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LYMPHOCYTE SELECTION METHOD

20260009795 ยท 2026-01-08

    Inventors

    Cpc classification

    International classification

    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.

    Claims

    1-12. (canceled)

    13. 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 distinct peptides comprising sequences derived from the protein antigen.

    14. The method of selection of claim 13, comprising a prior step of selecting lymphocytes from the cell population.

    15. The method of selection of claim 13, wherein the lymphocytes are B lymphocytes or T lymphocytes.

    16. The method of selection of claim 13, wherein the lymphocytes are B lymphocytes.

    17. The method of selection of claim 13, wherein the cell population is a peripheral blood mononuclear cell (PBMC) population.

    18. The method of selection of claim 13, wherein the at least two distinct peptides respectively comprise from 6 to 30 amino acid residues.

    19. The method of selection of claim 13, wherein the at least two distinct peptides comprise non-overlapping sequences derived from the protein antigen.

    20. The method of selection of claim 13, wherein the at least two distinct peptides are at a distance from each other of 3.10-9 m or less in the three-dimensional structure of the protein antigen.

    21. The method of selection of claim 13, comprising an additional step of isolating at least one lymphocyte binding to the at least two distinct peptides.

    22. The method of selection of claim 13, wherein the identification, and optionally isolation, step is carried out by flow cytometry, optionally with cell sorting.

    23. The method of selection of claim 13, wherein the protein antigen is derived from an infectious agent or from a human or animal protein.

    24. 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 carrying out the lymphocyte selection process as defined in claim 13.

    Description

    DESCRIPTION OF FIGURES

    [0058] FIG. 1 shows four flow cytometry graphs (panels) representing events sorted: [0059] as a function of diffracted light measured in front of the cytometer's laser beam (parameter FSC-A::FSC-A) and measured from the side (parameter SSC-A::SSC-A) (bottom left panel), [0060] as a function of the diffracted light measured in front of the cytometer laser beam (parameters FSC-H::FSC-H/FSC-A::FSC-A) (top left panel), [0061] as a function of light emitted by the fluorophores allophycocyanin (APC) and BV711 (parameters comp-VL4-A::CD27-BV711-A/comp-RL1-A::CD19-APC-A) (top right panel), [0062] as a function of light emitted by BV510 and PE fluorophores (parameters comp-VL2-A::S059-BV510-A/comp-BL2-A::S051-PE-A) (bottom right panel).

    [0063] FIG. 2 shows four flow cytometry graphs (panels) representing events sorted: [0064] as a function of diffracted light measured in front of the cytometer's laser beam (parameter FSC-H) and measured from the side (parameter SSC-H) (top left panel), [0065] as a function of the diffracted light measured in front of the cytometer's laser beam (FSC-A and FSC-H parameters) (bottom left panel), [0066] as a function of light emitted by the fluorophores allophycocyanin (APC) and BV711 (parameters Comp-BV711-A::CD27-/Comp-APC-A::CD19) (top right panel), [0067] as a function of light emitted by BV510 and PE fluorophores (parameters Comp-BV510-A::S059-/Comp-PE-A::S051-) (bottom right panel).

    EXAMPLES

    Example 1

    [0068] Peripheral blood mononuclear cells (PBMC) from an individual immunized against Covid-19 were purified by Ficoll.

    [0069] The cells were then labeled using the following antibodies: anti-CD19-APC, anti-CD27-BV711, which mark memory B cells.

    [0070] Next, cells were labeled with the two biotinylated peptides S051 and S059 following the recommendations from 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. Peptides S051 and S059 are disjoint (non-overlapping) in the Spike protein sequence, but neighboring three-dimensionally in that they are located within 25 Angstroms of each other in the three-dimensional structure of the Spike protein.

    [0071] The inventors analyzed 231,000 events by flow cytometry, from which they visually removed cell-free debris (bottom left panel of FIG. 1), then eliminated doublets (top left panel of FIG. 1). They then selected the CD19.sup.+ CD27.sup.+ events (top right panel of FIG. 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 FIG. 1).

    [0072] Statistics for the CD19+/CD27+ population are summarized in Table 1 below:

    TABLE-US-00001 TABLE 1 CD19+/ S051+/ S051/ S051+/ S051/ CD27+ S059+ S059+ S059 S059 cells cells cells cells cells Quantity 3554 4 9 20 3521 % among 0.11 0.25 0.56 99.1 CD19+/CD27+ cells % among PBMC 2.27 1.73 .Math. 10.sup.3 3.89 .Math. 10.sup.3 8.64 .Math. 10.sup.3 1.52

    [0073] The inventors were thus able to identify and isolate four distinct B lymphocytes recognizing both the distinct, non-overlapping S051 and S059 peptides. This indicates that the identified B cells carry antibodies recognizing both the S051 peptide and the S059 peptide, which are disjoint in the primary Spike structure. These antibodies therefore recognize a conformational epitope of the SARS-COV-2 Spike protein.

    Example 2

    1. Sorting of Specific Memory B Lymphocytes and Culture of Clones.

    [0074] Two 8 mL BD Vacutainer CPTT citrated tubes were collected from a healthy individual who had undergone a full vaccination regimen and 2 SARS-CoV-2 infections. These tubes allow very simple separation of peripheral blood mononuclear cells (PBMC). The purified PBMCs were then labeled with 2 fluorophore-coupled peptides, S051 and S059, 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 fluorochrome 510 (BV510) respectively, as shown in Example 1.

    [0075] Labeling of memory B cells was performed with labeled antibodies (anti-CD19-APC, anti-CD27-BV711).

    [0076] Sorting was then carried out on a flow cytometry platform (Cytek Aurora cell sorter) as shown in FIG. 2, and in the end, forty-eight single cells labeled with all fluorophores (PE+, BV510+, APC+, BV711+) were sorted on cell sheets of Feeder MS5-CD40L cells previously spread on 96-well flat-bottom plates.

    [0077] Media were changed 2 times a week for 4 weeks. (RPMI-1640 supplemented with 10% FCS, 55 UM 2-ME, 1% Pen Strep (100 units/ml penicillin, 100 g/ml streptomycin), 10 mM HEPES, 1 mM sodium pyruvate and 1% MEM NEAA with recombinant human IL-2 (Peprotech 200-02, 500 g; final culture concentration 50 ng/ml), recombinant human IL-4 (Peprotech 200-04, 100 g; final concentration 10 ng/ml), recombinant human IL-21 (Peprotech 200-21, 100 g; final concentration 10 ng/ml), and recombinant human BAFF (Peprotech 310-13, 100 g; final concentration 10 ng/ml).

    [0078] Supernatants were collected and frozen at 20 C. before being tested for the presence of antibodies.

    B. Test of Supernatants for the Presence of Specific IgG.

    [0079] The thus prepared supernatants were tested for the presence of IgG by ELISA. Positive wells were then tested by Western blot for the presence of IgG specific to the Spike protein. The presence of IgG is highlighted by an anti-IgG-HRP antibody and the optical density measured by luminescence.

    [0080] 30 supernatants tested from the 48 clones initially sorted showed variable levels of antibody, 7 of them at high levels (ranging from 4 to 15 luminescence units). 2 of these 7 supernatants responded positively for the presence of IgG specific to recombinant Spike protein deposited on nitrocellulose membrane.

    [0081] This shows that sorting memory B lymphocytes by flow cytometry using the 2 fluorescently-labeled peptides does indeed yield monoclonal antibodies recognizing a conformational epitope within the Spike protein.

    [0082] In addition, it is verified that these supernatants also recognize each of the two isolated peptides.