Method for the selection of lymphocytes

Abstract

The present invention relates to a method for selecting lymphocytes recognizing a conformational epitope of a protein antigen from a population of cells, comprising a step of identifying at least one lymphocyte binding to at least two separate peptides comprising sequences of the protein antigen, in particular at least three separate peptides comprising sequences of the protein antigen.

Description

[0001] METHOD FOR SELECTING LYMPHOCYTES

[0002] Field of invention

[0003] The present invention relates to a method for selecting lymphocytes, in particular B or T lymphocytes, as well as a method for preparing antibodies from the selected B lymphocytes.

[0004] Technical background

[0005] Interest in therapeutic monoclonal antibodies continues to grow year after year. Their highly specific affinity for antigens allows them to deliver highly effective medical treatments. Thus, between 2005 and 2017, the number of monoclonal antibodies approved by the US Food and Drug Administration increased from 2 to 64.

[0006] However, there remains a critical obstacle to overcome in preparing effective monoclonal antibodies.

[0007] Indeed, the selection of B lymphocytes from which monoclonal antibodies are derived is often done with respect to the primary structure of the target protein antigens, i.e. the linear epitopes to which the antibodies bind, and this while it is epitopes derived from the three-dimensional, tertiary or quaternary structure of the proteins which most often make it possible to obtain the best affinity for the antibodies and thus to best ensure their biological functions.

[0008] Thus, enabling the production of monoclonal antibodies specific to conformational epitopes, thus recognizing the three-dimensional structure of the target antigens, should improve the avidity of these antibodies, and therefore their use as tools for experimental use, as markers for diagnostic use, or as immunotherapeutic treatments.

[0009] In this context, Tsumoto et al. (2019) Immunotherapy 11:1 1 -127 propose to prepare monoclonal antibodies directed against conformational epitopes of an antigen by immunizing mice using DNA molecules encoding the antigen, isolating B lymphocytes and then fusing them with myeloma cells expressing this antigen to obtain hybridomas producing monoclonal antibodies specific to the three-dimensional structure of the antigen. However, while this process does indeed make it possible to obtain monoclonal antibodies directed against conformational epitopes, it is relatively complex to implement.

[0010] It therefore remains to provide a simple process to implement allowing the obtaining of monoclonal antibodies directed against conformational epitopes.

[0011] Summary of the invention

[0012] The present invention arises from the unexpected demonstration by the inventors that it was possible to select B lymphocytes specific for a conformational epitope of an antigen by using two distinct peptides derived from the antigen and three-dimensionally close in the structure of the antigen.

[0013] Thus, the present invention relates to a method for selecting lymphocytes, in particular B or T, recognizing a protein antigen, in particular a conformational epitope of a protein antigen, from a population of cells, comprising a step of identifying at least one lymphocyte, in particular B or T, binding to at least two distinct peptides comprising sequences derived from the protein antigen, in particular at least three distinct peptides comprising sequences derived from the protein antigen.

[0014] The present invention also relates to a method for preparing at least one antibody, or antibody fragment, directed against a protein antigen, in which the antibody, or antibody fragment, is prepared from at least one B lymphocyte obtained by implementing the method for selecting B lymphocytes as defined above.

[0015] The present invention also relates to a method for preparing at least one TCR, or TCR fragment, directed against a protein antigen, in which the TCR, or the TCR fragment, is prepared from at least one T lymphocyte obtained by implementing the method for selecting T lymphocytes as defined above.

[0016] The present invention also relates to the antibodies, or antibody fragments, and the TCRs, or TCR fragments, capable of being obtained by implementing the preparation methods according to the invention.

[0017] The present invention also relates to the antibodies, or antibody fragments, and the TCRs, or TCR fragments, capable of being obtained by implementing the preparation methods according to the invention for their use as a medicament or diagnostic agent.

[0018] The present invention also relates to the in vitro use of antibodies, or antibody fragments, and TCRs, or TCR fragments, capable of being obtained by implementing the preparation methods according to the invention for experimental or diagnostic purposes. detailed description of the invention

[0019] As used herein, the term "comprising" is synonymous with "including", "containing" or "encompassing", i.e., when an object "comprises" one or more characteristics, other characteristics than those mentioned may also be included in the object. Conversely, the expression "consisting of" means "made up of", i.e., when an object "consists of" one or more characteristics, the object cannot include other characteristics than those mentioned.

[0020] Lymphocytes

[0021] The lymphocytes according to the invention are preferably B lymphocytes and / or T lymphocytes. By "B lymphocyte" is meant any cell of the B lineage, such as, for example, a naive B lymphocyte, an activated B lymphocyte, a memory B lymphocyte, a plasmablast or a plasma cell, in particular a long-lived one. By T lymphocyte is meant any cell of the T lineage.

