Method for obtaining antibody molecules with high affinity

Abstract

Disclosed herein is a method for obtaining antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen, comprising: contacting a population of antibody-producing cells, including cells expressing antibody molecules against an antigen on their cell surface, with a first labeled form of the antigen to enable the antigen to bind to antibody molecules on the cell surface; subsequently contacting the cells with (i) an unlabeled form of the antigen, (ii) a second labeled form of the antigen, or (iii) both the unlabeled and second labeled forms of the antigen; and collecting cells that remain bound to the first labeled form of the antigen, thereby obtaining cells that express antibody molecules with high affinity to the antigen. This method makes it possible to obtain cells expressing high-affinity antibody molecules from a pool of antibody-producing cells expressing antibody molecules with different affinities.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority from U.S. Provisional Application No. 63 / 452,206, filed on 15 March 2023, and U.S. Provisional Application No. 63 / 559,544, filed on 29 February 2024, the contents of both of which are incorporated herein by reference. [Background technology]

[0002] High affinity binding is a desirable attribute for many therapeutic antibody molecules. The availability of high-affinity monoclonal antibodies is crucial for the development of targeted immunotherapies. However, very high-affinity antibody molecules are rare in many immunized animals, and standard methods for generating antibody molecules are inefficient for isolating high-affinity antibody molecules.

[0003] Typically, monoclonal antibodies are obtained from mouse hybridomas, most often resulting from the fusion of B lymphocytes from immunized mice with mouse myeloma cells. However, the isolation of rare, high-affinity antibodies using hybridoma technology is not efficient due to throughput limitations on hybridoma cultures.

[0004] Another approach to producing high-affinity antibody molecules involves the use of display techniques to generate lead antibody candidates from phage, yeast, or mammalian libraries. While direct DNA isolation from B cells expressing antibody molecules may be utilized, the DNA library is expressed in a cell expression system such as a phage, yeast, or bacterial system, and then "panning" or titrating to select high-affinity antibody molecules. Display techniques can provide high-quality protein libraries, although they offer limited diversity. Therefore, in generating high-affinity antibody molecules from such libraries, in vitro mutagenesis-based affinity maturation is often the next step.

[0005] Therefore, there is a need for an efficient method to obtain large quantities of antibody molecules with the required specificity and high binding affinity in an efficient manner, without requiring several rounds of screening (such as "panning") or site-directed mutagenesis. [Overview of the project] [Means for solving the problem]

[0006] Disclosed herein are methods for obtaining B cells and other antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen, and mammalian host cells produced by the disclosed methods. This disclosure is based on the observation that cells expressing antibody molecules with high affinity to a target antigen can be directly selected and enriched from a population of antibody-producing cells with varying affinities. Therefore, antibody molecules are directly selected from B cells and other antibody-producing cells before single-cell isolation and collection, for example, by using fluorescence-activated cell sorting (FACS). This provides a convenient method for obtaining cells expressing high-affinity antibody molecules from a pool of B cells and other antibody-producing cells expressing antibody molecules with different affinities.

[0007] In one embodiment, the present disclosure relates to a method for obtaining antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen, and the method is (a) To enable the antigen to bind to antibody molecules on the cell surface by bringing a population of antibody-producing cells expressing antibody molecules against an antigen on the cell surface into contact with a first labeled form of the antigen, wherein the first labeled form of the antigen is conjugated to a first detectable label. (b) Wash the cells to remove unbound antigens, (c) cells, (i) Unlabeled form of the antigen, (ii) A second labeling form of the antigen, (iii) Contacting the unlabeled form of the antigen with the second labeled form of the antigen, (d) Wash the cells to remove unbound antigens, (e) collecting cells that remain bound to the first labeled form of the antigen, thereby obtaining cells that express antibody molecules with high affinity for the antigen.

[0008] In some embodiments of the method, the first labeled form of the antigen is at a concentration of 0.001 nM to 1 μM. In some embodiments of the method, the first labeled form of the antigen is at a concentration of 0.05 nM to 10 nM. In some embodiments, the first labeled form of the antigen is at a concentration of 0.1 to 7.5 nM. In some embodiments, the first labeled form of the antigen is at a concentration of about 0.2 nM. In some embodiments, the first labeled form of the antigen is at a concentration of about 5 nM.

