)) can be used for antigenic epitope development.

[0102] In some embodiments, the antigenic epitope is based on a surface-exposed peptide that is initially (or early) cleaved from the protein as described above (e.g., the peptide from the first sample taken (first sampling point) as described above, or peptides ranked among the top eight peptides based on the order in which they appear during limited or restrictive proteolysis as described above (e.g., unique peptides ranked among the top eight)), regardless of the functional importance or predicted functional importance of the amino acid sequence of the cleaved peptide.

[0103] In some embodiments, the antigenic epitope is based on peptides ranked as the top eight peptides (e.g., a unique set of top eight peptides) that have amino acid sequences that are functionally important to the protein, or are predicted to be functionally important, in addition to the peptides themselves. These peptides do not necessarily have to be the same as (but may be) the set of eight peptides ranked as absolutely top based solely on their order of appearance (as described above).

[0104] In some embodiments, a region of interest on a protein that is functionally important or predicted to be so is identified or selected, and the antigenic epitope is based on a top eight peptide (e.g., a unique set of top eight peptides) ranked based on the order in which the peptides having an amino acid sequence cleaved from the region of interest appear during limited or restrictive proteolysis. These peptides do not necessarily have to be the same as (but may be) the set of eight peptides that are absolutely ranked to the top based solely on the order in which they appear (as described above).

[0105] In some embodiments, the antigenic epitope for antibody production is based on the amino acid sequence of a peptide that is initially (or early) cleaved from the protein by the action of a protease during limited proteolysis, and thus has a high rank (surface-exposed peptide) (e.g., the peptide from the first sample taken as described above (first sampling point) or peptides ranked among the top eight based on the order in which they appear during limited or restrictive proteolysis as described above (e.g., unique peptides ranked among the top eight)).

[0106] Therefore, in some embodiments, the method of the present invention includes selecting a surface-exposed peptide with a high rank for antigenic epitope development (e.g., the peptide from the first sample taken (first sampling point) as described above, or peptides ranked among the top eight based on the order in which they appear during limited proteolysis or restriction proteolysis as described above (e.g., unique peptides ranked among the top eight)), and producing an antibody against the antigenic epitope based on (or developed from) the surface-exposed peptide.

[0107] In some embodiments, the method of the present invention includes selecting a high-ranking surface-exposed peptide (e.g., the peptide from the first sample taken (first sampling point) as described above, or a peptide ranked among the top eight peptides (e.g., a unique peptide ranked among the top eight based on the order in which it appears during limited proteolysis or restrictive proteolysis as described above)), constructing an antigenic epitope based on the surface-exposed peptide, and producing an antibody against the antigenic epitope.

[0108] In some embodiments, the method of the present invention includes selecting a high-ranking surface-exposed peptide (e.g., the peptide from the first sample taken as described above (the first sampling point) or a peptide ranked among the top eight based on the order in which it appears during limited proteolysis or restrictive proteolysis as described above (e.g., a unique peptide ranked among the top eight)), associating it with a defined biological property (or biological function) of the protein, constructing an antigenic epitope based on the surface-exposed peptide, and producing an antibody against the antigenic epitope. Peptides having an amino acid sequence associated with a defined biological property (or function) of the protein are typically preferred.

[0109] Any means may be employed to identify the cleaved peptides (surface-exposed peptides). In some embodiments, the cleaved peptides are identified by mass spectrometry. In some embodiments, liquid chromatography is used in conjunction with mass spectrometry. Preferably, the cleaved peptides (surface-exposed peptides) are identified by LC-MS / MS (liquid chromatography-tandem mass spectrometry). Exemplary and preferred mass spectrometry methodologies are described in the examples. Tandem mass spectrometry spectra can be retrieved by MASCOT (Matrix Science, London, UK) against a suitable database, for example, as described in the examples.

[0110] As used herein, a digested, broken down, or truncated protein is a protein that has been cleaved by a protease at one or more sites along its length. Such protein cleavage results in one or more peptides (surface-exposed peptides) cleaved (i.e., detached) from the protein. Thus, a surface-exposed peptide is a peptide cleaved from a protein by the action of a protease. The term "surface-exposed" usually indicates that, in light of the full-length protein (i.e., the uncleaved protein), the portion of the protein corresponding to the cleaved (detached) peptide sequence is well exposed and accessible to the protease.

[0111] This invention provides a novel method for discovering therapeutic antibodies and a novel pharmacologically active antibody against the human TRPV1 protein.

[0112] The present invention relates to a method for detecting protein epitopes that are well exposed and can therefore be used as guides for antibody targeting.

[0113] Some methods of the present invention include identifying antigenic epitopes by identifying cleaved surface-exposed peptides having amino acid sequences that are functionally important (e.g., biologically important) to the protein, or are predicted to be so, and producing antigenic epitopes based on such surface-exposed peptides. In some embodiments, antibodies are produced against such antigenic epitopes.

[0114] Identifying whether a surface-exposed peptide cleaved from the protein is functionally important to the protein or has an amino acid sequence that is predicted to be functionally important can be done by any preferred means, and this can be easily done by those skilled in the art.

[0115] For example, in some embodiments, proteins digested, degraded, or truncated during limited or restriction proteolysis can be examined in a functional assay to assess whether their function or functional activity (e.g., biological function) has changed. This can be done by comparing the level of functional activity of the digested, degraded, or truncated protein with the level of functional activity of the protein not subjected to limited or restriction proteolysis (the level of functional activity of the protein not subjected to limited or restriction proteolysis can be considered a control level). If the biological function of the protein has changed after (or during) limited or restriction proteolysis, this indicates that the cleaved peptide (surface-exposed peptide) has been cleaved (cut) from a region of the protein that is functionally relevant to the protein (e.g., biologically important). Thus, the cleaved surface-exposed peptide can be associated with functional data for assessing its functional importance in the protein. The cleaved peptide can be identified, for example, in a parallel experiment, as described elsewhere herein (e.g., by LC-MS / MS) (e.g., the sequence of the cleaved peptide can be revealed). If the cleavage of peptides from proteins (surface-exposed peptides) results in a change in the functional activity of the protein, this indicates that surface-exposed peptides may be particularly useful for the production of antigenic epitopes in this invention. Alternatively, antigenic epitopes based on such surface-exposed peptides may be particularly useful and preferable for antibody production.

[0116] In one embodiment, the protein is TRPV1, and the assay for determining the functional importance of the cleaved peptide to TRPV1 is an inside-out patch clamp assay, as described elsewhere herein.

[0117] The "altered" function or functional activity, or the "change" in function or functional activity, can be any measurable change, preferably a significant change, more preferably a statistically significant change. The "altered" function or "change in function" can be an increase or decrease in function. Exemplary changes in function are changes of ≥2%, ≥3%, ≥5%, ≥10%, ≥25%, ≥50%, ≥75%, ≥100%, ≥200%, ≥300%, ≥400%, ≥500%, ≥600%, ≥700%, ≥800%, ≥900%, ≥1000%, ≥2000%, ≥5000%, or ≥10,000%. The change is typically evaluated against an appropriate control level of function or functional activity, for example, against the function or functional activity of an equivalent protein that has not been subjected to limited proteolysis or restriction proteolysis.

[0118] In some embodiments, the antigenic epitope is based on the amino acid sequence of a surface-exposed peptide that, when cleaved from the protein, results in a change in the protein's function or functional activity.

[0119] In some embodiments, whether a surface-exposed peptide sequence is functionally important (e.g., biologically important) is predicted or determined by utilizing bioinformatics and / or other information already known (e.g., in the scientific literature) about functionally important regions of the protein. Thus, the functional importance of a cleaved surface-exposed peptide to the protein can be predicted or determined in relation to known data about functionally important regions of the protein. If the amino acid sequence of a surface-exposed peptide is known to be functionally important (or is predicted to be functionally important), this indicates that the surface-exposed peptide may be particularly useful for antigenic epitope production in the present invention. Alternatively, antigenic epitopes based on such surface-exposed peptides may be particularly useful and preferred for antibody production.

[0120] Therefore, in some embodiments, the antigenic epitope is based on the amino acid sequence of a surface-exposed peptide that is known (or is predicted to be) functionally important based, for example, on bioinformatics analysis and / or other information already known (e.g., in the academic literature) about functionally important regions of the protein.

[0121] In some embodiments, the antigenic epitope is an antigenic epitope of TRPV1 based on the calmodulin-binding sequence of TRPV1 or the amino acid sequence of a surface-exposed peptide associated with (or corresponding to) the capsaicin-binding site of TRPV1.

[0122] In some embodiments, functional assays are performed to determine the functional importance of surface-exposed peptides, in addition to predicting or determining the functional importance of surface-exposed peptides by using bioinformatics and / or other information already known (e.g., in the scientific literature) about functionally important regions of proteins.

[0123] "Bioinformatics methods," "bioinformatics analysis," "bioinformatics data," and "bioinformatics information" include, but are not limited to, database searches (e.g., BLAST searches), structural modeling, or structural biology, and the data / information obtained therefrom.

[0124] Function (e.g., biological function) may include any biological or physiologically relevant function relating to the protein. Function (e.g., biological function) may include, but is not limited to, the ability of the protein to bind to targets or other binding partners (such as ligands or receptors), e.g., cofactors, signaling activity, enzymatic activity of the protein, ion channel activity, transporter activity, release, e.g., insulin release, and uptake mechanisms. Therefore, functionally relevant or functionally important regions of a protein may include, but are not limited to, regions that confer the ability of the protein to bind to targets or other binding partners (such as ligands or receptors), e.g., cofactors, signaling activity, enzymatic activity of the protein, ion channel activity, transporter activity, and regions that result in the release and uptake of molecules (e.g., insulin).

[0125] In one embodiment, the method of the present invention further comprises the steps of producing in silico a series of putative peptides (e.g., all putative peptides) that can be cleaved from a protein by one or more proteases (e.g., by using a computer program that can identify cleavage sites of the protein based on known recognition sequences of the one or more proteases); optionally, filtering the series of putative peptides produced in silico to obtain a filtered list of putative peptides by excluding peptides previously described (e.g., in sequence databases, e.g., BLAST search or other literature); comparing the filtered list of putative peptides with a list of peptides identified by limited or restriction proteolysis of the protein; identifying peptides common to both the filtered list and the list of peptides identified by limited or restriction proteolysis of the protein; identifying (or constructing) an antigenic epitope based on the peptides common to both lists; and optionally, producing an antibody against the antigenic epitope.

[0126] In another aspect, the present invention provides a method for identifying an antigenic epitope, the method being: (i) The first protein is subjected to limited proteolysis by contacting it with at least one protease, thereby forming at least one digested, degraded, or truncated first protein and at least one surface-exposed peptide cleaved from the first protein by the action of the protease. (ii) Identifying the amino acid sequence of a region (or part or portion) of a second protein that is identical or substantially homologous to the amino acid sequence of a surface-exposed peptide cleaved from the first protein, and (iii) producing an antigenic epitope based on the amino acid sequence of the region (or part or portion) of the second protein that is identical or substantially homologous to the amino acid sequence of the surface-exposed peptide cleaved from the first protein, and optionally (iv) Including producing antibodies against the antigenic epitope.

[0127] Exemplary types of substantially homologous sequences are discussed elsewhere in this specification. Such methods can facilitate the production of antigenic epitopes in a protein (second protein) based on limited or restriction proteolysis performed on a different protein (first protein). This may be particularly useful when the first and second proteins belong to the same protein family or are otherwise related. For example, data from limited or restriction proteolysis performed on TRPV1 can be used to identify the TRPV2 antigenic epitope. Determining (or identifying) a substantially homologous protein of the second protein can be done using any suitable means (e.g., a computer program), which will be familiar to those skilled in the art. Simply as an example, the EMBOSS Needle program provided by EMBL-EBI is a suitable computer program. EMBOSS Needle reads two input sequences and writes out an optimal global sequence alignment, which is a calculated result that finds the optimal alignment (including gaps) of the two sequences along their entire length using the Needleman-Wunsch alignment algorithm.

[0128] In some embodiments of the present invention, the antigenic epitopes are not based on surface-exposed peptides having amino acid sequences conserved with other proteins (e.g., evolutionarily conserved sequences or sequences identical or substantially homologous to the amino acid sequence of the surface-exposed peptide). This can minimize cross-reactivity (or nonspecific binding) of antibodies produced against such antigenic epitopes. In other words, antigenic epitopes based on unique amino acid sequences (or sequences not found in other proteins) can be used in some embodiments.

[0129] The present invention relates to a method for detecting protein epitopes that are functionally relevant and can therefore be used as guidelines for antibody targeting. More specifically, such a method includes a proteomics tool that reveals hotspot epitopes of a target protein. These epitopes, which may be used as antigens in antibody production, are referred to herein as antigenic epitopes.

[0130] In aspects of the present invention, proteins are digested, broken down, and / or truncated through protease activity, and in parallel, one or more functional assays on the digested, broken down, and / or truncated proteins are used to identify functionally important regions of the proteins.

[0131] In one embodiment, protein digestion, degradation, and / or truncation are performed concurrently by functional assays to identify functionally important regions of the protein that lead to epitope selection for antibody production.

[0132] In some embodiments, a single protease may be used to digest, degrade, and / or truncate a protein. In other embodiments, multiple proteases may be used to digest, degrade, and / or truncate target proteins one by one sequentially or in parallel and simultaneously. Examples of such proteases include, but are not limited to, Arg-C proteinase, Asp-N endopeptidase, clostrypain, glutamyl endopeptidase, Lys-C, Lys-N, trypsin, chymotrypsin, proteinase K, and thermolysin. Regions readily digested by several proteases should be exposed regions of the protein, while regions digested by only a single protease are likely located in more hidden regions. Alternatively, proteases may possess unique cleavage specificity and / or physicochemical and / or structural features that allow them to identify surface-exposed peptides of target proteins that other proteases cannot recognize. Therefore, the use of multiple proteases is preferable, as each different protease can generate complementary or unique information regarding the suitability of surface-exposed peptides as antigenic epitopes.

[0133] The sequential use of multiple proteases means that different proteases are incubated with the protein one after another; that is, one protease is incubated, then another protease is incubated at a later time, and one or more other different proteases are incubated at a later time, at the discretion of the protease

[0134] The sequential use of a single protease means that the same protease (e.g., the same concentration of protease) is incubated with the protein several times at several different (sequentially consecutive) time points, or that several samples are taken from the proteolytic digestion reaction over time, and the emergence of new or unique peptides produced in the reaction is detected and tracked over time.

[0135] Parallel use means that multiple separate single-protease digestion reactions are carried out, each with a different protease, or the same protease is used but under different protease degradation conditions, such as different protease concentrations and / or temperatures and / or time points, as described elsewhere herein.

[0136] Multiple proteases may be used to identify surface-exposed peptides that are overlapping, complementary, or unique. In this context, “overlapping” means that a surface-exposed peptide identified via limited or restriction proteolysis with one protease has an amino acid sequence that (partially or completely) overlaps with the amino acid sequence of a surface-exposed peptide identified via limited or restriction proteolysis with one or more other (i.e., different) proteases. In this context, “complementary” means that a surface-exposed peptide identified via limited or restriction proteolysis using one protease has an amino acid sequence that is adjacent to or near (or even partially overlaps with) the amino acid sequence of a surface-exposed peptide identified via limited or restriction proteolysis using one or more other (i.e., different) proteases, in relation to the whole protein sequence (i.e., the whole protein sequence before limited or restriction proteolysis). A "unique" surface-exposed peptide is a surface-exposed peptide that is identified only after limited or restrictive proteolysis using one or a few (a small number) of the tested proteases.