[0022] As will be apparent to those skilled in the art, B lymphocytes recognize the protein antigen through the antibodies they carry and T lymphocytes recognize the protein antigen through the T cell receptor (TCR).

[0023] Cells

[0024] The cell population is of any type likely to include lymphocytes, including B or T lymphocytes.

[0025] The cell population may originate from a single individual or from multiple individuals. Preferably, the cell population is obtained from a biological sample or collection from one or more individuals, such as a sample or collection of whole blood or bone marrow. The individual is preferably a human or an animal, especially a mammal. The individual is preferably immunized against the protein antigen, which may be of the same or different species as the individual, especially an infectious agent. Immunization may be natural, for example, when the protein antigen is derived from an infectious agent that has previously infected the individual, or it may be induced by administering to the individual the protein antigen or a portion thereof, or a nucleic acid, DNA or RNA, encoding the protein antigen or a portion thereof, i.e., by active immunization or vaccination.

[0026] Preferably, the cell population is a peripheral blood mononuclear cell (PBMC) population.

[0027] Preferably, the method for identifying lymphocytes, in particular B or T, as defined above, comprises a prior step of selecting lymphocytes, in particular B or T, from the cell population. As understood herein, the prior step of selecting lymphocytes, in particular B or T, may result in a subpopulation comprising only lymphocytes, in particular B or T, or enriched in lymphocytes, in particular B or T. This selection may in particular be carried out using ligands, in particular antibodies, targeting specific membrane markers of lymphocytes, in particular B or T, the antibodies being able to be coupled to magnetic beads or to luminophores, in particular fluorophores, to facilitate the detection, selection, isolation or purification of the lymphocyte-antibody complexes.

[0028] Peptides

[0029] As used herein, sequences derived from the protein antigen are portions of contiguous amino acid residue sequences of the total amino acid residue sequence of the protein antigen.

[0030] Preferably, the at least two distinct peptides respectively comprise from 6 to 30 amino acid residues, 6 to 25 amino acid residues, 6 to 20 amino acid residues, 6 to 15 amino acid residues, 10 to 30 amino acid residues, 10 to 20 amino acid residues, 12 to 20 amino acid residues, 10 to 18 amino acid residues, or 10 to 15 amino acid residues.

[0031] Preferably, the at least two distinct peptides comprise respectively at least 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Preferably, the at least two distinct peptides comprise respectively at most 30, 25, 20 or 15 amino acid residues.

[0032] Preferably, the at least two peptides comprise non-overlapping sequences from the protein antigen. As used herein, non-overlapping sequences are such that they do not overlap within the primary structure of the antigen, they are disjoint peptides.

[0033] Preferably, the at least two peptides are at a distance from each other of 3.10 -9 m, 2.5.10 -9 , 2.10 -9 m, 10 -9 m or less in the three-dimensional structure of the protein antigen.

[0034] The three-dimensional structure of the protein antigen can be obtained by X-ray crystallography, nuclear magnetic resonance (NMR), microscopy or by computer prediction of three-dimensional structure.

[0035] The distance between the at least two distinct peptides may be calculated in many ways well known to those skilled in the art. It may be the average of the distances between each amino acid residue of one of the peptides from each amino acid residue of the other peptide(s). Preferably, it is the minimum distance among the respective distances between each amino acid residue of one of the peptides from each amino acid residue of the other peptide(s).

[0036] As used here, the distance between two amino acid residues is the distance between the respective alpha carbon centers of each residue.

[0037] Protein antigen

[0038] The protein antigen according to the invention may be a protein or a protein complex of any type. It may in particular be a monomeric or multimeric protein, in particular homomultimeric or heteromultimeric. In one embodiment of the invention, in particular in the context of a method for selecting T lymphocytes as defined above, the protein antigen is a complex between a peptide, a polypeptide or an antigenic protein and an antigen-presenting protein, in particular a molecule of the major histocompatibility complex (MHC).

[0039] Preferably, the protein antigen comprises at most 5000, 2500, 1000 or 500 amino acid residues. Preferably, the protein antigen comprises at least 25, 50, 75, 100 or 200 amino acid residues.

[0040] Preferably, the protein antigen comprises from 25 to 5000, more preferably from 50 to 2500, even more preferably from 75 to 2000 amino acid residues.

