Methods for lymphocyte selection

Abstract

The present invention relates to a method for selecting lymphocytes which recognise a structural epitope of a protein antigen from a cell population, comprising the step of identifying at least one lymphocyte which binds to at least two distinct peptides comprising the sequence of the protein antigen, in particular at least three distinct peptides comprising the sequence of the protein antigen.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for selecting lymphocytes, in particular B lymphocytes or T lymphocytes, and to a method for preparing antibodies from the selected B lymphocytes.

[0002] 2. Background of the Invention Interest in therapeutic monoclonal antibodies continues to grow year by year. Indeed, their highly specific affinity for antigens allows them to provide highly effective medical treatments. Therefore, between 2005 and 2017, the number of monoclonal antibodies approved by the US Food and Drug Administration increased from 2 to 64.

[0003] However, the preparation of effective monoclonal antibodies remains a significant hurdle that must be overcome.

[0004] Indeed, while the selection of B lymphocytes derived from monoclonal antibodies is often based on the primary structure of the target protein antigen, i.e., the linear epitope to which the antibody binds, it is the epitopes derived from the three-dimensional, tertiary or quaternary structure of the protein that most often provide the best affinity for the antibody and therefore the most certain for its biological function.

[0005] Thus, the ability to obtain monoclonal antibodies specific for structural epitopes, capable of recognizing the three-dimensional structure of the target antigen, should improve the activity of these antibodies and thus their use as experimental tools, diagnostic markers or immunotherapy.

[0006] In this context, Tsumoto et al. (2019) Immunotherapy 11:119-127 propose to prepare monoclonal antibodies against structural epitopes of an antigen by immunizing mice with a DNA molecule encoding the antigen, isolating B lymphocytes, then fusing them with myeloma cells expressing this antigen, to obtain hybridomas that produce monoclonal antibodies specific to the three-dimensional structure of the antigen.

[0007] However, although this process produces monoclonal antibodies against conformational epitopes, it is relatively complicated to carry out.

[0008] Therefore, it remains a challenge to provide an easily implemented method for obtaining monoclonal antibodies against structural epitopes.

[0009] Summary of the Invention The present invention stems from the inventors' unexpected discovery that it is possible to select B lymphocytes specific for a structural epitope of an antigen using two different peptides that are derived from the antigen and that are three-dimensionally similar in structure to the antigen.

[0010] The present invention therefore relates to a method for selecting lymphocytes, in particular B or T lymphocytes, which recognise a protein antigen, in particular a structural epitope of the protein antigen, from a cell population, comprising the step of identifying at least one lymphocyte, in particular B or T lymphocyte, which binds to at least two different peptides comprising a sequence derived from the protein antigen, in particular at least three different peptides comprising a sequence derived from the protein antigen.

[0011] The present invention also relates to a method for preparing at least one antibody, or antibody fragment, against a protein antigen, wherein the antibody, or antibody fragment, is prepared from at least one B lymphocyte obtained by carrying out a method for selecting B lymphocytes as defined above.

[0012] The present invention also relates to a method for preparing at least one TCR, or a TCR fragment, against a protein antigen, wherein the TCR, or the TCR fragment, is prepared from at least one T lymphocyte obtained by carrying out a method for selecting T lymphocytes as defined above.

[0013] The present invention also relates to antibodies, or antibody fragments, and TCRs, or TCR fragments, obtainable by carrying out the preparation method according to the invention.

[0014] The present invention also relates to antibodies, or antibody fragments, and TCRs, or TCR fragments, obtainable by carrying out the preparation method according to the invention for their use as drugs or diagnostic agents.

[0015] The present invention also relates to the in vitro use of antibodies, or antibody fragments, and TCRs, or TCR fragments, obtainable by carrying out the preparation method according to the invention for experimental or diagnostic purposes.

[0016] Detailed Description of the Invention As used herein, the term "comprising" is synonymous with "including," "containing," or "encompassing," i.e., when an object "comprises" one or more features, features other than the mentioned features may be included in the object. Conversely, the phrase "consisting of" means "constituted by," i.e., when an object "consists of" one or more features, the object cannot include features other than the mentioned features.

[0017] Lymphocytes The lymphocytes according to the present invention are preferably B lymphocytes and / or T lymphocytes. Here, "B lymphocytes" refers to any cell of the B lineage, such as naive B lymphocytes, activated B lymphocytes, memory B lymphocytes, plasmablasts or plasma cells, especially long-lived plasma cells. T lymphocytes are any cell of the T lineage.