[0137] While not constrained by theory, regions of proteins cleaved by multiple proteases are likely to be located in well-exposed (e.g., surface-exposed) regions of the protein. Therefore, surface-exposed peptides from regions of proteins cleaved by multiple proteases may represent particularly useful surface-exposed peptides that underlie antigenic epitopes.

[0138] The use of multiple proteases is not limited to the use of two, three, four, or five proteases.

[0139] In some embodiments of the method of the present invention, the protease is selected from the group consisting of (or comprising) trypsin, Arg-C proteinase, Asp-N endopeptidase, clostripine, glutamyl endopeptidase, Lys-C, Lys-N, chymotrypsin, proteinase K, thermolysin, pepsin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, enterokinase, factor Xa, granzyme B, neutrophil elastase, proline endopeptidase, staphylococcal peptidase I, and thrombin.

[0140] In some preferred embodiments, the protease is selected from the group consisting of (or comprising) trypsin, Asp-N endopeptidase, chymotrypsin, pepsin, and proteinase K. In preferred embodiments, the protease is trypsin.

[0141] In yet another aspect of the present invention, a mixture of several proteases is used for a single or multiple exposures at a fixed concentration or at various concentrations of one or more proteases with time intervals between them. Thus, in some embodiments, a single mixture (admixture) of multiple proteases is used.

[0142] When multiple proteases are used, a ranking list is created for each protease.

[0143] This method will lead to a new fundamental understanding of protein function and a novel methodology / technology for the rapid and precise development of pharmacologically active antibodies that can be used to treat diseases in humans and / or animals. The method can be applied to soluble or membrane-bound proteins, extracellular or intracellular proteins.

[0144] The list of epitopes produced by the methods proposed herein is preferably categorized for selected bioinformatics data and functional assays. The methods preferably utilize input data from both experimental and bioinformatics information. In some embodiments, focus will be on membrane proteins and membrane-bound proteins. Examples of such proteins include, but are not limited to, the human nociceptor TRPV1, other ion channels of the TRP superfamily, several excitatory amino acid receptors including NMDA receptors, and G proteins. These proteins (e.g., ion channels) have the advantage of being directly investigated in detail using, for example, patch-clamp assays. Other classes of proteins of interest relate to oncogenic proteins, including the oncogenic small GTPases KRAS, NRAS, and HRAS. KRAS is a key protein in several metastatic malignancies, including pancreatic cancer, colon cancer, and lung cancer. GTPase activity can be investigated, for example, by measuring free 32P after GTP is hydrolyzed to GDP following radioisotope labeling of GTP, or by Western blotting after a pull-down assay. Furthermore, other interesting protein classes include immunomodulatory proteins involved in immunomodulation in cancer treatment, such as PD1, PDL1, and CD40, to name just a few.

[0145] The "protein" according to the present invention may be any protein.

[0146] In some embodiments of the present invention, the protein is a membrane-bound protein, a soluble (e.g., circulating) protein, an extracellular protein, or an intracellular protein.

[0147] In some embodiments, the protein is a membrane protein or a membrane-bound protein.

[0148] In some embodiments, the protein is an ion channel, for example, an ion channel of the TRP superfamily (e.g., TRPV1 or TRPV2). In preferred embodiments, the protein is TRPV1.

[0149] In some embodiments, the protein is an excitatory amino acid receptor. In some such embodiments, the protein is an NMDA receptor or a G protein.

[0150] In some embodiments, the protein is an oncogenic protein. In some such embodiments, the protein is an oncogenic low molecular weight G protein selected from the group consisting of KRAS, NRAS, and HRAS.

[0151] In some embodiments, the protein is an immunomodulatory protein. In some such embodiments, the protein is selected from the group consisting of PD1, PDL1, CD40, OX40, VISTA, LAG-3, TIM-3, GITR, and CD20.

[0152] In some embodiments, the protein is neither a urokinase plasminogen activator receptor (u-PAR), nor a transglutaminase 3 (TGase 3), nor a Neisseria meningococcal protein, nor a cannabinoid receptor (e.g., CB1).

[0153] In some embodiments, the protein is a eukaryotic protein. For example, in some embodiments, the protein is a mammalian protein, preferably a human protein.

[0154] In some embodiments, the protein is any protein from the human proteome. In other words, human proteins are preferred.

[0155] Using limited digestion protocols for single or multiple proteases as targets will lead to the discovery of novel antibodies against hotspot epitopes. Different proteases will produce a variety of cleaved peptides. In some embodiments, membrane proteins are degraded, and the effect of each truncation is explored for its impact on protein function. Rare spots found only in certain proteases will also be identified. The identified data will then be analyzed against selected bioinformatics data and also from functional assays of the truncated proteins to identify functionally important regions of those proteins.

[0156] Aspects of the embodiments relate to a method for identifying an antigenic epitope of a protein. The method comprises subjecting a protein to limited proteolysis by contacting the protein with at least one protease to form at least one digested, degraded, or truncated protein and at least one surface-exposed peptide. In another embodiment, the method also comprises searching for at least one digested, degraded, or truncated protein in a functional assay to test, confirm, or validate at least one biological function of the protein. Furthermore, the method comprises identifying the antigenic epitope of the protein as a surface-exposed peptide among the at least one surface-exposed peptide located in a region of the protein involved in the expression of the protein's biological function as determined based on the functional assay.

[0157] In one embodiment, exposing a protein to limited proteolysis or restrictive proteolysis involves contacting the protein with at least one protease at i) a selected temperature or temperature range, ii) a selected concentration or concentration range (relative to the protein concentration), and / or ii) a selected time. This then allows at least one protease to cleave the surface-exposed regions of the protein but not the flexible regions and / or internal regions of the protein.

[0158] Exposing a protein to limited proteolysis by contacting it with at least one protease suggests that the protein is subjected to mild proteolysis. As a result, particularly the flexible peptide portion exposed on the surface of the protein is cleaved from its amino acid sequence by the action of at least one protease. The temperature, concentration, and / or time used for proteolysis are typically dependent on the specific protease and the protein in question. Thus, in embodiments, a set of candidate proteolysis conditions is first tested to select or identify suitable temperatures, protease concentrations, and / or times used for digestion, as well as buffer conditions for degrading or truncating the protein to obtain at least one surface-exposed peptide. For example, to identify the most appropriate proteolysis conditions for the given combination of protein and protease, proteolysis can be carried out at multiple, i.e., at least two different reaction temperatures, at multiple phase-different protease concentrations (relative to the protein concentration), and / or at multiple phase-different reaction times, and under various buffer conditions, e.g., as shown in Figure 1.

[0159] Suitable protease conditions are, for example, temperature, concentration, and / or time that result in digestion, breakdown, or truncation of the protein into one or up to N surface-exposed peptides. A typical value for parameter N is 7, preferably 6 or 5, more preferably 4 or 3, or even more preferably 2 or 1.

[0160] In one embodiment, a functional assay tests, confirms, or validates at least one biological function of a protein. Non-limiting examples of such biological functions include the protein's ability to bind to a target such as a ligand or receptor, its enzymatic activity, or its ion channel activity.

[0161] In some embodiments, subjecting a protein to limited or restriction proteolysis involves contacting the protein with multiple proteases to form multiple digested, degraded, or truncated proteins and multiple surface-exposed peptides. In certain embodiments, the protein is contacted with multiple proteases sequentially, i.e., one after the other. In other specific embodiments, the protein is contacted with multiple proteases simultaneously and in parallel.

[0162] In one embodiment, identifying an antigenic epitope involves identifying a surface-exposed epitope among at least one surface-exposed peptide located in a region that, when cleaved or removed from the protein during limited or restrictive proteolysis, results in a lack of or significant alteration of the protein's biological function.

[0163] In one embodiment, the method also includes selecting at least one target region within a protein based on known data of bioinformatics and / or the biological function of the protein. In such cases, identifying an antigenic epitope includes identifying a surface-exposed peptide among at least one surface-exposed peptide located in the region of the protein within the at least one target region.

[0164] In this embodiment, bioinformatics and / or other known data on biological function are used to guide antigenic epitope selection. This means that only surface-exposed peptides located in one of the selected target regions are used as candidates when identifying or selecting antigenic epitopes. Therefore, the number of candidates can be reduced by removing or excluding surface-exposed peptides located in regions known to lack any biological function and / or regions known not to be involved in expressing the biological function of the protein.

[0165] Another embodiment relates to an antigenic epitope identified according to the above method for identifying an antigenic epitope of a protein.

[0166] In one embodiment, the present invention is LLSQDSVAASTEK (Sequence ID 2), LLSQDSVAASTEKTLR (Sequence ID 3), and This invention provides an antigenic epitope for TRPV1 that includes (or consists of) an amino acid sequence selected from the group consisting of QFSGSLKPEDAEVFKSPAASGEK (SEQ ID NO: 4) or a sequence substantially homologous to that sequence.

[0167] In another embodiment, the present invention is LLSQDSVAASTEKTLRLYDRRS (Sequence ID 5) and This provides an antigenic epitope for TRPV1 containing (or consisting of) an amino acid sequence selected from the group consisting of GRHWKNFALVPLLRE (SEQ ID NO: 6).

[0168] In one embodiment, the present invention provides an antigenic epitope of TRPV1 comprising (or consisting of) the amino acid sequence LVENGADVQAAAHGDF (SEQ ID NO: 7) or a sequence substantially homologous thereto.

[0169] In another embodiment, the present invention is DGPTGARLLSQ (Sequence ID 8) and This invention provides an antigenic epitope for TRPV1 that includes (or consists of) an amino acid sequence selected from the group consisting of DAEVFKSPAASGEK (SEQ ID NO: 9), or a sequence substantially homologous to that sequence.

[0170] In another embodiment, the present invention is SQDSVAASTEKTL (SEQ ID NO: 10) and This invention provides an antigenic epitope for TRPV1 that includes (or consists of) an amino acid sequence selected from the group consisting of SGSLKPEDAEVF (SEQ ID NO: 11) or a sequence substantially homologous to that sequence.

[0171] In one embodiment, the present invention is VSPVITIQRPGD (sequence number 12), VSPVITIQRPGDGPTGA(Sequence ID 13), LNLHDGQNTTIPLLL (Sequence ID 14), YTDSYYKGQ (Sequence ID 15) SLPSESTSH (Sequence ID 16), EDPGNCEGVKR (Sequence ID 17), DRQSAQPEEVYLR (SEQ ID NO: 18), and This invention provides an antigenic epitope for TRPV1 that includes (or consists of) amino acids selected from the group consisting of QSAQPEEVYLR (SEQ ID NO: 19) or a sequence substantially homologous thereto.

[0172] In some embodiments, the present invention provides antigenic epitopes of TRPV1 or sequences substantially homologous thereto, including the amino acid sequences presented in the second heading (headings marked with double asterisks (**)) in Tables 2, 3, 4, 5, and 6 of Example 3 herein. Such peptides are digested using higher proteolytic activity (or more severe or stronger proteolytic conditions) and are generally less desirable than peptides digested using lower proteolytic activity (or less severe or weaker proteolytic conditions) (e.g., shorter time and / or lower concentration, as presented in the first heading (headings marked with single asterisks (*) in Tables 2, 3, 4, 5, and 6)). However, they may be of particular interest if they are functionally important to the protein or are expected to be functionally important. Peptides presented under the second heading (**) in Tables 2, 3, 4, 5, and 6 can be considered peptides that were digested later. Peptides presented under the first heading (*) in Tables 2, 3, 4, 5, and 6 can be considered peptides that were digested first.

[0173] In relation to the antigenic epitope of TRPV1 described above, the substantially homologous sequence may be a sequence that includes one, two, three, four, five, or six (preferably one, two, or three) amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence having at least 70% sequence homology to a given amino acid sequence, or a sequence having at least six consecutive amino acids of a given amino acid sequence. Other examples of “substantially homologous” sequences are described elsewhere herein in relation to “substantially homologous” amino acid sequences to surface-exposed peptides, and those examples of “substantially homologous” sequences are also applicable to the specific peptide sequences described above. The specific peptide sequences described above are surface-exposed peptide sequences.

[0174] In some embodiments, the present invention provides antigenic epitopes comprising (or consisting of) the above-described peptide sequences (or sequences substantially homologous thereto) in the form of elongated, truncated, or cyclic peptides. Elongated, truncated, and cyclic peptides are discussed elsewhere herein in relation to elongated, truncated, and cyclic surface-exposed peptides, and that discussion is also applicable to the above-described peptide sequences. The specific peptide sequences described above are surface-exposed peptide sequences.

[0175] In one embodiment, the present invention is FAPQIRVNLNYRKGTG (Sequence ID 20), ASQPDPNRFDRDR (Sequence ID 21), LNLKDGVNACILPLL(Sequence ID 22) CTDDYYRGH (Sequence ID 23), LVENGANVHARACGRF (Sequence ID 24), EDPSGAGVPR (SEQ ID NO: 25), and This provides an antigenic epitope for TRPV2 comprising (or consisting of) amino acids selected from the group consisting of GASEENYVPVQLLQS (Sequence ID 26) or a substantially homologous sequence. Exemplary substantially homologous sequences are discussed elsewhere in this specification.

[0176] Further embodiments of the embodiments relate to a complex configured for use in the production of antibodies. The complex is defined as being conjugated to or mixed with a peptide carrier and comprising at least one antigenic epitope.

[0177] Therefore, in one embodiment, the present invention provides a complex comprising an antigenic epitope of the present invention or an antigenic epitope identified (or created) by the present invention. The complex may comprise the antigenic epitope and any separate entity (i.e., any entity different from the antigenic epitope), such as a label or a peptide carrier. Typically, the complex comprises the antigenic epitope and a peptide carrier, wherein the antigenic epitope is conjugated to the peptide carrier or mixed with the peptide carrier.

[0178] In some embodiments, the peptide carrier is selected from the group consisting of (or containing) keyhole limpet hemocyanin (KLH) and ovalbumin. The binding can be, for example, a covalent bond or a disulfide crosslink. In one embodiment, keyhole limpet hemocyanin is a preferred peptide carrier. In some embodiments, the antigenic epitope may have a cysteine ​​residue at its N-terminus or C-terminus (preferably the N-terminus). Such a cysteine ​​residue can facilitate the binding of the antigenic epitope to the peptide carrier (e.g., KLH).

[0179] A further embodiment of the embodiment relates to the use of antigenic epitopes and / or complexes thereof for the production of antibodies that specifically bind to proteins.

[0180] A further embodiment of the embodiments relates to a method for producing antibodies that specifically bind to proteins. The method includes producing antibodies against an antigenic epitope and / or a complex thereof, and isolating the antibodies. Isolating the antibodies may include isolating the antibodies from the cells (e.g., host cells) and / or from the growth medium / supernatant from which the antibodies were produced or created.

[0181] In certain embodiments, the method includes subjecting a protein to limited proteolysis by contacting the protein with at least one protease to form at least one digested, degraded, or truncated protein and at least one surface-exposed peptide. The method also includes searching for at least one digested, degraded, or truncated protein in a functional assay to test, confirm, or validate at least one biological function of the protein. Furthermore, the method includes identifying an antigenic epitope of the protein as a surface-exposed peptide located in a region of the protein involved in the expression of the protein's biological function as determined based on the functional assay, among the at least one surface-exposed peptide. The method further includes producing an antibody against the antigenic epitope and isolating the antibody.