[0041] The protein antigen may consist solely of amino acid residues or include aprotein components in addition to the amino acid residues. The protein may be substituted with at least one polysaccharide and / or at least one lipid. Furthermore, the protein may include one or more prosthetic groups, such as a heme group, a cofactor, a nucleic acid, or an iron-sulfur cluster.

[0042] The protein antigen can be derived from any type of organism. Preferably, the protein antigen is derived from an infectious agent or a human or animal protein.

[0043] The protein antigen can be obtained in particular by purification from the organism producing it naturally or by recombinant means, from cell cultures of any type (eukaryotic or prokaryotic).

[0044] Identification

[0045] As will be clear to those skilled in the art, the step of identifying at least one lymphocyte, in particular B or T, binding to at least two distinct peptides comprising sequences derived from the protein antigen comprises bringing the peptides into contact with the cell population to identify within the cell population at least one lymphocyte which binds to the at least two distinct peptides.

[0046] Preferably, the selection method as defined above comprises an additional step of isolating at least one lymphocyte, in particular B or T, binding to the at least two distinct peptides of the protein antigen.

[0047] The identification, and possibly the isolation, of lymphocytes, in particular B or T lymphocytes, binding to the at least two distinct peptides can be carried out by numerous techniques well known to those skilled in the art. For example, one can cite the successive passage of cells on chromatographic columns on which the peptides are fixed (a first peptide then selection, a second peptide then selection), or successive bindings-selections with magnetic beads carrying the peptides (a first peptide then selection, a second peptide then selection), or successive attachments-selections to plates presenting the peptides, or even in cell sorting associated with flow cytometry where the cells are identified on the one hand and then sorted. In the case of B lymphocytes, it is also possible to clone the cells by limiting dilution and to test the antibodies of the supernatant which recognize both the at least two peptides.Once lymphocytes have been selected and cloned for the recognition of at least two distinct peptides, their variable sequences can be sequenced directly or after amplification of the selected clone.

[0048] Preferably, the identification step, and possibly isolation, is carried out by flow cytometry, with possible cell sorting.

[0049] When the identification step, and possibly the isolation step, is carried out by flow cytometry, the at least two distinct peptides are preferably labeled with distinct luminophores, in particular fluorophores. This makes it possible to ensure that the distinct peptides are indeed recognized by the same lymphocyte, in particular B or T.

[0050] Furthermore, when the identification step, and possibly isolation, is implemented by flow cytometry, the identification of the binding of the peptides to the lymphocyte can be implemented by bringing the cell population into contact with the distinct peptides added all at the same time, i.e. during the same sorting, or sequentially, peptide by peptide, i.e. in several successive sorts, for example by isolating lymphocytes using a first peptide, then using a second peptide on the lymphocytes sorted with the first peptide, then possibly using one or more additional peptides on the lymphocytes sorted using the first two peptides, etc. Bringing the cell population into contact with the peptides added at the same time will generally require that each peptide be labeled with a different marker.Conversely, bringing the cell population and the peptides added sequentially into contact does not require labeling each peptide with a different marker, or even allows only one type of peptide labeling to be used.

[0051] Preferably, the method for selecting lymphocytes, in particular B lymphocytes, recognizing a conformational epitope of a protein antigen, from a population of cells according to the invention, comprises:

[0052] - a step of selecting lymphocytes from the cell population; - a step of identifying at least one lymphocyte, in particular B, binding to at least two distinct peptides, in particular of 6 to 30 amino acid residues, comprising sequences, in particular non-overlapping ones, derived from the protein antigen, the at least two distinct peptides being in particular at a distance between them of 3.10 9 m or less in the three-dimensional structure of the protein antigen;

[0053] - a step of isolation of at least one lymphocyte identified in the previous step.

[0054] Antibody - TCR

[0055] As used herein, the term "antibody" includes whole antibodies as well as antibody fragments comprising at least one antigen-binding portion, such as VL and / or VH, Fab, F(ab')2, and scFv fragments. The antibody according to the invention may be derived from a single species, chimeric, humanized, or human. The antibody according to the invention may be monospecific or bispecific. Furthermore, the antibody according to the invention may be monomeric or multimeric, in particular dimeric or pentameric. The antibody according to the invention may be of isotype A, D, E, G, or M, preferably G.

[0056] The antibody, or TCR, directed against a protein antigen is preferably an antibody, or TCR, recognizing a conformational epitope of the protein antigen.

[0057] The antibody, or TCR, directed against a protein antigen is preferably an antibody, or TCR, specific for a three-dimensional structure of the protein antigen.

[0058] The antibody directed against a protein antigen is preferably a monoclonal antibody.

[0059] The antibody directed against a protein antigen can be prepared from a purified B lymphocyte, by numerous techniques well known to those skilled in the art.