[0018] As will be appreciated by those skilled in the art, B lymphocytes recognize protein antigens via the antibodies they carry, and T lymphocytes recognize protein antigens via the T cell receptor (TCR).

[0019] cell The cell population may be of any type that may contain lymphocytes, particularly B or T lymphocytes.

[0020] The cell population may be derived from a single individual or from multiple individuals. Preferably, the cell population is obtained from one or more individual biological samples or specimens, such as whole blood or bone marrow samples or specimens. The individual is preferably a human or an animal, in particular a mammal. The individual is preferably immunized against a protein antigen, which may be of the same species as the individual or of a different species from the individual, in particular an infectious pathogen. The immunity may be natural, for example when the protein antigen is derived from an infectious pathogen that previously infected the individual, or may be induced by administering to the individual the protein antigen or a part thereof, or a nucleic acid, DNA or RNA encoding the protein antigen or a part thereof, i.e. by active immunization or vaccination.

[0021] Preferably, the cell population is a population of peripheral blood mononuclear cells (PBMCs).

[0022] Preferably, the method for identifying lymphocytes, in particular B or T lymphocytes, as defined above, comprises a prior step of selecting lymphocytes, in particular B or T lymphocytes, from a cell population. As understood herein, the prior step of selecting lymphocytes, in particular B or T lymphocytes, may result in a subpopulation that contains only lymphocytes, in particular B or T lymphocytes, or is enriched for lymphocytes, in particular B or T lymphocytes. This selection may be performed using a ligand, in particular an antibody, that targets a membrane marker specific for lymphocytes, in particular B or T lymphocytes, and the antibody may be coupled to a magnetic bead or a luminophore, in particular a fluorophore, to facilitate detection, selection, isolation or purification of lymphocyte-antibody complexes.

[0023] peptide As understood herein, a sequence derived from a protein antigen is a contiguous amino acid residue sequence that is a portion of the entire amino acid residue sequence of the protein antigen.

[0024] Preferably, the at least two different peptides each contain 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.

[0025] Preferably, the at least two different peptides each comprise at least 6, 7, 8, 9, 10, 11 or 12 amino acid residues.

[0026] Preferably, the at least two different peptides each contain no more than 30, 25, 20 or 15 amino acid residues.

[0027] Preferably, at least two peptides comprise non-overlapping sequences from the protein antigen. As understood herein, non-overlapping sequences are such that they do not overlap within the primary structure of the antigen, i.e., they are discontinuous peptides.

[0028] Preferably, at least two peptides are at least 3.10 apart from each other in the three-dimensional structure of the protein antigen. -9 m, 2.5.10 -9 , 2.10 -9 m, 10 -9 It is less than a metre away.

[0029] The three-dimensional structure of a protein antigen can be obtained by X-ray crystallography, nuclear magnetic resonance (NMR), microscopy or computer-assisted three-dimensional structure prediction.

[0030] The distance between at least two different peptides can be calculated in many ways well known to those skilled in the art. It can be the average of the distances between each amino acid residue of one peptide and each amino acid residue of the other peptide. Preferably, it is the minimum distance among the distances between each amino acid residue of one peptide and each amino acid residue of the other peptide.

[0031] As understood herein, the distance between two amino acid residues is the distance between the respective α-carbon centres of each residue.

[0032] Protein antigens The protein antigen according to the invention can be any type of protein or protein complex, in particular a monomeric or multimeric protein, in particular a homo- or heteromultimeric protein. In one embodiment of the invention, in particular in the context of the method for selecting T lymphocytes as defined above, the protein antigen is a peptide, a polypeptide or a complex of an antigenic protein and an antigen-presenting protein, in particular a major histocompatibility complex (MHC) molecule.

[0033] Preferably, the protein antigen comprises at most 5000, 2500, 1000 or 500 amino acid residues.

[0034] Preferably, the protein antigen comprises at least 25, 50, 75, 100 or 200 amino acid residues.

[0035] Preferably, the protein antigen contains 25 to 5,000, more preferably 50 to 2,500, and even more preferably 75 to 2,000 amino acid residues.