[0182] The production of antibodies against antigenic epitopes can be carried out in accordance with techniques known in the art, including hybridoma techniques and phage display techniques, as already described herein.

[0183] Further embodiments of the embodiments relate to antibodies against antigenic epitopes and / or complexes thereof, wherein the antibodies specifically bind to the protein.

[0184] Therefore, in one aspect, the present invention provides an antibody produced (or prepared) by the method of the present invention.

[0185] In another aspect, the present invention provides an antibody against the antigenic epitope of the present invention. In another view, the present invention provides an antibody that binds to the antigenic epitope of the present invention. In yet another view, the present invention provides an antibody that specifically binds to the antigenic epitope of the present invention.

[0186] As an example, the present invention provides an antibody against an antigenic epitope comprising (or comprising) an amino acid sequence selected from the group consisting of LLSQDSVAASTEKTLRLYDRRS (SEQ ID NO: 5) and GRHWKNFALVPLLRE (SEQ ID NO: 6). In one embodiment, the antibody against the antigenic epitope comprising (or comprising) the amino acid sequence LLSQDSVAASTEKTLRLYDRRS (SEQ ID NO: 5) is an antagonist (inhibitor) antibody against TRPV1, and preferably has one or more functional properties related to the antibody OTV1 described in the Examples section. This epitope corresponds to an amino acid sequence located in the N-terminal intracellular domain of TRPV1. In one embodiment, the antibody against the antigenic epitope comprising (or comprising) the amino acid sequence GRHWKNFALVPLLRE (SEQ ID NO: 6) is an agonist antibody against TRPV1, and preferably has one or more functional properties related to the antibody OTV2 described in the Examples section. This epitope corresponds to an amino acid sequence located in the C-terminal intracellular domain of TRPV1.

[0187] In some embodiments, the antibody may target an intracellular TRPV1 epitope (or domain). In some such embodiments, the antibody may be an antagonist antibody against an intracellular TRPV1 epitope (or domain). In other such embodiments, the antibody may be an agonist antibody against an intracellular TRPV1 epitope (or domain).

[0188] In some embodiments, the binding of an antibody to a protein results in a lack of or significant alteration of the protein's biological function.

[0189] Therefore, the antibody may be a functional antibody, for example, an agonist antibody or an antagonist antibody (e.g., an antagonist antibody or agonist antibody against TRPV1 or TRPV2). An antagonist antibody can bind to a protein and inhibit or reduce the protein's function. An agonist antibody can bind to a protein and enhance or increase its function. In the case of TRPV1 or TRPV2 (or any other ion channel), the function in question may be ion transport activity. For example, the ability of an antibody to block (reduce) or enhance (increase) its binding to capsaicin or calmodulin may be evaluated. Antibodies having such ability form a preferred embodiment of the present invention.

[0190] Relevant aspects of the embodiments are defined as antibodies in accordance with their use as pharmaceuticals as described above.

[0191] Antibodies and / or complexes against antigenic epitopes can be obtained by immunizing animals with one or more antigenic epitopes and / or one or more complexes according to the embodiments. The animals to be immunized may be selected from a group including humans, mice, rats, rabbits, sheep, non-human primates, goats, horses, and poultry.

[0192] The antibodies according to the embodiment may also be obtained by an in vitro immunoassay using one or more antigenic epitopes and / or one or more complexes according to the embodiment.

[0193] The antibody according to the present invention may be either a polyclonal antibody or a monoclonal antibody.

[0194] An antibody can be a ligand, a Fab (antigen-binding fragment) fragment, an F(ab)'2 fragment (a fragment containing two Fabs), an ScFv fragment (a single-strand variable fragment), one or more fragments of an antibody such as a bispecific antibody or a tetraspecific antibody, or a complete antibody.

[0195] The antibodies of the present invention can generally bind (e.g., specifically bind) to the full-length form of proteins, for example, to the full-length form of proteins in their native form (e.g., intracellular or on cells).

[0196] In some embodiments, the antibody is an antibody against one of the proteins (or types of proteins) described elsewhere herein.

[0197] Antibodies and antigenic epitopes may be isolated or purified. As used in this context, the terms “isolated” or “purified” refer to molecules isolated or purified from their natural environment, or from an organism with substantially no constraints on that natural environment, for example (if such molecules certainly exist in nature), or to molecules produced by technical means, i.e., molecules produced recombinantly and synthetically. Thus, the terms “isolated” or “purified” typically refer to antibodies or antigenic epitopes that substantially contain no cellular material or other proteins from their source of origin. In some embodiments, such isolated or purified molecules substantially contain no culture medium if produced by recombinant technology, and substantially contain no precursor chemicals or other chemicals if chemically synthesized.

[0198] The functional effects of antibodies produced by the present invention on their target proteins may be evaluated, and those skilled in the art will be able to easily determine a suitable assay to use, for example, based on the properties of the target protein. For example, if the antibody is against TRPV1 (or any other ion channel), the functional effects of the antibody can be evaluated, for example, using the electrophysiological and / or YO-PRO uptake assay described in Example 2 herein.

[0199] The method of the present invention can be used to produce antibodies that can be isolated, prepared, or manufactured for a variety of downstream applications. Therefore, further embodiments of the present invention provide a method for preparing or manufacturing and / or isolating antibodies.

[0200] If one or more antibodies have been produced, prepared, selected, identified, isolated and / or purified by the methods of the present invention, these antibodies, or their components, fragments, variants, or derivatives, may be prepared together with at least one pharmacologically acceptable carrier or excipient, if desired. Such prepared molecules, or their components, fragments, variants, or derivatives, are also encompassed by the present invention. Alternatively, these molecules may take the form of nucleic acids encoding the antibodies, which may then be incorporated into a suitable expression vector and / or contained in a suitable host cell. Thus, nucleic acid molecules encoding the antibodies or expression vectors containing such nucleic acid molecules form further embodiments of the present invention.

[0201] When a specific antibody, or its components, fragments, variants, or derivatives, is produced or fabricated in accordance with the present invention, the expression vector encoding the antibody can be readily used (or adapted for use) to produce a sufficient amount of the molecule by expression in a suitable host bacterium or system, and by appropriately isolating the antibody from the host cell or system, or its growth medium or supernatant. For polyclonal antibodies, the antibody can be isolated or purified from the serum of an immunized animal.

[0202] Therefore, a further aspect of the present invention provides a method for producing or manufacturing an antibody, comprising the steps of producing or manufacturing an antibody according to the method of the present invention described above, manufacturing or manufacturing the antibody, or its components, fragments, variants, or derivatives, and optionally formulating the manufactured antibody with at least one pharmacologically acceptable carrier or excipient.

[0203] Examples of antibody variants or derivatives include peptoid equivalents, molecules with a non-peptide synthetic skeleton, and polypeptides. In polypeptides related to or derived from the original identified polypeptide, the amino acid sequence is modified by single or multiple amino acid substitutions, additions, and / or deletions, which may alternatively or additionally include substitutions or additions of amino acids chemically modified by deglycosylation or glycosylation, for example. Conveniently, such derivatives or variants may have at least 60, 70, 80, 90, 95, or 99% sequence homology to the original polypeptide from which they are derived.

[0204] Since the present invention relates to the production of antibodies, the variants or derivatives further include the conversion of an antibody molecule from one format to another (e.g., conversion from Fab to scFv or vice versa, or conversion between any of the formats of antibody molecules described elsewhere herein, e.g., conversion to any other type of antibody fragment described herein), or the conversion of an antibody molecule to a particular class of antibody molecules (e.g., conversion of an antibody molecule to IgG or its subclasses particularly suitable for therapeutic antibodies, e.g., IgG1 or IgG3), or the humanization or chimeric formation of any antibody.

[0205] The variants or derivatives further include the binding of the antibody with additional functional components that may be useful for downstream applications of the antibody. For example, the antibody may be bound to a component that directs the antibody to a specific site in the body, or to a detectable portion useful for imaging or other diagnostic applications, or to a payload such as a radioisotope, toxin, or chemotherapeutic agent in the form of an immune complex.

[0206] Clearly, a primary requirement for such components, fragments, variants, or derivatives that bind to a partner molecule or target entity is that they retain or enhance their original functional activity in terms of binding ability.

[0207] Antibody molecules produced, prepared, or manufactured using the methods of the present invention can be used in any method requiring antibodies specific to a target entity (e.g., antibodies specific to a particular antigen). Thus, antibodies can be used as molecular tools, and further embodiments of the present invention provide reagents comprising such antibodies as defined herein. In addition, such molecules can be used in in vivo therapeutic or prophylactic applications, in vivo or in vitro diagnostic or imaging applications, or in vitro assays.

[0208] Some specific embodiments of the present invention are described below. 1. A method for producing antibodies against a protein, (i) Subjecting the protein to limited proteolysis by contacting it with at least one protease, thereby forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and identifying the antigenic epitope of the protein by producing an antigenic epitope based on the surface-exposed peptide, and (ii) A method comprising producing an antibody against the antigenic epitope.

[0209] 2. A method for producing antibodies against a protein, (i) Forming at least one digested, degraded, or truncated form of the protein and at least one surface-exposed peptide cleaved from the protein by the action of the protease, and subjecting the protein to limited proteolysis or restriction proteolysis by contacting the protein with at least one protease, (ii) Identifying an antigenic epitope by identifying a surface-exposed epitope in the region of the protein where the peptide is cleaved or removed from the protein during the limited proteolysis, resulting in a lack of or significant change in the biological function of the protein, or A method comprising: (iii) selecting at least one target region within the protein based on known data of bioinformatics and / or the biological function of the protein; identifying an antigenic epitope by identifying a surface-exposed epitope with the at least one surface-exposed peptide within the at least one target region; and (iii) producing an antibody against the antigenic epitope.

[0210] 3. The method according to Embodiment 1 or Embodiment 2, wherein the at least one protease is used under conditions such that up to 8, 7, or 5 surface-exposed peptides, or up to 8, 7, or 5 unique surface-exposed peptides, are cleaved from the protein in a sample of proteolytic digestion material by the action of the protease, and a plurality of samples are optionally collected or processed sequentially or in parallel simultaneously at various times and / or various concentrations of the protease.

[0211] 4. The method according to any one of Embodiments 1 to 3, wherein at least one protease is used under conditions that result in a maximum of 8, 7, or 5 surface-exposed peptides being cleaved from the protein by the action of the protease. 5. The method according to any one of Embodiments 1 to 4, wherein the dynamic activity of at least one protease is reduced to such an extent that the surface-exposed peptide is cleaved in the sample by one or up to several at a time, for example, up to eight, up to seven, or up to five at a time, and optionally multiple samples are collected or processed sequentially or in parallel simultaneously.

[0212] 6. The method according to any one of Embodiments 1 to 5, wherein the cleaved surface-exposed peptides are ranked based on the order of appearance after contact with the at least one protease, with the surface-exposed peptide being cleaved first or at the lowest concentration of the protease being given a higher rank, and the surface-exposed peptide being cleaved later or at the highest concentration of the protease being given a lower rank, and optionally, surface-exposed peptides being cleaved in the middle being ranked according to the order of their appearance. 7. The method of Embodiment 6, comprising selecting a surface-exposed peptide with a high rank for antigenic epitope development and producing an antibody against the antigenic epitope. 8. The method of Embodiment 6, comprising selecting a high-rank surface-exposed peptide, constructing an antigenic epitope based on the surface-exposed peptide, and producing an antibody against the antigenic epitope.

[0213] 9. The method of Embodiment 6, comprising selecting a high-rank surface-exposed peptide, relating the surface-exposed peptide to defined biological properties of the protein, constructing an antigenic epitope based on the surface-exposed peptide, and producing an antibody against the antigenic epitope. 10. The method according to any one of Embodiments 1 to 9, wherein a single protease is used to digest, break down, and / or truncate the protein. 11. The method according to any one of Embodiments 1 to 9, wherein multiple proteases are used to digest, break down, and / or truncate the protein. 12. The method of Embodiment 11, wherein the plurality of proteases are used one by one in sequence, used in parallel and simultaneously, or used in a single cocktail of the plurality of proteases.

[0214] 13. The method according to any one of Embodiments 1 to 13, wherein the plurality of proteases used to identify overlapping, complementary, or unique surface-exposed peptides are selected from the group consisting of trypsin, Arg-C proteinase, Asp-N endopeptidase, clostripine, glutamyl endopeptidase, Lys-C, Lys-N, chymotrypsin, proteinase K, thermolysin, pepsin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, enterokinase, factor Xa, granzyme B, neutrophil elastase, proline-endopeptidase, staphylococcal peptidase I, and thrombin.

[0215] 15. The method according to any one of Embodiments 1 to 14, wherein the protease is trypsin. 16. The method according to any one of Embodiments 1 to 15, wherein the protein is a membrane protein present in cell-derived proteoliposomes. 17. The method according to any one of Embodiments 1 to 16, wherein the proteoliposomes are immobilized on a flow cell to form a stationary phase for membrane proteins. 18. The method according to any one of Embodiments 1 to 15, wherein the protein is bound to the surface or is present in a protein-containing lipid vesicle suspended in a solution. 19. The method according to any one of Embodiments 1 to 15, wherein the protein is bound to the surface of intact cells or suspended in a solution.

[0216] 20. The method according to any one of Embodiments 1 to 15, wherein the protein is in solution. 21. The method according to any one of Embodiments 1 to 20, wherein the protein is any protein of the human proteome. 22. The method according to any one of Embodiments 1 to 21, wherein the protein is a membrane-bound protein, a soluble protein, an extracellular protein, or an intracellular protein. 23. The method according to any one of Embodiments 1 to 22, wherein the protein is a membrane protein or a membrane-bound protein.

[0217] 24. The method according to any one of Embodiments 1 to 23, wherein the protein is an ion channel of the TRP superfamily. 25. The method of Embodiment 24, wherein the protein is TRPV1 or TRPV2. 26. The method according to any one of Embodiments 1 to 23, wherein the protein is an excitatory amino acid receptor. 27. The method of Embodiment 26, wherein the protein is an NMDA receptor or a G protein. 28. The method according to any one of Embodiments 1 to 23, wherein the protein is a carcinogenic protein.

[0218] 29. The method of Embodiment 28, wherein the protein is a carcinogenic low molecular weight GTPase selected from the group consisting of KRAS, NRAS, and HRAS. 30. The method according to any one of Embodiments 1 to 23, wherein the protein is an immunomodulatory protein. 31. The method of Embodiment 30, wherein the protein is selected from the group consisting of PD1, PDL1, CD40, OX40, VISTA, LAG-3, TIM-3, GITR, and CD20.

[0219] 32. The method according to any one of Embodiments 1 to 31, wherein the cleaved peptide is identified by mass spectrometry. 33. The method of Embodiment 24, wherein the cleaved peptide is identified using LC-MS / MS. 34. The method according to any one of Embodiments 2 to 33, wherein the biological function is selected from the group consisting of the ability of the protein to bind to a target such as a ligand or receptor, the enzymatic activity of the protein, ion channel activity, transporter activity, and release and uptake mechanisms such as insulin release.