[0060] For example, the B lymphocyte, possibly after clonal multiplication, can be fused with a myeloma cell to give a hybridoma producing monoclonal antibodies. Also for example, the portions of the B lymphocyte genome coding for all or part of the antibody, in particular its variable parts, can be cloned, then expressed, recombinantly, by cultured cells, possibly after insertion into a chimeric, humanized or human antibody structure.

[0061] The TCR directed against a protein antigen can be prepared from a T lymphocyte by many techniques well known to those skilled in the art.

[0062] For example, portions of the T cell genome coding for all or part of the TCR, including its variable parts, can be cloned and then expressed recombinantly by other cells. In particular, TCRs can be produced as chimeric fused proteins to stabilize them and facilitate their use, for example with constant parts of immunoglobulins.

[0063] The invention is further explained with the aid of the following non-limiting Examples and figures.

[0064] Description of figures

[0065] Figure 1

[0066] Figure 1 shows four flow cytometry graphs (panels) representing sorted events:

[0067] - as a function of the diffracted light measured in front of the cytometer laser beam (parameter FSC-A::FSC-A) and measured on the side (parameter SSC-A::SSC-A) (bottom left panel),

[0068] - as a function of the diffracted light measured in front of the cytometer laser beam (FSC-H::FSC-H / FSC-A::FSC-A parameters) (top left panel),

[0069] - as a function of the light emitted by the fluorophores allophycocyanin (APC) and BV71 1™ (comp-VL4-A::CD27-BV71 lA / comp-RLl-A::CD19-APC-A parameters) (top right panel),

[0070] - as a function of the light emitted by the BV510™ and PE fluorophores (comp-VL2-A::S059-BV510-A / comp-BL2-A::S051-PE-A parameters) (bottom right panel).

[0071] Figure 2

[0072] Figure 2 shows four flow cytometry graphs (panels) representing sorted events: - as a function of the diffracted light measured in front of the cytometer laser beam (FSC-H parameter) and measured on the side (SSC-H parameter) (top left panel),

[0073] - as a function of the diffracted light measured in front of the cytometer laser beam (FSC-A and FSC-H parameters) (bottom left panel),

[0074] - as a function of the light emitted by the fluorophores allophycocyanin (APC) and BV71 1™ (Comp-BV71 1-A::CD27- / Comp- APC-A::CD19 parameters) (top right panel),

[0075] - as a function of the light emitted by the BV510™ and PE fluorophores (Comp- BV510-A::S059- / Comp- PE-A::S051- parameters) (bottom right panel).

[0076] EXAMPLES

[0077] Example 1

[0078] Peripheral blood mononuclear cells (PBMCs) from an individual immunized against Covid-19 were purified by Ficoll.

[0079] The cells were then labeled using the following antibodies: anti-CD19-APC, anti-CD27-BV71 1 which allow the labeling of memory B lymphocytes.

[0080] Then, the cells were labeled with both biotinylated peptides S051 and S059 using the recommendations of the supplier Thermo Scientific™ (EZ-Link™ Sulfo-NHS-LC-Biotinilation kit) and coupled to fluorescent Streptavidin-BV510™ and Streptavidin-phycoerythrin (PE) conjugates. The S051 peptide consists of residues 201 to 215 of the SARS-CoV-2 Spike protein and the S059 peptide consists of residues 233 to 247 of the SARS-CoV-2 Spike protein. The S051 and S059 peptides are disjoint (non-overlapping) in the Spike protein sequence, but three-dimensionally neighbors in that they are located within 25 Angstroms of each other in the three-dimensional structure of the Spike protein.

[0081] The inventors analyzed 231,000 events by flow cytometry, from which they visually removed acellular debris (bottom left panel of Figure 1), then removed doublets (top left panel of Figure 1). They then selected CD19 events + CD27 + (top right panel of Figure 1) corresponding to memory B lymphocytes. Among these cells, 4 have antibodies on their surface with affinity for both S051 and S059 (bottom right panel of Figure 1).

[0082] Statistics for the CD19+ / CD27+ population are summarized in Table

[0083] 1 below:

[0084]

[0085] Table 1

[0086] The inventors were thus able to identify and isolate four distinct B lymphocytes recognizing both the distinct and non-overlapping S051 and S059 peptides. This indicates that the identified B lymphocytes carry antibodies recognizing both the S051 peptide and the S059 peptide which are disjoint in the primary structure of Spike, these antibodies therefore recognize a conformational epitope of the SARS-CoV-2 Spike protein.

[0087] Example 2

[0088] 1. Sorting of specific memory B lymphocytes and culturing of clones.