[0009] In some embodiments, the first detectable label is a first fluorescent label.

[0010] In some embodiments, the antigen is a protein in monomeric form. In some embodiments, the antigen is a protein in multimeric form, such as, inter alia, dimeric, trimeric, tetrameric, pentameric, hexameric, or mixtures thereof. In some embodiments, the antigen is a protein that exists in both monomeric and multimeric forms.

[0011] In some embodiments, the first labeled form of the antigen is the monovalent form of the antigen. In such embodiments, the antigen can be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms, and there is no multimerization of the antigen in monovalent form. In some embodiments, the first labeled form of the antigen is the polyvalent form of the antigen. In such embodiments, the antigen can be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms, and multiple units of the antigen are presented in the polyvalent form. In some embodiments, the first labeled form of the antigen is a mixture of the monovalent and polyvalent forms of the antigen. In embodiments where the polyvalent form of the antigen is used, the polyvalent form of the antigen can be provided by a polyvalent molecule to which the antigen is conjugated or linked. In some embodiments, the polyvalent molecule is a streptavidin multimer (e.g., a tetramer), which can be conjugated with biotin to which the antigen is conjugated. In some embodiments, the polyvalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming domain molecule such as Fordon.

[0012] In some embodiments, the unlabeled form of the antigen that can be used in the tracking step is the monovalent form of the antigen, and the antigen can be a protein in the monomeric form, a protein in the multimeric form, or a protein in both the monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is the multivalent form of the antigen, and the antigen can be a protein in the monomeric form, a protein in the multimeric form, or a protein in both the monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen comprises a mixture of the monovalent form and the multivalent form of the antigen, and the antigen can be a protein in the monomeric form, a protein in the multimeric form, or a protein in both the monomeric and multimeric forms. In embodiments where the multivalent form of the antigen is used, the multivalent form of the antigen can be provided by a multivalent molecule to which the antigen is bound or linked. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which can be bound by biotin to which the antigen is conjugated. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the multivalent molecule is a trimer of a trimer-forming domain molecule such as a foldon.

[0013] In some embodiments, the second labeling form of the antigen that can be used in the tracking step is the monovalent form of the antigen, and the antigen can be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In some embodiments, the second labeling form of the antigen is the polyvalent form, and the antigen can be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In some embodiments, the second labeling form of the antigen comprises a mixture of the monovalent and polyvalent forms, and the antigen can be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In embodiments where the polyvalent form of the antigen is used, the polyvalent form of the antigen can be provided by a polyvalent molecule to which the antigen is conjugated or linked. In some embodiments, the polyvalent molecule is a streptavidin multimer (e.g., a tetramer), which can be conjugated with biotin to which the antigen is conjugated. In some embodiments, the polyvalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming domain molecule such as Fordon. The second labeling form of the antigen is labeled with a second detectable label that is different from the first detectable label. In some embodiments, the second detectable label is a second fluorescent label, which, if used, is different from the first fluorescent label.

[0014] In embodiments where an unlabeled form of the antigen is used for tracking, the unlabeled form may be a monovalent form of the antigen, a polyvalent form of the antigen, or a mixture thereof, and the antigen may be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is the monovalent form of the antigen. In some embodiments, the antigen in the unlabeled form has a molar ratio of at least 2 to 150 times, and for example, up to 2500 times, relative to the first labeled form of the antigen. For example, the antigen in the unlabeled form has a molar ratio of at least 2 to at least 120 times, for example, up to 2500 times, including 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, 75 times, 100 times, 125 times, or 150 times relative to the first labeled form of the antigen. In some embodiments, depending on the concentration of the first labeled form of the antigen, the unlabeled form of the antigen in the follow-up step can be at a concentration of 0.4 nM to 600 nM.

[0015] In embodiments where the second labeled form of the antigen is used for tracking, the second labeled form of the antigen can be a monovalent form, a polyvalent form, or a mixture thereof, a monomeric form, and a polymeric form, and the antigen can be a protein in monomeric form, a protein in polymeric form, or a protein in both monomeric and polymeric forms. In some embodiments, the second labeled form of the antigen is the polyvalent form of the antigen. In some embodiments, the antigen in the second labeled form has a molar ratio of at least 2 to 150 times, and for example, up to 2500 times, relative to the first labeled form of the antigen. For example, the antigen in the second labeled form has a molar ratio of at least 2 to at least 120 times, for example, up to 2500 times, including 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times, 75 times, 100 times, 125 times, or 150 times relative to the first labeled form of the antigen. In some embodiments, the second labeled form of the antigen is at a concentration of 15 to 600 nM, depending on the concentration of the first labeled form of the antigen.