[0220] 35. The method according to any one of Embodiments 1 to 34, wherein the production of an antibody against an antigenic epitope is performed by hybridoma technology, phage display technology, or by immunizing an animal with the antigenic epitope. 36. The method according to any one of Embodiments 1 to 35, wherein the antibody is monoclonal or polyclonal. 37. An antibody produced by any one of the methods described in Embodiments 1 to 36.

[0221] 38.LLSQDSVAASTEK (Sequence ID 2), LLSQDSVAASTEKTLR (Sequence ID 3), and An antigenic epitope of TRPV1 comprising an amino acid sequence selected from the group consisting of QFSGSLKPEDAEVFKSPAASGEK (SEQ ID NO: 4) or a sequence substantially homologous to that sequence, An antigenic epitope in which the substantially homologous sequence is a sequence having one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence having at least 70% sequence homology to a given amino acid sequence, or a sequence having at least six consecutive amino acids of a given amino acid sequence.

[0222] 39.LLSQDSVAASTEKTLRLYDRRS(Sequence ID 5) and An antigenic epitope of TRPV1 containing an amino acid sequence selected from the group consisting of GRHWKNFALVPLLRE (SEQ ID NO: 6). 40. An antigenic epitope of TRPV1 comprising the amino acid sequence of LVENGADVQAAAHGDF (SEQ ID NO: 7) or a sequence substantially homologous thereto, An antigenic epitope in which the substantially homologous sequence is a sequence that includes one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence that has at least 70% sequence homology to a given amino acid sequence, or a sequence that has at least six consecutive amino acids of a given amino acid sequence.

[0223] 41. DGPTGARLLSQ (Sequence ID 8) and An antigenic epitope of TRPV1 comprising an amino acid sequence selected from the group consisting of DAEVFKSPAASGEK (SEQ ID NO: 9) or a sequence substantially homologous to that sequence, An antigenic epitope in which the substantially homologous sequence is a sequence that includes one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence that has at least 70% sequence homology to a given amino acid sequence, or a sequence that has at least six consecutive amino acids of a given amino acid sequence.

[0224] 42. SQDSVAASTEKTL (SEQ ID NO: 10) and An antigenic epitope of TRPV1 comprising an amino acid selected from the group consisting of SGSLKPEDAEVF (SEQ ID NO: 11) or a sequence substantially homologous to that sequence, An antigenic epitope in which the substantially homologous sequence is a sequence that includes one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence that has at least 70% sequence homology to a given amino acid sequence, or a sequence that has at least six consecutive amino acids of a given amino acid sequence.

[0225] 43.VSPVITIQRPGD(Sequence ID 12), VSPVITIQRPGDGPTGA(Sequence ID 13), LNLHDGQNTTIPLLL (Sequence ID 14), YTDSYYKGQ (Sequence ID 15) SLPSESTSH (Sequence ID 16), EDPGNCEGVKR (Sequence ID 17), DRQSAQPEEVYLR (SEQ ID NO: 18), and An antigenic epitope of TRPV1 comprising an amino acid selected from the group consisting of QSAQPEEVYLR (SEQ ID NO: 19) or a sequence substantially homologous thereto, An antigenic epitope in which the substantially homologous sequence is a sequence that includes one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence that has at least 70% sequence homology to a given amino acid sequence, or a sequence that has at least six consecutive amino acids of a given amino acid sequence.

[0226] 44.FAPQIRVNLNYRKGTG (Sequence ID 20), ASQPDPNRFDRDR (Sequence ID 21), LNLKDGVNACILPLL (Sequence ID 22), CTDDYYRGH (Sequence ID 23), LVENGANVHARACGRF (Sequence ID 24), EDPSGAGVPR (SEQ ID NO: 25), and An antigenic epitope of TRPV2 comprising an amino acid selected from the group consisting of GASEENYVPVQLLQS (Sequence ID 26) or a sequence substantially homologous thereto, An antigenic epitope in which the substantially homologous sequence is a sequence that includes one, two, or three amino acid substitutions or deletions compared to a given amino acid sequence, or a sequence that has at least 70% sequence homology to a given amino acid sequence, or a sequence that has at least six consecutive amino acids of a given amino acid sequence. 45. An antibody against any one antigenic epitope from Embodiments 38 to 44.

[0227] As outlined above, we have developed a methodology for identifying surface-exposed antigenic epitopes that produce pharmacologically active antibodies using kinetically controlled proteolysis. Ideally, the proteolysis step is performed very slowly so that the protease cleaves one or a few peptides at that point. The first peptide to be cleaved is surface-exposed and readily accessible to the antibody, and is therefore generally more advantageous than subsequent peptides. These peptides can then be correlated for sequence-based functional significance using curated bioinformatics data and functional assays performed on truncated proteins.

[0228] However, the present invention also provides improved methods to the above methods that enable further optimization of epitope design and / or identification of additional epitopes. Such improved methods are referred to as Method A and Method B.

[0229] With respect to other methods described herein, these modified methods can utilize several proteases in parallel to maximize the number of epitopes achieved. Five proteases have been tested on the ion channel TRPV1, and the range of useful proteases can be broadened if the specified proteases have different cleavage specificities and produce more unique peptides from native and minimally digested proteins.

[0230] Overall, these improved methods should enhance antibody development and generate novel, therapeutically pharmacologically active antibodies to combat disease. Pharmacologically active antibodies that act both intracellularly and extracellularly can be produced using the methods described herein.

[0231] Unlike many known techniques for discovering novel antibodies, the method of the present invention does not blindly focus on affinity rather than functionality from the outset, but rather allows for the design of antibodies to bind to specific sites and perform specific functions of arbitrary choice from the beginning. A subset of antibodies exhibiting good binding characteristics are then tested for pharmacological and biological effects.

[0232] Method A Therefore, in one aspect, the present invention provides a method for identifying an epitope on a protein that can be bound by an antibody, and the method is (i) After the protein has been exposed to limited or restrictive proteolysis by the one or more proteases, identify the sites where the protein has been cleaved by the one or more proteases, and (ii) Searching for multiple epitopes on the protein located between cleavage sites, overlapping with cleavage sites, or adjacent to cleavage sites using an antibody against the epitopes, thereby identifying one or more epitopes that can be bound by the antibody.

[0233] Any suitable protease can be used, and suitable proteases that can be used in such a method are described elsewhere in this specification. Thus, one or more proteases can be used as described elsewhere in this specification. When multiple proteases are used, in some embodiments they can be used in parallel as described elsewhere in this specification. Limited proteolysis or restrictive proteolysis is also described elsewhere in this specification. Any limited or restrictive proteolysis conditions described herein can be used according to this embodiment (Method A).

[0234] The sites identified in part (i) of the above method are sometimes referred to as cleavage sites (sites where a protease cleaves or is expected to cleave and cleave a surface-exposed peptide). Any suitable method / technique can be used to identify the sites (cleavage sites) where one or more proteases have cleaved (or will cleave) the protein. One preferred technique is mass spectrometry. Findings of the peptide sequences released (or cleaved) from the protein by limited or restrictive proteolysis (e.g., identified by mass spectrometry) provide information about the cleavage sites. In this regard, terminal residues of the released peptide (cleaved peptide) provide information about the cleavage sites in the protein (e.g., in native or full-length proteins).

[0235] To avoid misunderstanding, the “cleavage site” in step (ii) of the above method (Method A) can be considered as a site (or location) in the amino acid sequence of a protein (e.g., a native protein, full-length protein, or wild-type protein) that corresponds to the site that has been cleaved (or detached) (or will be cleaved) according to step (i) (corresponding to the site identified in step (i)). Thus, step (ii) of Method A typically involves searching for multiple epitopes on a native protein (or full-length protein, or wild-type protein) that are located between, overlap with, or adjacent to cleavage sites using an antibody against the epitopes, thereby identifying one or more epitopes that can be bound by the antibody.

[0236] To avoid misunderstanding, "between cleavage sites" preferably means between adjacent cleavage sites. Therefore, "between cleavage sites" preferably means between a given cleavage site and the next (or previous) cleavage site in the amino acid sequence of a protein (e.g., a natural protein, full-length protein, or wild-type protein). Therefore, "between cleavage sites" means between adjacent cleavage sites in the primary amino acid sequence.

[0237] In some embodiments, the method may further include (before step (ii)) the step of generating (or synthesizing) a plurality of isolated epitopes (e.g., two or more, three or more, five or more, ten or more, twenty or more, fifty or more) having sequences corresponding to epitopes (or sequences) on the protein located between cleavage sites, overlapping with cleavage sites, or adjacent to cleavage sites, and generating (causing to produce) antibodies against (binding to) the isolated epitopes. Such antibodies can then be used in step (ii) of the method to search for a plurality of epitopes on the protein (e.g., in natural or full-length proteins) located between cleavage sites, overlapping with cleavage sites, or adjacent to cleavage sites. Any suitable method / technique for generating isolated epitopes or antibodies can be used (as described elsewhere in this specification, for example), and those skilled in the art will be familiar with them.

[0238] In some embodiments, epitopes have varying lengths and / or arrangements. Therefore, within a group of epitopes, there may be epitopes with different lengths and / or arrangements. In other embodiments, epitopes have the same (or similar) length and typically different arrangements. Therefore, in some embodiments, within a group of epitopes, the epitopes have the same (or similar) length.

[0239] The epitope can be of any suitable length. In some embodiments, the isolated epitope is 7-8 amino acids long or has a length as described elsewhere herein.

[0240] In a preferred embodiment, the epitope overlaps with (or includes or surrounds) the cutting site.

[0241] Typically, an epitope (or at least a part of any given epitope) will be within the range of 50 amino acids of the cleavage site, i.e., at amino acids +50 to -50 relative to the cleavage site. Preferably, an epitope (or at least a part of any given epitope) will be within the range of 20 amino acids of the cleavage site, i.e., at amino acids +20 to -20 relative to the cleavage site, within the range of 10 amino acids of the cleavage site, i.e., at amino acids +10 to -10 relative to the cleavage site, within the range of 5 amino acids of the cleavage site, i.e., at amino acids +5 to -5 relative to the cleavage site.

[0242] In some embodiments, the plurality of epitopes is a set (or group) of epitopes, and the sequence of each epitope within the set is offset by one or several (e.g., one, two, or three) amino acids, preferably by only one amino acid, from another epitope within the set. In other words, in some embodiments, in a set of epitopes (a plurality of epitopes), each epitope sequence is shifted by one or several (e.g., one, two, or three) amino acids, preferably by only one amino acid, relative to another epitope sequence in the set. Thus, the plurality of epitopes can be a nested set of epitopes as shown, for example, in FIG. 16d. Typically, such a nested set of epitopes will include up to about 50 amino acids of the protein sequence in either direction (or both directions) relative to (or surrounding) the cleavage site. Preferably, such a nested set of epitopes will include up to about 20 or 10 or 5 amino acids of the protein sequence in either direction (or both directions) relative to (or surrounding) the cleavage site.

[0243] When a nested set of epitopes is used, in a preferred embodiment, a significant number of the epitopes include the cleavage site, preferably substantially all of the epitopes of the nested set include the cleavage site, and more preferably all of the epitopes of the nested set include the cleavage site.

[0244] In some embodiments, a step of actively performing limited or restricted proteolysis is carried out. In other embodiments, the active step of performing limited or restricted proteolysis is not carried out. Rather, the identification of the sites where one or more proteases cleaved the protein is based on data from a previously performed limited or restricted proteolysis experiment (e.g., archival data, e.g., mass spectrometry data including the sequences of released peptides released from the protein of a previous limited or restricted proteolysis experiment). Preferred and suitable methods for performing limited or restricted proteolysis are also described elsewhere in this specification.

[0245] Probing multiple epitopes means that multiple (e.g., two or more, three or more, five or more, ten or more, twenty or more, fifty or more, or two or more but up to four, five, ten, twenty, or fifty) epitopes (or potential epitopes) on a protein (e.g., a native or full-length protein) are analyzed (or evaluated) with respect to their ability to be bound by an antibody generated against (or that binds to) an isolated epitope corresponding to that epitope (or potential epitope) on the protein.

[0246] As described above, searching for multiple epitopes on a protein that are between, overlap with, or are adjacent to cleavage sites can be done using an antibody against that epitope (i.e., the antibody functions as a probe). In practice, searching using an antibody (e.g., a Fab fragment or other antibody fragment) is preferred. However, alternatively, other binding entities may be used as probes (e.g., other affinity probes may be used). Affibodies are an example of an affinity probe that can be used.

[0247] Preferred proteins are described elsewhere in this specification.

[0248] In one embodiment, the present invention provides an epitope (or antigenic epitope) identified by a method (Method A) for identifying an epitope on a protein that can be bound by an antibody as described above. In one embodiment, the present invention provides an antibody that binds to such an epitope on a protein. In some embodiments, antibodies that bind to the vicinity of the cleavage site described herein, for example, within a range of 5, 10, 20, or 50 amino acids of the cleavage site, are preferred. Those skilled in the art are familiar with methods or techniques for producing antibodies against a given epitope and can use any suitable method (for example, as described elsewhere in this specification). Preferred types of antibodies are also described elsewhere in this specification.

[0249] In some embodiments, Method (Method A) further comprises the step of generating (or producing or preparing) an antibody against (or conjugating to) the epitope identified by Method A (identified in step (ii)). Optionally, a further step may be taken to formulate the antibody with at least one pharmaceutically acceptable carrier or excipient.

[0250] Accordingly, in one embodiment, the present invention provides a method for producing or manufacturing an antibody that binds to an epitope identified by Method A (identified in step (ii)). Optionally, a further step may be taken to formulate the produced or manufactured antibody with at least one pharmaceutically acceptable carrier or excipient. Methods for producing or manufacturing antibodies are described elsewhere in this specification and are applicable to this embodiment of the present invention with necessary modifications.

[0251] In one embodiment, the present invention provides a complex comprising at least one epitope identified by Method A, conjugated to or mixed with a peptide carrier. The complex is described elsewhere herein, and its considerations apply to this embodiment of the present invention with necessary modifications.

[0252] Antibodies that bind to the epitopes identified by Method A (or the epitopes identified by Method A or conjugates containing such epitopes) can be used for therapeutic purposes.

[0253] Antibodies that target multiple epitopes on a protein (e.g., one or more antibodies, or a group or series of antibodies, or a large number of antibodies) can be tested for their ability to bind to the protein, for example, to evaluate their binding affinity to the protein or other functional effects (e.g., as described elsewhere in this specification). Thus, antibodies can be screened to identify the best binding agents. Thus, particularly useful epitopes (e.g., those targeted by antibodies) can be identified, for example, those that are particularly suitable for targeting by high-affinity antibodies, or whose targeting results in a significant or measurable functional effect (e.g., antagonist or agonist effect) on the target protein. Thus, the optimal epitope (e.g., for antibody targeting, e.g., for therapeutic applications) can be identified. Thus, from another perspective, the present invention provides a method for optimizing epitope design or for selecting the optimal epitope (e.g., for antibodies produced against or targeting it). The method may enable the determination of the optimal length and position of the epitope relative to the cleavage site.