[0036] Protein antigens may consist of amino acid residues only or may contain aprotic moieties in addition to amino acid residues. The protein may be substituted with at least one polysaccharide and / or at least one lipid. Furthermore, the protein may contain one or more prosthetic groups such as heme groups, coenzymes, nucleic acids or iron-sulfur clusters.

[0037] The protein antigen may be derived from any type of organism. Preferably, the protein antigen is derived from an infectious agent or a human or animal protein.

[0038] In particular, the protein antigen can be obtained by purification from the organism which naturally produces it, or recombinantly from any type of cell culture (eukaryotic or prokaryotic).

[0039] Identification As will be apparent to one skilled in the art, the step of identifying at least one lymphocyte, particularly a B or T lymphocyte, that binds at least two different peptides comprising sequences derived from a protein antigen comprises contacting a peptide with a cell population to identify within the cell population at least one lymphocyte that binds at least two different peptides.

[0040] Preferably, the selection method as defined above comprises the additional step of isolating at least one lymphocyte, in particular a B or T lymphocyte, which binds to at least two different peptides of the protein antigen.

[0041] The identification and possibly isolation of lymphocytes, particularly B or T lymphocytes, that bind at least two different peptides can be achieved by many techniques well known to those skilled in the art. Examples include sequential passing of cells over a chromatography column with immobilized peptides (selected with a first peptide, then selected with a second peptide), or sequential binding / selection with magnetic beads carrying peptides (selected with a first peptide, then selected with a second peptide), or sequential attachment / selection with plates displaying peptides, or cell sorting combined with flow cytometry, where cells are first identified and then sorted. In the case of B lymphocytes, it is also possible to clone the cells by limiting dilution and test the supernatant for antibodies that recognize both of the two or more peptides. Once lymphocytes have been selected and cloned to recognize at least two different peptides, their variable sequences can be sequenced directly or after amplification of the selected clones.

[0042] Preferably, the steps of identification and optionally isolation are carried out with flow cytometry, optionally with cell sorting.

[0043] If the identification and optionally isolation steps are carried out by flow cytometry, the at least two different peptides are preferably labeled with different luminophores, in particular fluorophores, to ensure that the different peptides are recognized by the same lymphocytes, in particular B or T lymphocytes.

[0044] Furthermore, when the identification and, optionally, isolation steps are performed by flow cytometry, identification of peptides that bind to lymphocytes can be performed by adding different peptides all at the same time (i.e., during the same sorting) or sequentially for each peptide (i.e., during multiple successive sortings) and contacting the cell population. For example, it can be performed by isolating lymphocytes using a first peptide, then using a second peptide on lymphocytes selected with the first peptide, and then using one or more additional peptides on lymphocytes selected with the first peptide, second peptide, etc. When contacting a cell population with peptides added simultaneously, it is generally necessary to label each peptide with a different marker. In contrast, when contacting a cell population with peptides added sequentially, it is not necessary to label each peptide with a different marker, and it is even possible to use only one type of peptide label.

[0045] Preferably, the method for selecting lymphocytes, in particular B lymphocytes, which recognize a structural epitope of a protein antigen from a cell population according to the present invention comprises the steps of: - selecting lymphocytes from the cell population; - identifying at least one lymphocyte, in particular a B lymphocyte, which binds to at least two different peptides, in particular 6 to 30 amino acid residues, comprising sequences, in particular non-overlapping sequences, derived from a protein antigen, wherein the at least two different peptides are in particular within 3.10 of each other in the three-dimensional structure of the protein antigen; -9 m or less away,process; - isolating at least one lymphocyte identified in the previous step; The method includes:

[0046] Antibody-TCR As used herein, the term "antibody" refers to whole antibodies as well as V L and / or V HThe present invention encompasses antibody fragments comprising at least one antigen-binding portion, such as Fab, F(ab')2, and scFv fragments. The antibodies according to the invention may be of a single species or may be chimeric, humanized, or human origin. The antibodies may be monospecific or bispecific. Furthermore, the antibodies according to the invention may be monomeric or multimeric, in particular dimeric or pentameric. The antibodies according to the invention may be of isotype A, D, E, G or M, preferably G.

[0047] The antibody or TCR against a protein antigen is preferably an antibody or TCR that recognizes a structural epitope of the protein antigen.

[0048] The antibody or TCR against a protein antigen is preferably an antibody or TCR specific for the three-dimensional structure of the protein antigen.