[0016] In some embodiments, the tracking step and the subsequent washing are repeated at least once before collecting cells that remain bound to the first labeled form of the antigen.

[0017] In embodiments in which an unlabeled form of the antigen and a second labeled form of the antigen are used for tracking, the unlabeled form may be a monovalent form, a polyvalent form of the antigen, or a mixture of a monovalent form and a polyvalent form, and the second labeled form of the antigen may be a monovalent form, a polyvalent form of the antigen, or a mixture of a monovalent form and a polyvalent form. In some embodiments, tracking is performed with an unlabeled monovalent form of the antigen and a second labeled form of the antigen which is polyvalent. In some embodiments of combination tracking, the unlabeled form of the antigen has a molar ratio of at least 2 to at least 150 times, and for example, up to 2500 times, relative to the first labeled form of the antigen, including 2x, 3x, 4x, 5x, 10x, 15x, 20x, 25x, 50x, 75x, 100x, 125x, or 150x, and the second labeled form of the antigen has a molar ratio of at least 2 to at least 150 times, and for example, up to 2500 times, relative to the first labeled form of the antigen, including 2x, 3x, 4x, 5x, 10x, 15x, 20x, 25x, 50x, 75x, 100x, 125x, or 150x, and for example, up to 2500 times.

[0018] In some embodiments, the first detectable label is a fluorescent label, and fluorescence-activated cell sorting is used to collect cells that remain bound to the first labeled form of the antigen. In some embodiments, the first detectable label is a first fluorescent label, and the second detectable label is a second fluorescent label different from the first fluorescent label, and two-dimensional fluorescence-activated cell sorting is used to collect cells that remain bound to the first labeled form of the antigen.

[0019] In some embodiments, antibody-producing cells are mammalian cells or yeast (such as S. cerevisiae or Pichia) that produce antibody molecules on their cell surface. In some embodiments, antibody-producing mammalian cells are primary antibody-producing cells or immortalized cell lines that produce antibody molecules on their cell surface. In some embodiments, primary antibody-producing cells are obtained from the spleen, lymph nodes, peripheral blood, and / or bone marrow of a mammalian subject (e.g., human, rabbit, pig, cattle, horse, and rodent). In some embodiments, immortalized cell lines are selected from Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) 293 cells, and hybridoma cells that produce antibody molecules on their cell surface.

[0020] In some embodiments, the cells collected in step (e) are enriched for cells expressing high-affinity antibody molecules. In some embodiments, the high affinity of the obtained antibody molecule has a KD of less than 25 nM. In some embodiments, the high affinity of the obtained antibody molecule is in the range of 0.1 pM to about 25 nM (KD). In some embodiments, the high affinity is less than 10 nM (KD). In some embodiments, the high affinity is less than 5 nM (KD). In some embodiments, the high affinity is less than 1 nM (KD). In some embodiments, the high affinity is less than 0.1 nM (KD). In some embodiments, the high affinity is less than 0.01 nM (KD). In some embodiments, the high affinity is less than 5 pM (KD). In some embodiments, the high affinity is less than 1 pM (KD). In some embodiments, at least 40% (e.g., 40%, 50%, 60%, 70%, 80%, 90% or more) of the collected cells express high-affinity antibody molecules, e.g., antibody molecules with a KD of 0.1 pM to 25 nM. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in the cell population after follow-up increases by at least 30% (e.g., 30%, 40%, 50%, 75%, 100%, 200% or more) compared to the frequency in the cell population before or without follow-up. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in the cell population after tracking increases by at least 30% (e.g., 30%, 40%, 50%, 75%, 100%, 200% or more) compared to the frequency in the cell population before tracking or without tracking.

[0021] In some embodiments, the method further includes isolating nucleic acids encoding antibodies from collected cells, which remain bound to a first labeled form of the antigen.