[0254] When using limited proteolysis as a tool to verify accessible regions for antibody binding, it relies on the release of peptides from the protein, i.e., the protease cleaving at two accessible sites surrounding a sequence of a suitable size for detection, for example, by mass spectrometry. The information obtained from such experiments is verification of the accessibility of the two cleavage sites that were digested and caused the release of peptides. However, the size and location of the accessible region surrounding the cleavage site may be unknown. Method A verifies the accessibility of the region surrounding the cleavage site using an antibody (or other binding protein). For example, the optimal epitope length and location relative to the cleavage site can be determined by developing antibodies that target epitopes surrounding the cleavage site and then testing their binding affinity and / or function. This method may involve developing a group or a number of antibodies targeting epitopes of different lengths and different sequence positions relative to the cleavage site. Typically, each epitope will be shifted by only one amino acid relative to each other and will contain -20 to +20 amino acids around the cleavage site. Different proteases can be used in parallel to experimentally validate accessible cleavage sites using limited proteolysis. Since different proteases may require larger or smaller accessible regions to enable binding to and digestion of the cleavage site, the optimal epitope design may differ depending on the cleavage site validated by the different proteases. Therefore, the optimal epitope design can be determined for each type of protease by exploring the cleavage sites validated by the methodology described above (Method A).

[0255] Preferred features of other methods described herein can be applied to this embodiment of the present invention (Method A) with necessary modifications.

[0256] As described above, due to the presence of some non-released peptides, previous methods may miss some potential Ab-binding sites; therefore, we developed a further improved method. This can occur, for example, when a protease cleaves only one of two cleavage sites surrounding a particular amino acid sequence. The improved method (Method B) described below develops a novel search algorithm based on both Fab-protease homology binding and multi-protease digestion datasets in silico, enabling the discovery of unique and novel antibody-binding sites, as well as generating new structural data for native and partially digested proteins. Thus, this technique (along with Method A above) can provide a comprehensive tool for exploring protein structure and function.

[0257] Method B In another aspect, the present invention provides a method for identifying an epitope on a protein that can be bound by an antibody, the method being: (i) In silico, protease digestion of the protein using one or more proteases to identify sites on the protein that are predicted to be cleaved by the one or more proteases; optionally, protein homology modeling to predict which of the predicted protease cleavage sites in silico is most likely to be exposed; and / or optionally, docking of antibody fragments or proteases in silico to predict which cleavage sites are most likely to be cleaved in vitro. (ii) Protease digestion of the protein in vitro using one or more proteases. (iii) Identify the peptide released from the protein by in vitro protease digestion in step (ii), and thereby identify the cleavage site. (iv) Compare the predicted cleavage site in the in silico identified in step (i) with the cleavage site identified in step (iii). (v) Search for one or more epitopes in protein regions that include or are adjacent to the protease cleavage sites predicted in the in silico identified in step (i), but which are not the cleavage sites identified in step (iii), using one or more antibodies, and (vi) The procedure includes determining whether or not the one or more antibodies bind to the one or more epitopes, thereby identifying the epitopes on proteins that can be bound by the antibodies.

[0258] The protease digestion step in in silico is carried out by the method described above (Method B). However, alternatively, other methods or techniques for predicting the protease digestion of a protein may be used. For example, the amino acid sequence of a protein can be visually checked, and predicted cleavage sites (i.e., sites predicted to be cleaved by the protease) can be identified based on knowledge of the specificity and rules of a given protease. Any method or technique for predicting the protease digestion of a protein can be used based on knowledge of the specificity and rules of a given protease. Therefore, in some embodiments of this method, a computer-based method is preferred, but computer-based prediction is not essential.

[0259] Any suitable protease can be used, and suitable proteases are described elsewhere in this specification. Therefore, one or more proteases can be used, as described elsewhere in this specification.

[0260] This method involves performing optional modeling (e.g., in silico modeling), such as protein homology modeling, to predict which of the predicted protease cleavage sites in in silico are most likely to be exposed (e.g., solvent exposure or surface exposure).

[0261] This method optionally includes performing in silico docking (or binding) of an antibody fragment (such as a Fab fragment or other antibody fragments described elsewhere in this specification) or predicting proteolytic cleavage in silico to predict which cleavage sites are likely to be cleaved in vitro. Those skilled in the art would be able to readily perform such docking analysis or modeling in silico.

[0262] Modeling (such as homology modeling) can be performed using any suitable means, for example, the homology modeling engine of MOE software (Molecular Operating Environment (MOE) 2015.10. Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7. 2016).

[0263] Software such as MOE software can be used to construct and model protein models and / or perform protein-protein docking modeling (docking in silico). This software enables prediction of protein-protein binding arrangements and can generate docked protein structures. Thus, a model of a protein docked (or bound) to an antibody (or antibody fragment) or a model of a protein docked (or bound) to a protease can be generated.

[0264] In a preferred embodiment, in vitro, proteolytic digestion of a protein by one or more proteases is limited or restricted proteolysis. Limited or restricted proteolysis is described elsewhere in this specification.

[0265] When multiple proteases are used, the proteases are preferably used separately (e.g., in parallel).

[0266] Identifying the peptides (peptide sequences) released from the protein by in vitro protease digestion in the steps of the above method (Method B) can be performed by any suitable method or technique, for example, by mass spectrometry (e.g., LC-MS / MS). Once the peptides (peptide sequences) released from the protein are identified, the findings of the peptide sequences released from the protein by limited or restrictive proteolysis (e.g., identified by mass spectrometry) provide information about the cleavage sites, so the sites (cleavage sites) where one or more proteases cleaved the protein can be easily identified. In this regard, the terminal residues of the released peptides (cleaved peptides) provide information about the cleavage sites in the protein (e.g., in native or full-length proteins).

[0267] Step (v) of Method B refers to searching for one or more epitopes in a protein region that contains or is adjacent to a cleavage site that is predicted in the in silico identified in step (i), but is not identified in step (iii), using one or more antibodies. As is evident elsewhere in this specification, and to avoid misunderstanding, a cleavage site (where one or more epitopes overlap or are adjacent) can be thought of as a site (or location) in the amino acid sequence of a protein (e.g., a native protein, full-length protein, or wild-type protein) that is predicted (identified) to be cleaved in step (i), but is not identified in step (iii).

[0268] Searching for one or more (e.g., multiple) epitopes means that one or more epitopes (or potential epitopes) on a protein (e.g., a native or full-length protein) are analyzed (or evaluated) in terms of their ability to be bound by antibodies generated against (or bound to) isolated epitopes corresponding to those epitopes (or potential epitopes) on the protein. In a preferred embodiment, multiple (or a series of) epitopes are searched.

[0269] Therefore, step (v) of method B typically involves searching for one or more epitopes in a region of native (or full-length or wild-type) protein that contains or is adjacent to a predicted protease cleavage site in in silico identified in step (i), but is not a cleavage site identified in step (iii), using one or more antibodies. Alternatively, step (v) of method B typically involves searching for native (or full-length or wild-type) protein in a region of said protein that contains or is adjacent to a predicted protease cleavage site in in silico identified in step (i), but is not a cleavage site identified in step (iii), using one or more antibodies.

[0270] In some embodiments, the method may further include (before step (v)) the step of generating (or synthesizing) one or more isolated epitopes (e.g., multiple, e.g., two or more, three or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to three, four or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to three, four or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to four, five or more, ten or more, twenty or more, fifty or more, e.g., up to fifty, four or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to fifty, four or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to fifty, four or more, five or more, ten or more, twenty or more, fifty or more, e.g., up to fifty, four or more, five or more, ten or more, fifty Such antibodies may then be used in step (v) of the above method for searching for one or more epitopes on the protein (e.g., in the natural or full-length protein). Any suitable methods or techniques (e.g., as described elsewhere herein) for generating isolated epitopes or antibodies may also be used, and these will be familiar to those skilled in the art.

[0271] In some embodiments, epitopes have varying lengths and / or arrangements. Therefore, within a group of epitopes, there may be epitopes with different lengths and / or arrangements. In other embodiments, epitopes have the same (or similar) length and typically different arrangements. Therefore, in some embodiments, within a group of epitopes, the epitopes have the same (or similar) length.

[0272] The epitope can be of any suitable length. In some embodiments, the isolated epitope is 7-8 amino acids long or has a length as described elsewhere herein.

[0273] In a preferred embodiment, the epitope includes (or overlaps with or surrounds) the cleavage site.

[0274] Typically, the epitope (or at least a portion of any given epitope) will be within a range of 50 amino acids of the cleavage site (a protease cleavage site predicted in the in silico identified in step (i), but not the cleavage site identified in step (iii)), i.e., +50 to -50 amino acids relative to the cleavage site. Preferably, the epitope (or at least a portion of any given epitope) will be within a range of 20 amino acids of the cleavage site (a protease cleavage site predicted in the in silico identified in step (i), but not the cleavage site identified in step (iii)), i.e., +20 to -20 amino acids relative to that cleavage site, or within a range of 10 amino acids of the cleavage site, i.e., +10 to -10 amino acids relative to that cleavage site, or within a range of 5 amino acids of the cleavage site, i.e., +5 to -5 amino acids relative to that cleavage site.

[0275] In some embodiments, multiple epitopes are a set (or group) of epitopes, where the sequence of each epitope in the set is offset by one or several amino acids (e.g., one, two, or three), preferably one amino acid, from another epitope in the set. In other words, in some embodiments, in a set of epitopes (multiple epitopes), each epitope sequence is shifted by one or several amino acids (e.g., one, two, or three), preferably one amino acid, from another epitope sequence in the set. Thus, multiple epitopes can be nested sets of epitopes, as shown, for example, in Figure 16d. Typically, such a nested set of epitopes will contain up to about 50 amino acids of the protein sequence in either direction (or both directions) relative to (or surrounding) the cleavage site (the in silico predicted protease cleavage site identified in step (i), but not the cleavage site identified in step (iii)). Preferably, such a nested set of epitopes will contain up to about 20 amino acids of the protein sequence in either direction (or both directions) relative to (or surrounding) the cleavage site (the in silico predicted protease cleavage site identified in step (i), but not the cleavage site identified in step (iii)). In some embodiments, such a nested set of epitopes will contain up to about 6 amino acids of the protein sequence in either direction (or both directions, preferably both directions) relative to (or surrounding) the cleavage site (the in silico predicted protease cleavage site identified in step (i), but not the cleavage site identified in step (iii)).

[0276] When nested sets of epitopes are used, in a preferred embodiment, a substantial number of epitopes include cleavage sites, preferably substantially all epitopes in the nested set include cleavage sites, and more preferably all epitopes in the nested set include cleavage sites.

[0277] As described above, searching for multiple epitopes on a protein located between, overlapping, or adjacent to cleavage sites can be done using an antibody against the epitope (i.e., the antibody functions as a probe). In practice, searching using an antibody (e.g., a Fab fragment or another antibody fragment) is preferred. However, alternatively, other binding entities may be used as probes (e.g., other affinity probes may be used). Affibodies are an example of affinity probes that can be used.

[0278] Preferred proteins are described elsewhere in this specification.

[0279] In another embodiment, as an alternative to actively performing in vitro protease digestion in step (ii) of method B, the active step of protein digestion (in vitro step) is omitted, and instead, the identification of peptides released from the protein, and therefore the identification of cleavage sites in step (iii), is performed based on data from previously performed proteolysis experiments (e.g., archived data, e.g., mass spectrometry data containing the sequences of peptides released from proteins in previous proteolysis experiments). However, in the preferred method, a step of actively performing limited or restrictive proteolysis is performed.

[0280] In one embodiment, the present invention provides an epitope (or antigenic epitope), such as an isolated epitope, identified by a method (Method B) for identifying an epitope on a protein that can be bound by an antibody as described above. In one embodiment, the present invention provides an antibody that binds to such an epitope on a protein. In some embodiments, antibodies that bind to the vicinity of the cleavage site described herein, for example, within a range of 5, 10, 20, or 50 amino acids of the cleavage site, are preferred. Those skilled in the art are familiar with epitopes (e.g., isolated epitopes) and methods or techniques for producing antibodies against a given epitope and can use any suitable method (for example, as described elsewhere in this specification). Preferred types of antibodies are also described elsewhere in this specification.

[0281] In one embodiment, the present invention provides an antibody that binds to an epitope on a protein that includes, or is adjacent to, a protease cleavage site predicted in silico but not a cleavage site identified in vitro by proteolysis (e.g., limited or restriction proteolysis), or an adjacent (preferably included) cleavage site.

[0282] Antibodies targeting epitopes (preferably multiple epitopes) on a protein (e.g., a group or series of antibodies, or a large number of antibodies) can be tested for their ability to bind to the protein, for example, to evaluate their binding affinity to the protein or other functional effects (e.g., as described elsewhere in this specification). Thus, antibodies can be screened to identify the best binding agents. Thus, particularly useful epitopes (e.g., those targeted by antibodies) can be identified, for example, those that are particularly suitable for targeting by high-affinity antibodies, or whose targeting results in a significant or measurable functional effect (e.g., antagonist or agonist effect) on the target protein. Thus, the optimal epitope (e.g., for antibody targeting) can be identified. Thus, from another perspective, the present invention provides a method for optimizing epitope design or selecting the optimal epitope (e.g., for antibodies produced against or targeting it). The method may enable the determination of the optimal length and position of the epitope relative to the cleavage site.

[0283] In some embodiments, the method (Method B) further includes the step of generating (or producing or preparing) an antibody against (or conjugating to) the epitope identified by Method B (identified in step (vi)). Optionally, a further step may be taken to formulate the antibody with at least one pharmaceutically acceptable carrier or excipient.

[0284] Accordingly, in one embodiment, the present invention provides a method for producing or manufacturing an antibody that binds to an epitope identified by method B (identified in step (vi)). Optionally, a further step may be taken to formulate the produced or manufactured antibody with at least one pharmaceutically acceptable carrier or excipient. Methods for producing or manufacturing antibodies are described elsewhere in this specification and are applicable to this embodiment of the present invention with necessary modifications.

[0285] In one embodiment, the present invention provides a complex comprising at least one epitope identified by Method B, conjugated to or mixed with a peptide carrier. The complex is described elsewhere herein, and its considerations apply to this embodiment of the present invention with necessary modifications.

[0286] Antibodies that bind to the epitopes identified by Method B (or conjugates containing the epitopes identified by Method B or such epitopes) can be used for therapeutic purposes.

[0287] Method B can be used to identify epitopes on the protein surface that are accessible / cleaved by proteases but not released. This method can employ a search algorithm based on protease digestion and homology modeling (e.g., Fab-protease homology binding to the target protein) in silico to predict protease cleavage sites on the protein surface. The method utilizes in vitro protease digestion using several proteases (e.g., in parallel) at arbitrary selection. A microfluidic platform can be used for digestion. Mass spectrometry (MS), preferably LS-MS / MS, can be used to identify peptides released from the target protein by the protease. Experimentally determined cleavage sites are elucidated, for example, from peptide maps obtained by MS. Predicted cleavage sites on the protein surface in silico can be compared with experimentally observed cleavage sites. Predicted cleavage sites not experimentally observed in silico can be searched for using antibodies against sequences encompassing the cleavage site (e.g., amino acids -20 to +20 around the cleavage site). Antibodies can be ranked by binding strength (e.g., affinity) and / or activity (e.g., antagonist or agonist activity against the target protein). Antibodies can be tested for binding to both native and digested proteins. If the antibody binds to the cleavage site for both native and digested proteins, it can be concluded that the protease did not cleave there. Conversely, if the antibody binds to the native protein but not to the digested protein, it can be concluded that the site was indeed digested by the protease in vitro, but the release of the peptide could not be detected. This assumes that the antibody is unable to bind to the sequence at the time of cleavage.