[0049] The antibody against the protein antigen is preferably a monoclonal antibody.

[0050] Antibodies to protein antigens can be prepared from purified B lymphocytes using a number of techniques well known to those of skill in the art.

[0051] By way of example, B lymphocytes can be fused, optionally after clonal expansion, with myeloma cells to produce monoclonal antibody-producing hybridomas. By way of example, portions of the B lymphocyte genome that encode all or part of an antibody, particularly the variable portions thereof, can be cloned and recombinantly expressed by cultured cells, possibly after insertion into a chimeric, humanized or human antibody construct.

[0052] TCRs against protein antigens can be prepared from T lymphocytes by a number of techniques well known to those skilled in the art.

[0053] By way of example, all or part of the TCR, in particular the part of the T lymphocyte genome that encodes the variable part thereof, can be cloned and then recombinantly expressed by other cells. In particular, to stabilize the TCR and facilitate its use, for example with the constant part of an immunoglobulin, the TCR can be produced in the form of a fusion chimeric protein.

[0054] The invention is further illustrated by the following non-limiting examples and figures. [Brief description of the drawings]

[0055] [Figure 1] Figure 1 shows the diffraction pattern of the fluorophores allophycocyanin (APC) and BV711TM (FV711TM) as a function of the diffracted light measured in front of the cytometer laser beam (parameter FSC-A::FSC-A) and from the side (parameter SSC-A::SSC-A) (lower left panel) and as a function of the diffracted light measured in front of the cytometer laser beam (parameter FSC-H::FSC-H / FSC-A::FSC-A) (upper left panel). Four flow cytometry graphs (panels) showing events sorted as a function of light emitted by the -BV510TM and PE fluorophores (parameters comp-VL2-A::S059-BV510-A / comp-BL2-A::S051-PE-A) (lower right panel) and as a function of light emitted by the -BV510TM and PE fluorophores (parameters comp-VL4-A::CD27-BV711-A / comp-RL1-A::CD19-APC-A) (upper right panel). [Diagram 2]FIG. 2 shows four flow cytometry graphs (panels) representing the sorted events - as a function of the diffracted light measured in front of the laser beam of the cytometer (parameter FSC-H) and from the side (parameter SSC-H) (top left panel), - as a function of the diffracted light measured in front of the laser beam of the cytometer (parameters FSC-A and FSC-H) (bottom left panel), - as a function of the light emitted by the fluorophores allophycocyanin (APC) and BV711™ (parameters Comp-BV711-A::CD27- / Comp-APC-A::CD19) (top right panel), and as a function of the light emitted by the BV510™ and PE fluorophores (parameters Comp-BV510-A::S059- / Comp-PE-A::S051-) (bottom right panel). EXAMPLES

[0056] Example 1 Peripheral blood mononuclear cells (PBMCs) were purified with Ficoll from Covid-19 immunized individuals.

[0057] Cells were then labeled with the following antibodies: anti-CD19-APC, anti-CD27-BV711, which mark memory B cells.

[0058] Second, our supplier, Thermo Scientific TM company (EZ-Link TM Following the recommendations of the Sulfo-NHS-LC-Biotinilation kit, cells were labeled with two biotinylated peptides, S051 and S059, and fluorescent streptavidin-BV510. TMand streptavidin-phycoerythrin (PE) conjugate. The S051 peptide consists of residues 201-215 of the SARS-CoV-2 spike protein, and the S059 peptide consists of residues 233-247 of the SARS-CoV-2 spike protein. Peptides S051 and S059 are discontinuous (non-overlapping) in the sequence of the spike protein, but are three-dimensionally adjacent in that they are located within 25 angstroms of each other in the three-dimensional structure of the spike protein.

[0059] We analyzed 231,000 events by flow cytometry, visually removing cell-free debris (lower left panel of FIG. 1), removing doublets (upper left panel of FIG. 1), and detecting CD19, which represents memory B lymphocytes. + CD27 + Events were selected (upper right panel of FIG. 1). Of these cells, four had antibodies with affinity to both S051 and S059 on their cell surface (lower right panel of FIG. 1).

[0060] The statistics of the CD19+ / CD27+ population are summarized in Table 1 below: [Table 1]

[0061] The inventors were able to identify and isolate four different B lymphocytes that recognize both distinct, non-overlapping S051 and S059 peptides, indicating that the identified B cells have antibodies that recognize both the S051 and S059 peptides, which are discontinuous in the primary structure of the spike. Thus, these antibodies recognize structural epitopes of the SARS-CoV-2 spike protein.