[0089] Two 8mL BD Vacutainer® CPT™ citrated tubes were collected from a healthy individual who had completed a vaccination course and had undergone 2 SARS-CoV-2 infections. These tubes allow for the very simple separation of peripheral blood mononuclear cells (PBMC). The purified PBMC were then labeled with two fluorophore-coupled peptides, S051 and S059, peptides identified in Example 1 as part of a conformational epitope of the Spike protein. These two peptides, S051 and S059, were coupled to phycoerythrin (PE) and brilliant violet 510 (BV510™), respectively, as indicated in Example 1.

[0090] The labeling of memory B lymphocytes was carried out by labeled antibodies (anti-CD19-APC, anti-CD27-BV71 1™).

[0091] Sorting was then performed on a flow cytometry platform (Cytek Aurora cell sorter) as shown in Figure 2, and ultimately, forty-eight single cells labeled with all fluorophores (PE+, BV510+, APC+, BV71 1 +) were sorted onto cell lawns of Feeder MS5-CD40L cells previously plated on 96-well flat-bottom plates.

[0092] The media were changed twice a week for 4 weeks. (RPMI-1640 supplemented with 10% FCS, 55 µM 2-ME, 1% Pen Strep (100 units / ml penicillin, 100 µg / ml streptomycin), 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM NEAA with recombinant human IL-2 (Peprotech 200-02, 500 µg; final concentration for culture of 50 ng / ml), recombinant human IL-4 (Peprotech 200-04, 100 µg; final concentration of 10 ng / ml), recombinant human IL-21 (Peprotech 200-21, 100 µg; final concentration of 10 ng / ml), and recombinant human BAFF (Peprotech 310-13, 100 µg; concentration final of 10 ng / ml).

[0093] Supernatants were collected and frozen at -20°C before being tested for the presence of antibodies.

[0094] B. Testing of supernatants for the presence of specific IgG.

[0095] The supernatants thus prepared were tested for the presence of IgG by ELISA. The positive wells were then tested by Western blot for the presence of IgG specific to the Spike protein. The presence of IgG is demonstrated using an anti-IgG-HRP antibody and the optical density measured by luminescence.

[0096] 30 tested supernatants from the 48 initially sorted clones showed varying levels of antibodies, including 7 with high levels (ranging from 4 to 15 luminescence units). 2 of these 7 supernatants responded positively for the presence of IgG specific to the recombinant Spike protein deposited on a nitrocellulose membrane.

[0097] This highlights that sorting memory B lymphocytes by flow cytometry using the two fluorescently labeled peptides does indeed allow the production of monoclonal antibodies recognizing a conformational epitope within the Spike protein.

[0098] Furthermore, it is verified that these supernatants also recognize each of the two isolated peptides.

Claims

DEMANDS 1. Method for selecting lymphocytes recognizing a conformational epitope of a protein antigen from a population of cells, comprising a step of identifying at least one lymphocyte binding to at least two distinct peptides comprising sequences from the protein antigen, in particular at least three distinct peptides comprising sequences from the protein antigen.

2. Selection method according to claim 1, comprising a prior step of selecting lymphocytes from the cell population.

3. A method according to claim 1 or 2, wherein the lymphocytes are B lymphocytes or T lymphocytes.

4. A method according to any one of claims 1 to 3, wherein the lymphocytes are B lymphocytes.

5. Selection method according to any one of claims 1 to 4, wherein the cell population is a population of peripheral blood mononuclear cells (PBMCs).

6. Selection method according to any one of claims 1 to 5, wherein the at least two distinct peptides comprise respectively from 6 to 30 amino acid residues.

7. Selection method according to any one of claims 1 to 6, wherein the at least two distinct peptides comprise non-overlapping sequences from the protein antigen.

8. A selection method according to any one of claims 1 to 7, wherein the at least two distinct peptides are at a distance of 3.10 from each other -9 m or less in the three-dimensional structure of the protein antigen.

9. A selection method according to any one of claims 1 to 8, comprising an additional step of isolating at least one lymphocyte binding to at least two distinct peptides.

10. A selection method according to any one of claims 1 to 9, wherein the identification step, and optionally the isolation step, is carried out by flow cytometry, optionally with cell sorting.

11. Selection method according to any one of claims 1 to 10, wherein the protein antigen is derived from an infectious agent or from a human or animal protein.

12. A method for preparing at least one antibody, or antibody fragment, directed against a protein antigen, wherein the antibody, or antibody fragment, is prepared from at least one B lymphocyte obtained by implementing the lymphocyte selection method as defined in any one of claims 1

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