[0288] The objective of this method is to elucidate protein structures using novel procedures and algorithms with antibodies used to identify antibody-binding sites and / or epitopes that are accessible / cleaved by proteases but not released. The method is based on in silico digestion and, optionally, modeling of protein structures, and / or simulated docking of antibody fragments (e.g., Fab fragments) and / or proteases to target proteins. Microfluidic protease digestion (e.g., multiple proteases used in parallel as described elsewhere herein) can be used with MS-MS detection. This procedure enables the discovery of unique and novel antibody-binding sites and can generate new structural data for native and partially digested proteins.

[0289] The evaluation of proteolysis using mass spectrometry relies on the release of peptides from the protein, i.e., the cleavage of a protease at two sites surrounding a sequence of a size suitable for detection by mass spectrometry. However, some target regions of a protein may not meet these criteria. A protease may cleave at only one site, creating a cleavage without releasing a peptide. Two cleavages are required for peptide release. Since the peptide is not released, no evidence of binding event or proteolytic activity based on MS is obtained. The single cleavage remains undetected. Other reasons for undetection may include glycosylation of the peptide, or the peptide remaining bound to the protein by ionic or covalent bonds. One way to circumvent this problem is to produce antibodies against sequences containing such cleavage sites. Given the very similar size between the antibody Fab region and the protease (Figure 1), the protease is useful for surface exploration for antibody binding sites, and vice versa.

[0290] If antibody binding is confirmed to a site on a native protein, we know that, based on size similarity, the protease should also bind to that site. If the same antibody binding test is performed on a digested protein, the absence of binding to the site after proteolysis indicates that the target sequence was actually cleaved because the specific epitope recognized by the antibody was destroyed by the protease.

[0291] The workflow is designed to identify silently undetected sectional areas, and is outlined in Figure 16.

[0292] We use one or more different proteases in the digestion of protein sequences in silico. This takes into account the specificity and rules of proteases, for example, that trypsin cleaves only at the C-terminal position of arginine or lysine. Rules and exceptions regarding digestion are given for most proteases; see, for example, Peptidecutter (Expasy, SIB Swiss Institute of Bioinformatics). Therefore, computer programs such as Peptidecutter can be used to perform protease digestion in silico. Optionally, modeling (e.g., protein homology modeling) can be used to estimate which cleavage sites are likely to be exposed to the solvent, and by combining this step with docking of antibody fragments (e.g., Fab fragments) in silico or proteases along the surface, it is possible to predict which cleavage sites are likely to be digested in vitro (Figure 16a). Homology models can be generated using the highly flexible and intuitive homology modeling engine of MOE software (Molecular Operating Environment (MOE) 2015. 10. Chemical Computing Group Inc., 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7. 2016). For example, a homology model of human TRPV1 can be constructed using the low-temperature EM structures of rat TRPV1 (deletion mutants, PDB entries 3J5P29 and 5IRZ30). Based on these structures, we constructed a human TRPV1 homology model using MOE for method development.

[0293] Following the prediction of protease digestion sites, such as surface protease digestion sites, in vitro proteolysis experiments are performed. For membrane-bound proteins, proteoliposomes containing native proteins can be digested in a microfluidic flow cell (LPI, Nanoxis Consulting AB). Flow cell technology allows for flexible chemistry, such as limited proteolysis, on membrane proteins contained in the stationary phase (Jansson ET, Trkulja CL, Olofsson J, et al. Microfluidic flow cell for sequential digestion of immobilized proteoliposomes. Anal Chem. 2012;84(13):5582-5588), and can be subjected to several rounds of a series of processes with solution and various types of chemical regulation, such as by enzymes. The cell membrane can be inverted, allowing direct examination of both the intracellular and extracellular domains of transmembrane proteins. Soluble proteins can be subjected to limited proteolysis using standard in-solution methods.

[0294] To cover as many sequences as possible, multiple proteases with varying specificities can be used in parallel reactions. Established limiting conditions, such as protease concentrations in the range of 2–5 μg / mL and digestion for 5 minutes, can be used to limit proteolysis to the protein surface (Figure 16b). Other limiting conditions (limited or restrictive proteolysis conditions) are discussed elsewhere in this specification, and any of these can be used according to this embodiment (Method B). The released peptides can be identified by mass spectrometry (e.g., LC-MS / MS), preferably using a high-resolution mass spectrometer (e.g., Q Exactive, Thermo Fisher) and Mascot peptide / protein identification. With a peptide map at hand, we can determine which cleavage sites are physically accessible by the protease.

[0295] To identify sites that are accessible / cleaved by proteases but not released, we compare experimentally confirmed cleavage sites with a list of predicted sites and select missing predicted sites from MS data (Figure 16c). To construct polyclonal antibodies (pAbs), peptide sequences containing these sites, preferably 7-8 amino acids in length, can be synthesized and used. The reason for selecting this length is to minimize the polyclonality of the pAb by minimizing the target sequence, while ensuring the sequence is not so short as to be immunogenic.

[0296] Using a single amino acid frameshift, a linear sequence of this length (e.g., 6 amino acids) can be selected within a set distance on each side of the cleavage site. These can then be used to construct a series of sequence target pAbs, which can then be screened for binding to native, intact proteins, for example, using ELISA.

[0297] The observed binding event means that the site is accessible by the antibody, and therefore should also be accessible by the protease, and vice versa. This also suggests that the protease should be cleaved at that site, but the peptide is not released. Verification can be achieved by using the same set of antibodies on digested proteins, and the decrease in binding to the cleavage site confirms that proteolysis has occurred (Figure 16e). This assumes that the antibody does not bind to the broken sequence, but antibodies are generally very specific to the amino acid configuration used as an epitope.

[0298] This methodology has the potential to be used not only to determine protease cleavage but also as a tool to detect truncation, excision, or loss of local domain structures. For example, verification of truncation is useful when truncation cannot be evaluated by mass spectrometry because the released sequence is too short or too long for MS detection, or when the peptide involves glycosylation or remains bound to the protein by one or more ionic or covalent bonds. We correlated peptide excision with a functional assay of TRPV1. Furthermore, changes in local structure caused by ligand binding or protein-protein interactions can be explored using these antibodies. Antibodies produced by this method can also be used as sequence-targeted functional antibodies for therapeutic use.

[0299] To avoid misunderstanding, the use of a search algorithm is not essential in the method of the present invention, but such algorithms may be used. For example, algorithms can be used to analyze (or process) one or more datasets in silico (e.g., protease digestion data and / or protein homology modeling data and / or protein structure and function data contained in (or predicted by) computer models in silico), one or more Fab-protease homology conjugation datasets (e.g., those generated by protein-protein docking in silico), one or more other protein-protein docking models in silico (such as protein-antibody docking models, protein-antibody fragment docking models, and / or protein-protease docking models), and / or one or more protease digestion (e.g., multiprotease digestion) datasets (e.g., mass spectrometry data). To find (or predict) protein structural regions that are functionally accessible by antibodies and are functionally relevant to protein function (for example, perturbations in those regions that alter protein function), search algorithms can combine and process inputs from one or more different datasets.

[0300] Preferred features of other methods described herein can be applied to this embodiment of the present invention (Method B) with necessary modifications.

[0301] In embodiments in which multiple proteases are used, suitable examples are described elsewhere in this specification. In some embodiments, preferred proteases are one or more selected from the group consisting of (or comprising) trypsin, Asp-N, chymotrypsin, pepsin, proteinase K, Lys-C, Arg-C, clostripine, glutamyl endopeptidase, Lys-N, and thermolysin.

[0302] Other features and advantages of the present invention are evident from the following examples. The provided examples illustrate various components and methodologies useful for carrying out the present invention. These examples do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and adopt other components and methodologies useful for carrying out the present invention. [Examples]

[0303] Example 1 This embodiment describes a successful method for discovering and developing the action of a polyclonal antibody OTV1 acting on the intracellular side of the human TRPV1 ion channel, based on our proposed invention and encompassing methods. The antibody is pharmacologically active and exhibits strong inhibitory activity against the protein when stimulated with the agonist capsaicin. To our knowledge, this is the first discovery of an inhibitory antibody targeting the intracellular domain of TRPV1. This demonstrates that the concept has a high probability of being practically usable, and that better and more optimized antibodies could be identified if a more abundant starting matrix of epitopes derived from multiple protease datasets were available. The antibody was selected from numerous hits of limited proteolysis and bioinformatics analysis. The antibody was initially selected and showed strong evidence of efficacy. This is a significant advance, as it directly provides a unique epitope that can be targeted by a pharmacologically active antibody, eliminating the need for a screening step and complementing current attempts to identify antibodies.

[0304] The target epitope region was selected and further optimized based on limited digestion of the target protein using the LPI microfluidics platform's optimization protocol. Polyclonal antibodies were produced by modifying the target peptide epitope with a cysteine ​​residue and binding it to keyhole limpet hemocyanin (KLH). Specific antibodies were produced by immunizing SPF rabbits with KLH conjugated to the specific peptide. The antibodies were purified and subjected to ELISA testing according to a standard protocol. Antibody titers against the linear epitope were measured by ELISA, resulting in a concentration of 0.25 μg / ml. The efficacy of the antibody against native TRPV1 was investigated using inside-out patch clamp recording, which allows the intracellular side of TRPV1 to be exposed to the antibody solution. Inside-out recording was performed using a patch clamp recording microfluidics instrument (Dynaflow, Cellectricon AB, Gothenburg, Sweden). Current amplitude was measured by exposing patches containing several ion channels to capsaicin both with and without antibody. The control was exposed to 1 μM capsaicin for 30 seconds, followed by 70 seconds in buffer, and then again to 1 μM capsaicin for 30 seconds. The antibody-treated patches were exposed to 1 μM capsaicin for 30 seconds, followed by 70 seconds in 0.14 mg / ml antibody, and then 30 seconds in 1 μM capsaicin with 0.14 mg / ml antibody. For all measurements, the activity with antibody was compared to the activity after exposure to buffer only to rule out any effects of desensitization or enhancement. The current-time integral area was calculated, and the ratio of the integral areas for the second and first currents was calculated and compared between treatments. For antibody-treated cells, a 50% reduction in current response was observed compared to the buffer-only treatment (Figure 3). Statistical significance was calculated using Student's t-test (p>0.05).

[0305] Example 2 The therapeutic monoclonal antibody market is growing rapidly and is expected to reach a value of approximately US$125 billion in 2020. Novel monoclonal antibodies are gaining regulatory approval one after another, and immunotherapy-based monoclonal antibodies such as PD1 inhibitors are currently attracting considerable attention because they significantly improve outcomes for certain types of refractory metastatic cancer. However, the discovery of novel antibodies for therapeutic purposes is largely reliant on screening and is largely trial and error. Further investigation is needed to examine the biological effects of subsets of antibodies that exhibit favorable binding characteristics, focusing on affinity. The details of binding interactions, antigenic determinants, and mechanisms of action remain unclear.

[0306] We present a method for selecting antigen epitopes based on a kinetic challenge of limited proteolysis using microfluidics and mass spectrometry. The proteolysis step is performed slowly enough that, after exposure to protease, the antigen separates into one or a few peptides at that point. The first peptides produced are readily accessible to polyclonal or monoclonal antibodies and are therefore preferred over later-produced peptides that reside in more difficult-to-reach regions of the protein. These peptides are then ranked and correlated with their sequence-based functional significance using selected bioinformatics data. Highly ranked peptides that detach quickly from the target protein and possess functional significance are used for epitope development, immunization, and subsequent antibody production. The truncated proteins can also be used for pharmacological testing. This method relies on sequence-based information and is based on the pharmacological mechanism of action for antibody discovery, and can be used for intracellular, circulating, and extracellular targets. Using this method, we have developed two antibodies: one that activates the calmodulin binding sequence, and another that inhibits the capsaicin binding site at the N-terminus of the intracellular region of the human TRPV1 ion channel.

[0307] Two key parameters in developing therapeutic antibodies are binding affinity and biological efficiency. Antibodies are large proteins of approximately 150 kDa and primarily bind to antigenic sites on the protein surface. The positions of amino acids near the surface of the native protein structure can guide the identification and prediction of these sites. We used limited proteolysis to explore the surface exposure and flexibility of proteins. Limited proteolysis is a method of limiting protease activity by controlling temperature, concentration, and / or digestion time. Under such conditions, only flexible regions that can locally unfold and accommodate proteases, surface-exposed regions, and regions with few local interactions such as hydrogen bonds and disulfide bridges will be digested. To maximize the acquisition of structural information, we used several proteases in parallel. Regions that are easily digested by several proteases should be located in the most exposed and accessible regions of the protein and should be highly suitable for subsequent antibody development. Regions digested by only a single protease are likely to be located in hidden regions of the protein and are difficult to access. The physiological and chemical properties of proteases capable of reaching and digesting those regions could potentially guide antibody development in such cases. We ranked digested peptides based on their digestibility, depending on which parameters were used to limit proteolysis. These parameters could include the time of digestion, the concentration, or the temperature used. We then associated the digested peptides from each protease to identify those peptides derived from the most accessible regions of the protein.

[0308] In conventional antibody development, biological efficiency is generally investigated after clear binding between the antibody and antigen has been confirmed. We believe that antibody development will benefit from an early mechanistic-driven approach, which focuses immunization on accessible sites in or near the biologically active site, rather than creating antibodies that target all possible antigen-determining sites. This approach minimizes the risks associated with screening procedures and optimizing antibodies that have high binding affinity to regions distant from the biologically active site. We wanted to find accessible epitopes that also have functional importance to the target protein. This was done by comparing ranked peptides from proteolytic degradation with bioinformatics data.

[0309] We demonstrated a mechanism-driven approach using the human TRPV1 ion channel as a model protein. TRPV1 is an ion channel highly sensitive to noxious stimuli such as low pH, high temperature (T>42°C), capsaicin, and several inflammatory mediators. The TRPV1 ion channel is mainly located in nociceptive neurons of the peripheral nervous system and is arranged in a tetrameric conformation. Each of its four monomers consists of six transmembrane domains, with both its N-terminus and C-terminus facing the intracellular side of the cell membrane. The pore region is contained in the fifth and sixth transmembrane domains. The intracellular portion of TRPV1 holds many regulatory regions that are important for thermal activation, sensitization, and desensitization.

[0310] Epitope Production Proteoliposomes containing TRPV1 were obtained from CHO cells and subjected to proteolysis in an LPI flow cell using trypsin and Asp-N separately. Protease activity was limited to digesting only a few peptides using room temperature and low concentrations. The digested peptides were then detected by liquid chromatography and tandem mass spectrometry (LC-MS / MS). Three peptides were detected after proteolysis with trypsin, and one peptide was detected after proteolysis with Asp-N. By comparing these peptides with known functional data, several peptides were associated with functionally important regions, as shown in Table 1. Two peptides, aa96-117 and aa785-799, were selected for further antibody development and named OTV1 and OTV2, respectively. Visualizations of the epitopes within the TRPV1 structure can be seen in Figures 4 and 5. The peptide sequence of OTV1 contains arg115 (arg114 for rTRPV1), which has been shown to be important for activation with capsaicin or protons. Both proteases digested a region close to this amino acid, increasing the likelihood that it is an exposed region of the protein's tertiary structure. The peptide sequence of OTV2 contains calmodulin binding sites aa786~aa798 (aa785~aa797 for rTRPV1) and was digested only by trypsin. The TRPV1 portion does not have an Asp-N digestion site that cleaves the N-terminal side of Asp and Cys. Synthetic peptides aa96~117 and aa785~799 were conjugated to limpet hemosinian (KLH) and further used to produce polyclonal antibodies by immunization of rabbits injected with KLH-conjugated peptides. The produced antibodies tend to aggregate when frozen and over time in solution. As a result, newly thawed antibodies were sonicated using the tip before use, and all experiments were performed within 30 minutes of tip sonication.