[0062] Example 2 1. Sorting and clonal culture of specific memory B lymphocytes. Two 8mL BD Vacutainer tubes were used from healthy individuals who had received a full vaccination regimen and two SARS-CoV-2 infections. (R) (Registered Trademark) CPT TM Citrate tubes were collected, which allow for the isolation of peripheral blood mononuclear cells (PBMCs) very simply. The purified PBMCs were then labeled with two fluorophore-conjugated peptides, S051 and S059, identified in Example 1 as part of the structural epitope of the spike protein. These two peptides, S051 and S059, were conjugated to phycoerythrin (PE) and brilliant violet fluorochrome 510 (BV510), respectively, as shown in Example 1. TM ) was attached.

[0063] Memory B cells were labeled with labeled antibodies (anti-CD19-APC, anti-CD27-BV711 TM ) was used.

[0064] Sorting was then performed using a flow cytometry platform (Cytek Aurora cell sorter) as shown in Figure 2, and finally, 48 single cells labeled with all fluorophores (PE+, BV510+, APC+, BV711+) were sorted onto a cell sheet of Feeder MS5-CD40L cells pre-spread in a 96-well flat-bottom plate.

[0065] The medium was changed twice a week for 4 weeks (RPMI-1640 supplemented with 10% FCS, 55uM 2-ME, 1% Pen Strep (100units / ml penicillin, 100μg / ml streptomycin), 10mM HEPES, 1mM sodium pyruvate, 1% MEM NEAA, and supplemented with recombinant human IL-2 (Peprotech 200-02, 500μg; final culture concentration 50ng / ml), recombinant human IL-4 (Peprotech 200-04, 100μg; final concentration 10ng / ml), recombinant human IL-21 (Peprotech 200-21, 100μg; final concentration 10ng / ml), and recombinant human BAFF (Peprotech 310-13, 100μg; final concentration 10ng / ml).

[0066] Supernatants were harvested and stored frozen at -20°C before being tested for the presence of antibodies.

[0067] B. Testing of supernatants for the presence of specific IgG. The prepared supernatants 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 was highlighted by anti-IgG-HRP antibody and absorbance was measured by luminescence.

[0068] Thirty supernatants tested from the 48 clones initially screened showed a range of antibody levels, seven of which were high (ranging from 4 to 15 luminescence units). Two of these seven supernatants tested positive for the presence of IgG specific for the recombinant spike protein deposited on a nitrocellulose membrane.

[0069] This indicates that flow cytometric sorting of memory B lymphocytes using two fluorescently labeled peptides can indeed yield monoclonal antibodies that recognize structural epitopes within the spike protein.

[0070] Furthermore, these supernatants were confirmed to recognize each of the two isolated peptides.

Claims

1. A method for selecting lymphocytes that recognize a structural epitope of a protein antigen from a cell population, comprising the step of identifying at least one lymphocyte that binds to at least two different peptides containing sequences derived from the protein antigen, particularly at least three different peptides containing sequences derived from the protein antigen.

2. The method according to claim 1, comprising a preliminary step of selecting lymphocytes from a cell population.

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

4. The method according to claim 1, wherein the lymphocytes are B lymphocytes.

5. The method according to claim 1, wherein the cell population is a population of peripheral blood mononuclear cells (PBMCs).

6. The method according to claim 1, wherein each of the at least two different peptides comprises 6 to 30 amino acid residues.

7. The method according to claim 1, wherein the at least two different peptides include non-overlapping sequences derived from a protein antigen.

8. The at least two different peptides are relative to each other in the three-dimensional structure of the protein antigen. －９ The method according to claim 1, wherein the distance is less than or equal to m.

9. The method according to claim 1, further comprising the additional step of isolating at least one lymphocyte bound to the at least two different peptides.

10. The method according to claim 1, wherein the steps of identification and optionally isolation are carried out by flow cytometry, optionally accompanied by cell sorting.

11. The method according to claim 1, 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 against a protein antigen, wherein the antibody or antibody fragment is prepared from at least one B lymphocyte obtained by carrying out a lymphocyte selection process as defined in any one of claims 1 to 11.

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