[0311] Table 1 - Asp-N and trypsin-digested peptides, and their biological relationships

[0312] [Table 1]

[0313] immunocytochemistry Immunocytochemistry was performed to visualize the antibody distribution within TRPV1-expressing CHO cells (Figure 6). Uninduced cells were used as a control for nonspecific binding. Cells were fixed and stained with either OTV1 or OTV2, followed by staining with goat anti-rabbit Alexa488 secondary antibody. Clear staining of the cell membrane was observed only in induced cells and was seen with both OTV1 and OTV2. Nonspecific binding of the secondary antibody was negligible (data not shown).

[0314] Electrophysiology Functional effects of OTV1 on capsaicin-induced TRPV1 activity and calmodulin / Ca 2+ The effect of OTV2 on ion-dependent desensitization was evaluated using inside-out patch-clamp recordings. A membrane patch containing several ion channels was excised from CHO cells to allow the antibody to expose the intracellular region of TRPV1. For OTV1, TRPV1 was activated with capsaicin, then treated with OTV1, and subsequently activated with capsaicin in the presence of OTV1. The control was activated with capsaicin, treated with buffer, and then activated with capsaicin again. Compared to treatment with buffer alone, treatment with OTV1 showed a 50% reduction in capsaicin-mediated current (Figure 7). Calmodulin / Ca 2+ We investigated OTV2's ability to inhibit addiction desensitization. TRPV1 was activated with capsaicin, followed by calmodulin, Ca 2+ , and then treated with OTV2, followed by calmodulin, Ca 2+ , and were activated with capsaicin in the presence of OTV2. The control was activated with capsaicin and calmodulin and Ca 2+ Processed with calmodulin and Ca 2+ It was activated with capsaicin in the presence of [unspecified substance]. Calmodulin desensitizes TRPV1 in the presence of calcium. Treatment with OTV2 reduced this effect by 45% (Figure 7).

[0315] TRPV1-mediated YO-PRO uptake assay The efficacy of antibodies within all cells was investigated by measuring TRPV1-mediated YO-PRO uptake using laser scanning confocal microscopy after delivery via electroporation. Cells were electroporated using a neon transfection system (Life Technologies) in the presence of either OTV1, OTV2, or a buffer. Cells electroporated with OTV1 or the buffer were subjected to capsaicin and YO-PRO in phosphate-buffered saline containing a calcium chelator. Subsequently, the increase in intracellular fluorescence due to TRPV1-mediated YO-PRO uptake was monitored. A 60% decrease in uptake rate was observed in cells treated with OTV1 during the first 12 seconds of activation. The highest uptake rate was observed at 20 seconds in OTV1-treated cells compared to 8 seconds in the control (Figure 8). Cells electroporated with OTV2 or the buffer were subjected to capsaicin and YO-PRO in phosphate-buffered saline containing calcium, and desensitization was performed via endogenous calmodulin induced by the added calcium. An 80% increase in uptake rate was observed 15 seconds after activation of cells treated with OTV2. Antibody internalization by electroporation was confirmed using immunocytochemistry (Figure 9).

[0316] We have developed a microfluidics method for antibody production that identifies the location of exposed and accessible antigen-determining sites within and / or near functionally important regions of a target protein. Accessible regions are examined using kinetically limited proteolysis within an LPI flow cell. The target protein is preserved in its native state while the complexity of its environment is carefully regulated, for example, by allowing the presence of cofactors. This results in better access to antigen-determining sites compared to binding assays using purified proteins. This method is well-suited for transmembrane targets that are difficult to purify without surfactants and use in binding assays by other methods. Both intracellular and extracellular domains can be targeted using this method.

[0317] Knowledge of the location and biological function of antigen determination sites is crucial for predicting and evaluating nonspecific binding and cross-reactivity with other proteins. To minimize cross-reactivity, epitopes located in highly conserved regions can be excluded from the analysis of potential epitope candidates.

[0318] The antibodies developed herein are polyclonal, although they do not result from immunization with whole proteins. Our method is comparable to conventional protocols for the production of monoclonal antibodies using hybridomas and subsequent screening procedures. We combine the best of both worlds by first experimentally validating the biological efficiency of several promising epitope candidates using polyclonal antibodies, then producing monoclonal antibodies using the best epitope(s), followed by a screening procedure based on high binding affinity.

[0319] Verification of antibody internalization Antibody internalization using electroporation was validated by immunocytochemistry 24 hours after electroporation. Cells were electroporated in phosphate-buffered saline in the presence of 0.14 mg / ml OTV1 or 0.27 mg / ml OTV2. The electroporated cells were then cultured for 24 hours in glass-bottom dishes (Willco Wells). Two different controls were prepared. One set was not electroporated, while the rest were treated similarly and subjected to the same antibody solution. The other set was not subjected to OTV1 and OTV2. The latter was used to quantify nonspecific binding of the secondary antibody. After 24 hours of culture, the cells were carefully washed with phosphate-buffered saline to remove any residual antibodies that could enter the cells during fixation. The cells were then fixed and permeabilized using the Image-iT® fixation / permeabilization kit (Invitrogen). The fixed and permeabilized cells were incubated with goat anti-rabbit Alexa488 secondary antibody (Invitrogen) at room temperature for 30 minutes. After the final washing step, cells were visualized, and fluorescence intensity was compared between electroporated cells, unelectroporated cells, and cells exposed only to the secondary antibody (Figure 9). A clear difference in intensity values ​​was observed between electroporated and unelectroporated cells. Statistical analysis was performed using Student's t-test, with p<0.05 considered statistically significant. Low levels of primary antibody were observed in unelectroporated cells, likely residual antibodies introduced during fixation and permeabilization.

[0320] In this specification, we present a method for producing highly affinity and biologically active antibodies by combining microfluidics and limited proteolysis. We demonstrated this method using the human TRPV1 ion channel and developed two antibodies. Both antibodies induced predicted changes in the TRPV1 response based on the functional importance of their respective epitope regions.

[0321] Materials and Methods Reagents Cell culture medium (DMEM / Ham's F12 containing glutamine), fetal bovine serum, and actase were purchased from PAA. Zeosin, Na4BAPTA, K4BAPTA, and goat anti-rabbit Alexa488 secondary antibody were purchased from Invitrogen. Sequencing-grade modified trypsin and sequencing-grade Asp-N were purchased from Promega. All other chemicals were purchased from Sigma. The following buffers were used: A: 300mM NaCl, 10mM Tris, pH 8.0, B: 20mM NH4HCO3, pH 8.0, C: 140mM NaCl, 5mM KCl, 1mM MgCl2, 10mM HEPES, 10mM D-glucose, 10mM Na4BAPTA pH7.4, D:140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 10mMK4BAPTA pH7.2, E:140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, pH7.2, F:140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, pH7.4, G:120mM KCl, 2mM MgCl2, 10mM HEPES, 10mM K4BAPTA, pH 8.0

[0322] cell culture Adherent Chinese hamster ovary (CHO) cells possessing the tetracycline-regulated expression system (T-REx) were cultured in culture flasks or culture dishes (Nunc) with and without glass slides in medium (DMEM / F12 containing glutamine) supplemented with 10% fetal bovine serum, zeosin (350 μg / ml), and blastosidine (5 μg / ml). For 18-24 hours prior to use, cells were cultured in medium (DMEM / F12 containing glutamine) supplemented with 10% fetal bovine serum and doxycycline (1 μg / ml) to induce human TRPV1 expression. Cell lines were always tested for mycoplasma infection.

[0323] Proteoliposome preparation Proteoliposomes were prepared in buffer A as already described in reference [1]. Each proteoliposome preparation was derived from several different culture flasks.

[0324] Digestive protocol Single digestion in a flow cell was performed as described in reference [1]. 5 μg / ml trypsin and 5 μg / ml Asp-N were dissolved in buffers G and B, respectively. Digestion with each protease was performed in a flow cell at room temperature for 5 minutes. Further digestion in the eluent was inhibited by adding 12% formic acid to the final concentration.

[0325] Liquid chromatography-tandem mass spectrometry Peptide samples derived from the digestion of CHO-proteoliposomes were analyzed at the Proteomics Core Facility at the University of Gothenburg (Gothenburg, Sweden) using the method described in reference [1]. All tandem mass spectrometry spectra were searched in MASCOT (Matrix Science, London, UK) with reference to UniProtKB. For UniProtKB references, digestion with trypsin was based on release 2013_04 (human, [homo sapiens]), and digestion with Asp-N was based on release 2015_06 (human, [homo sapiens]). Thermo Proteome Discoverer version 1.3 (Thermo Scientific) was used to confirm MS / MS based on peptide and protein identification. A false positive rate of 0.01 at the peptide level was used and determined by searching reversed databases.

[0326] Antibody development Synthetic peptides aa96-117 and aa785-799 were synthesized and purified, including an additional cysteine ​​residue at the N-terminus, based on the amino acid sequence of hTRPV1. The peptides were then conjugated to keyhole limpet hemocyanin (KLH) via the cysteine ​​residue. Polyclonal antibodies were produced by immunizing SPF (Specific Pathogen Free) rabbits with the KLH-conjugated peptide. The antibodies were purified and subjected to ELISA testing. Both synthetic peptides and polyclonal antibodies were produced by Innovagen AB (Lund, Sweden).

[0327] Fresh antibodies were used, processed within 30 minutes of tip sonication. The antibodies were sonicated three times at 14% amplitude with a 1-minute pause in between, using a Vibra Cell VCX600 from Sonics & Materials Inc. (Newtown, Connecticut, USA). The total sonication time was 40 seconds, with a pulse time of 0.5 seconds and a 0.5-second pause to reduce heating by the probe.

[0328] Electrophysiology Inside-out recording was performed using a patch-clamp microfluidic system (Dynaflow, Cellectricon AB, Gothenburg, Sweden) with a HEKA EPC10 (Heka Elektronik, Germany) patch-clamp amplifier. The chamber and pipette solution contained buffer C. The patch was maintained at +60mV, and the current signal was recorded at a sampling frequency of 20kHz and with a 5kHz low-pass filter.

[0329] For OTV1, the current amplitude was measured by subjecting patches containing several ion channels to capsaicin with and without antibody. The control was subjected to 1 μM capsaicin in buffer D for 30 seconds, then to buffer D for 70 seconds, and then to 1 μM capsaicin in buffer D for 30 seconds. Patches treated with OTV1 were subjected to 1 μM capsaicin in buffer D for 30 seconds, then to 0.14 mg / ml antibody in buffer D for 70 seconds, and subsequently to 1 μM capsaicin with 0.14 mg / ml antibody in buffer D for 30 seconds. For OTV2, the current amplitude was measured by subjecting patches to capsaicin with and without antibody and calmodulin / Ca 2+ . The control was subjected to 1 μM capsaicin in buffer E for 30 seconds, then to 0.5 μM calmodulin and 50 μM Ca 2+ in buffer E for 70 seconds, and then again to 1 μM capsaicin in buffer E for 30 seconds. Patches treated with antibody were subjected to 1 μM capsaicin in buffer E for 30 seconds, then to 0.14 mg / ml antibody, 0.5 μM calmodulin, and 50 μM Ca 2+ in buffer E for 70 seconds, and then to 1 μM capsaicin with 0.14 mg / ml antibody, 0.5 μM calmodulin, and 50 μM Ca 2+ in buffer E for 30 seconds. Measurements with a large shift in seal resistance after treatment were excluded from further analysis.

[0330] Data analysis Electrophysiology For all measurements, the activity after antibody treatment was compared to the activity after treatment with buffer only to eliminate any effects of desensitization or enhancement due to repeated activation. For data including current traces, the current-time integrated area was calculated for capsaicin activation for OTV1 between addition and removal, and for OTV2 between full activation (after 10 seconds) and removal, using Fitmaster (HEKA Elektronik, Germany) and Matlab (Mathworks, Massachusetts, USA). The ratio of the integrated areas of the second and first currents was calculated and compared between treatments. For OTV2, since the effect decreased in a time-dependent manner, the data points were grouped into two categories (<15 minutes before tip sonication and <30 minutes after tip sonication).

[0331] Statistical analysis was performed using one-way ANOVA, and, where applicable, Dunnett's post-hoc test and Student's t-test. A p-value of <0.05 was considered statistically significant. Data are presented as mean ± SEM.

[0332] Electroporation Antibody delivery to the cell substrate was performed using a neon transfection system (Life Technologies). Adherent CHO cells were detached using actase and washed with buffer F. 10⁵ cells were pelleted and resuspended in either buffer F, 0.14 mg / ml OTV1 in buffer F, or 0.27 mg / ml OTV2 in buffer F. 10 μl of cell / antibody suspension was transferred using a neon pipette tip and subjected to electroporation at the system's pipette station. A protocol optimized for antibody delivery [5] was used, in which the cells were exposed to 1550 V for 10 milliseconds in three pulses. The electroporated cells were transferred to a glass-bottom dish (Willco Wells).

[0333] Imaging Immunocytochemistry-mediated antibody localization and TRPV1-mediated YO-PRO uptake were measured using region of interest (ROI) analysis from fluorescence microscopy images. Microscopy images were formed using a Thorlabs CLS system (Thorlabs Inc., New Jersey, USA) equipped with a Galvo:Resonant scanner and a high-sensitivity GaAsP PMT, which were recorded using ThorImageLS software. The scanner unit was mounted on a Leica DMIRB microscope with an oil-immersion 63×A 1.47 Leica HCX PL APO objective lens. Fluorescence detection was performed from single cells excited at 488 nm using a Coherent Sapphire 488 LP laser (Coherent Inc., California, USA). Emissions were collected in the 500–550 nm range. ROI data were analyzed using ImageJ and Matlab (Mathworks, Massachusetts, USA).

[0334] immunocytochemistry Cells were cultured in glass-bottom dishes (Willco Wells), and TRPV1 expression was induced in some dishes 18–24 hours before use. Both dishes containing TRPV1-expressing cells and dishes containing uninducible cells were washed, fixed, and permeabilized with buffer F using the Image-iT® fixation / permeabilization kit (Invitrogen). The fixed and permeabilized cells were exposed to 25 μg / ml antibody in buffer F at 37°C for 30 minutes, then washed with buffer F, and incubated with goat anti-rabbit Alexa488 secondary antibody at room temperature for 30 minutes. After the final washing step, the cells were visualized, and antibody distribution was compared between induced and uninducible cells.

[0335] TRPV1-mediated YO-PRO uptake A glass-bottom dish containing 10 μl of electroporated cells was placed on the microscope. Recording was initialized at a rate of 0.5 Hz. For OTV1, 20 μl droplets containing capsaicin, YO-PRO, and K4BAPTA in buffer F were carefully pipetted onto the electroporated cells to minimize detachment. The final concentrations were 1 μM for capsaicin, 1 μM for YO-PRO, and 10 mM for K4BAPTA. For OTV2, capsaicin, YO-PRO, and Ca were added to buffer F. 2+ A 20 μl droplet containing the following was similarly pipetted onto the electroporated cells. The final concentrations were 1 μM for capsaicin, 1 μM for YO-PRO, and Ca 2+ The concentration became 50 μM.

[0336] The above embodiments should be understood as a few illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the invention. In particular, different partial solutions in different embodiments can be combined with other configurations, where technically possible.

[0337] References1 Jansson, ET; et. al., Anal. Chem. 2012, 84: 5582-55882 International Publication No. 2006 / 0686193 European Patent Application No. 2174908 (EP 2174908)4 Trkulja, CL, et al., J. Am. Chem. Soc. 2014, 136: 14875-148825 Freund, G. et al., MAbs, 2013, 5: 518-522

[0338] Example 3 Limited Digestion and Mass Spectrometry of Ion Channel TRPV1 Expressed in CHO Cells Using Multiple Proteases This example describes the use of multiple proteases in parallel to identify a protease-specific set of peptides derived from TRPV1. The proteases used in this example were trypsin, Asp-N, pepsin, proteinase K, and chymotrypsin. When compared to each other, the protease-specific sets of peptides may overlap, complement, or be unique. Various proteolytic activities were obtained by using different protease concentrations, and in a few cases by using different incubation times.

[0339] Materials and Methods: Cell Culture In short, CHO cells were cultured according to Trkulja et al. (J.Am.Chem.Soc.2014, 136, 14875-14882). In short, adherent Chinese hamster ovary (CHO) cells with a tetracycline-regulated expression system (T-REx) were cultured in T175 or T500 culture flasks (Nunc) or on glass dishes in medium (DMEM / F12 containing glutamine) supplemented with 10% FBS, zeosin (350 μg / mL), and blastosidine (5 μg / mL). Before use (18-24 hours), cells were cultured in medium (DMEM / F12 containing glutamine) supplemented with 10% FBS and doxycycline (1 μg / mL) to induce human TRPV1 expression. Cell lines were always tested for mycoplasma infection. After cell collection, the cells were frozen and stored at -80°C. The cells were further processed as follows.

[0340] Cell lysis and homogenization The cell suspension was centrifuged at 580xg for 3 minutes. The supernatant was discarded, and 4 ml of ice-cold phosphate-buffered saline was added to the tube. The cell pellet was carefully suspended, and then the tube was filled to 14 ml with ice-cold phosphate-buffered saline. The cell suspension was centrifuged again at 580xg for 3 minutes, and this procedure was repeated twice.

[0341] The cell pellet (approximately 800 μl) was suspended in about 6 ml of lysis buffer (10 mM NaHCO3, pH 7.4) and kept on ice for 10 minutes.

[0342] Next, the cells in the lysis buffer were transferred to one Dounce homogenizer (7 ml) for each cell suspension. Then, the cells were homogenized using a narrow-gap pestle in 20 strokes. After homogenization, the lysed cells were centrifuged at 580xg for 3 minutes. The supernatant was collected and the cell pellet was discarded. The supernatant was subjected to a second centrifugation step at 580xg for 3 minutes, and the remaining cell pellet (small amount) was discarded.

[0343] The supernatant was collected and transferred to a Beckmann centrifuge tube (50 ml), and lysis buffer was added until the volume reached 20 ml. The supernatant was centrifuged at 7300xg for 10 minutes to remove mitochondria and cellular debris. The supernatant was divided into two Falcon tubes (10 ml each) and frozen in a -80°C freezer for further processing.

[0344] Ultracentrifugation The supernatant was thawed on ice and transferred to two Beckman clear ultracentrifuge tubes (Beckman Coulter, item number 344057). The tubes were filled with ice-cooled buffer (10 mM Tris, 300 mM NaCl, pH 8), carefully balanced, and centrifuged at 100,000 xg (32900 rpm) for 45 minutes using an SW55 Ti rotor (Beckman Coulter). The supernatant was discarded, the pellet was suspended in ice-cooled buffer (10 mM Tris, 300 mM NaCl, pH 8), and the tubes were again filled with the same ice-cooled buffer. After carefully balancing and centrifuging at 100,000 xg (32900 rpm) for 45 minutes, the supernatant was discarded, and the pellet was suspended in ice-cooled buffer (10 mM Tris, 300 mM NaCl, pH 8) at a rate of approximately 800 μl per pellet. A total of approximately 1.6 ml of membrane preparation was collected and frozen at -80 degrees Celsius.

[0345] Ultrasonic treatment of the tip The frozen membrane preparations were thawed on ice and combined, then sonicated in an ice-cold conical vial using a sonicator (Vibracell). First, the membrane preparations were diluted to 4 ml with ice-cold buffer (10 mM Tris, 300 mM NaCl, pH 8) and subjected to 30 seconds of sonication using 15% amplitude and a 0.5-second pulse / pause cycle. Next, the conical vial and membrane preparations were cooled on ice for several minutes, and then the membrane preparations were subjected to another 30-second cycle using 15% amplitude and a 0.5-second pulse / pause cycle, and this was repeated. The resulting membrane preparations (proteoliposomes) were dispensed into 310 μl portions and frozen at -80°C.

[0346] protease All proteases were purchased from Promega. All solutions were prepared using Fischer Scientific LC-MS grade water. Catalog number V1621 Asp-N, sequencing grade, 2 μg Catalog number V1959 Pepsin, 250 mg Catalog number V3021 Proteinase K, 100 mg Catalog number V1062 Chymotrypsin, sequencing grade, 25 μg Catalog number V5111 Sequencing grade modified trypsin, 20 μg trypsin Trypsin was dissolved in 100 mM ammonium bicarbonate and Ambic. pH 8 Asp-N Asp-N was dissolved in 100 mM ammonium bicarbonate and Ambic. pH 8 Pepsin Pepsin was dissolved in 100 mM ammonium bicarbonate and Ambic. pH 8 Proteinase K Proteinase K was dissolved in 100 mM ammonium bicarbonate and Ambic. pH 8. Chymotrypsin Chymotrypsin was dissolved in 100 mM Tris-HCl and 10 mM CaCl2. pH 8

[0347] LPI process The experiment was conducted using digestive LPI HexaLane tips. Within each tip, one lane was used for one digestion. Briefly, dispensed proteoliposomes were thawed at room temperature, manually injected into the lanes using a 100 μl pipette, and fixed for 1 hour.

[0348] The lanes were also washed manually using a 100 μl pipette. Each well was washed with 200 μl of washing buffer. (The same buffer was used as the digestion buffer for all protocols except the pepsin digestion protocol. In the pepsin digestion protocol, 100 mM Ambic pH 8 was used as the washing buffer. This was to avoid keeping the flow cell at a low pH for an extended period.) Next, the lanes were washed with 4 × 100 μl of washing buffer using a 100 μl pipette.

[0349] Next, proteases were injected into the lanes and cultured according to the details below. Incubation (digestion) was performed at room temperature. After digestion, peptides were eluted from the lanes using 200 μl of digestion buffer (2 × 100 μl). Protease activity was stopped by acidifying the solution with 4 μl of formic acid. As a result, the peptide solution had a pH of approximately 2. This procedure was performed for all samples except pepsin. For pepsin, 16 μl of ammonia solution (25%) was added instead to make the solution basic (pH 9).

[0350] The following digestion conditions were applied to each lane, one at a time. Trypsin 0.5 μg / ml for 2.5 minutes 0.5 μg / ml for 5 minutes 2 μg / ml for 5 minutes 5 μg / ml for 5 minutes 10 μg / ml for 5 minutes 20 μg / ml for 5 minutes Asp-N 20 μg / ml for 5 minutes 2μg / ml 24 hours Chymotrypsin 5 μg / ml for 5 minutes 10 μg / ml for 5 minutes 20 μg / ml for 5 minutes Proteinase-K 5 μg / ml for 5 minutes 10 μg / ml for 5 minutes 20 μg / ml for 5 minutes pepsin 2 μg / ml for 5 minutes 5 μg / ml for 5 minutes 10 μg / ml for 5 minutes 20 μg / ml for 5 minutes

[0351] The samples were labeled and frozen at -80°C.

[0352] MS analysis Trypsin peptides were desalted and dried using a PepClean C18 spin column (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's guidelines, and redissolved in 3% gradient-grade acetonitrile (Merck KGaA, Darmstadt, Germany) containing 15 μl of 0.1% formic acid (Sigma-Aldrich, St. Louis, Missouri). 2 μl of the sample was injected into an Easy-nLC autosampler (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and analyzed using an Interface Q Exactive hybrid mass spectrometer (Thermo Fisher Scientific). The peptides were passed through a pre-column (45 × 0.075 mm inner diameter) and then separated using a chamber-packed reverse-phase column, 200 × 0.075 mm, 3 μm Reprosil-Pur C18-AQ particles (Dr. Maisch, Ammerbuch, Germany). The nano-LC (liquid chromatography) gradient was set to 200 nl / min, starting with 7% acetonitrile (ACN) containing 0.2% formic acid, increasing to 27% ACN over 25 minutes, then to 40% over 5 minutes, and finally to 80% ACN over 5 minutes, with the 80% ACN level held for 10 minutes.

[0353] In data-dependent cation mode analysis, ions were generated under a voltage of 1.8 kV and a capillary temperature of 320 degrees Celsius and sprayed into the mass spectrometer. In the orbitrap, a full-scan (MS1) spectrum was obtained with an AGC target value of 1e6 in the m / z range of 400–1,600 and a charge range of 2–6, at a resolution of 70,000 and a maximum duration of 250 ms. MS / MS spectra were obtained using high-energy collisional dissociation (HCD) from m / z 110 to 30%, with a precursor isolation mass width of 2 Da up to an AGC target value of 1e5 during an injection time of 110 ms, at a resolution of 35,000, for the top 10 most abundant parent ions. Dynamic exclusion for 30 seconds after MS / MS selection allowed for the detection of as many precursors as possible.

[0354] Summary of results Figure 10 shows the location of the peptide detected after limited trypsin-mediated proteolysis in a 3D model of TRPV1. The sequences of the peptide detected after limited trypsin-mediated proteolysis are shown in Table 2 below. The peptide digested with 0.5 μg / ml trypsin for 2.5 minutes is shown first. Peptides digested with 0.5 μg / ml trypsin for 5 minutes, 2 μg / ml trypsin for 5 minutes, 5 μg / ml trypsin for 5 minutes, 10 μg / ml trypsin for 5 minutes, and 20 μg / ml trypsin for 5 minutes are collected for presentation and shown next.

[0355] [Table 2]

[0356] Figure 11 shows the location of the peptides detected after limited proteolysis by Asp-N in a 3D model of TRPV1. The sequences of the peptides detected after limited proteolysis by Asp-N are shown in Table 3 below. The peptide digested with 20 μg / ml Asp-N for 5 minutes is shown first. The peptide digested with 2 μg / ml Asp-N for 24 hours is shown next.

[0357] [Table 3]

[0358] Figure 12 shows the location of the peptide detected after chymotrypsin-mediated limited proteolysis in a 3D model of TRPV1. The sequences of the peptide detected after chymotrypsin-mediated limited proteolysis are shown in Table 4 below. The peptide digested with 5 μg / ml chymotrypsin for 5 minutes is shown first. Peptides digested with 10 μg / ml chymotrypsin for 5 minutes and 20 μg / ml chymotrypsin for 5 minutes are collected for presentation and shown next.

[0359] [Table 4]

[0360] Figure 13 shows the location of the peptide detected after limited pepsin-mediated proteolysis in a 3D model of TRPV1. The sequences of the peptide detected after limited pepsin-mediated proteolysis are shown in Table 5 below. The peptide digested with 5 μg / ml chymotrypsin for 2 minutes is shown first. Peptides digested with 5 μg / ml pepsin for 5 minutes, 10 μg / ml pepsin for 5 minutes, and 20 μg / ml pepsin for 5 minutes are collected for presentation and shown next.

[0361] [Table 5]

[0362] Figure 14 shows the location of the peptide detected after limited proteolysis with proteinase K in a 3D model of TRPV1. The sequences of the peptide detected after limited proteolysis with proteinase K are shown in Table 6 below. The peptide digested with 5 μg / ml proteinase K for 5 minutes is shown first. Peptides digested with 10 μg / ml proteinase K for 5 minutes and 20 μg / ml proteinase K for 5 minutes are collected for presentation and shown next.

[0363] [Table 6]

[0364] In Tables 2, 3, 4, 5, and 6, the terms "start point" and "end point" refer to the positions of amino acid residues in the TRPV1 sequence.

[0365] Data evaluation was performed under Results Filters (peptides), with a significance threshold of 0.01 set in Mascot.

[0366] Trypsin led to an increase in peptide count, and along with the increase in protease concentration, it led to an increase in reliability.

[0367] Both pepsin and chymotrypsin produced a large number of peptides at both low and high concentrations.

Claims

1. A method for identifying epitopes on proteins that can be bound by antibodies, (i) After the protein has been subjected to limited proteolysis or restriction proteolysis by one or more proteases, identify the sites where one or more proteases cleave the protein. (ii) To generate a plurality of isolated epitopes having sequences homologous to a plurality of potential epitopes on the protein, and to generate antibodies that bind to the isolated epitopes. Here, the multiple potential epitopes on the protein are potential epitopes that overlap with the cleavage site identified in step (i), or potential epitopes in a region adjacent to the cleavage site, and the antibody is used to search for the multiple potential epitopes on the protein. (iii) A method comprising searching for a plurality of potential epitopes on the protein with an antibody that binds to the isolated epitope, thereby identifying one or more epitopes that can bind to the antibody.

2. The method according to claim 1, wherein a single protease is used.

3. The method according to claim 1, wherein multiple proteases are used.

4. The method according to any one of claims 1 to 3, wherein the protease is selected from the group consisting of trypsin, Arg-C proteinase, Asp-N endopeptidase, clostripine, glutamyl endopeptidase, Lys-C, Lys-N, chymotrypsin, proteinase K, thermolysin, pepsin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, enterokinase, factor Xa, granzyme B, neutrophil elastase, proline-endopeptidase, staphylococcal peptidase I, and thrombin.

5. The method according to any one of claims 1 to 4, wherein the protease is selected from the group consisting of trypsin, Asp-N endopeptidase, chymotrypsin, pepsin, and proteinase K.

6. The method according to any one of claims 1 to 5, wherein the protein is a membrane protein present in cell-derived proteoliposomes.

7. The method according to claim 6, wherein the proteoliposomes are immobilized on a flow cell to constitute a stationary phase of membrane proteins.

8. The method according to any one of claims 1 to 7, wherein the site where one or more proteases cleave the protein is identified by mass spectrometry.

9. The method according to any one of claims 1 to 8, wherein each of the potential epitopes is within the range of 20 amino acids of the cleavage site.

10. The method according to any one of claims 1 to 9, wherein the plurality of isolated epitopes constitute a set of isolated epitopes, and the sequence of each isolated epitope in the set is offset by one, two, or three amino acids relative to another isolated epitope in the set.

11. The method according to any one of claims 1 to 10, further comprising the step of producing an antibody against an epitope identified according to any one of claims 1 to 10.

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