[0022] In some embodiments, the method further comprises transfecting host cells with nucleic acids encoding antibody heavy chains or their variable domains, and antibody light chains or their variable domains, and growing the transfected cells under conditions that support antibody expression by the transfected cells. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells.

[0023] In another embodiment, the disclosure relates to mammalian host cells produced by isolating nucleic acids encoding antibodies from collected cells expressing high-affinity antibody molecules, transfecting host cells with nucleic acids encoding antibody heavy chains or their variable domains, and nucleic acids encoding antibody light chains or their variable domains, and growing the transfected host cells under conditions that support the expression of antibody molecules by the host cells. In some embodiments, the host cells are CHO cells.

[0024] This patent file or application file includes at least one drawing made in color. A copy of this patent or publication of this patent application with the color drawing will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0025] [Figure 1]This is an illustration showing the overall picture of the method used in this disclosure. Beads or cells coated with antibodies of various affinity are exposed to and conjugated with A647 conjugate antigen (i.e., a sorting agent or a first labeled form of the antigen) at concentrations, for example, 5 nM or 0.2 nM, followed by a follow-up step. Three exemplary tracking options are illustrated: A. Cold (unlabeled) monovalent tracking with an unlabeled monovalent antigen at a concentration of at least 2 to up to 2500 times that of the labeled sorting antigen; B. Fluorophore-labeled (or "hot") polyvalent tracking of a biotin-labeled antigen pre-bound with streptavidin (SA)-phycoerythrin (PE) (with antigen at a concentration of at least 4 to up to 2500 times that of the labeled sorting antigen, and with biotin-labeled antigen to SA-PE in a 4:1 ratio); or C. A combination of an unlabeled monovalent antigen at a concentration of at least 2 to up to 2500 times that of the labeled sorting antigen, and a hot polyvalent agent using a biotin-labeled antigen pre-bound with SA-PE (with antigen at a concentration of at least 2 to up to 2500 times that of the labeled sorting antigen, and with biotin-labeled antigen to SA-PE in a 4:1 ratio). [Figure 2]This is an overview of the method used in this disclosure, which uses human IL-13 as an antigen. Beads coated with IL-13 antibodies of various affinity are exposed to A647 conjugate human IL-13 (i.e., a sorting agent) at concentrations, for example, 5 nM or 0.2 nM, to conjugate, and then undergo a follow-up step. Three exemplary tracking options are illustrated: A. Cold (unlabeled) monovalent tracking with unlabeled human IL-13 at concentrations of at least 4 to up to 2500 times the labeled sorting antigen; B. Fluorophore-labeled (hot) multivalent tracking of biotin-labeled human IL-13 pre-conjugated with streptavidin (SA)-phycoerythrin (PE) (with human IL-13 at concentrations of at least 4 to up to 2500 times the labeled sorting antigen, and with biotin-labeled human IL-13 to SA-PE in a 4:1 ratio); or C. A combination of unlabeled monovalent tracking agent human IL-13 at concentrations of at least 4 to up to 2500 times the labeled sorting antigen, and a hot multivalent tracking agent using biotin-labeled human IL-13 pre-conjugated with SA-PE (with human IL-13 at concentrations of at least 4 to up to 2500 times the labeled sorting antigen, and with biotin-labeled human IL-13 to SA-PE in a 4:1 ratio). [Figure 3] This is a representative univariate histogram overlay of A647 staining of untracked IL13 antibody-coated beads detected by flow cytometry. Beads from four different groups were coated with IL13 antibodies having different dissociation constants (described in the insertion box), exposed to 5 nM A647-conjugated human IL13, and subsequently washed twice. A similar experiment was performed with 0.2 nM A647-conjugated human IL13, yielding similar results (not shown). [Figure 4]This is a representative univariate histogram overlay of A647 staining of IL13 antibody-coated beads with cold monomer tracking detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants (described in the insertion box), exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice with 4x (i.e., 10x) unlabeled human IL13 for 45 minutes each, with two washes in between, after which A647 was detected by flow cytometry. Similar experiments were performed with the same four different groups of beads coated with IL13 antibody, exposed to 0.2 nM A647-conjugated human IL13, washed, and then incubated twice with 20 nM (i.e., 10x) unlabeled human IL13, yielding similar results (not shown). [Figure 5] This is a representative univariate histogram overlay of A647 staining of IL13 antibody-coated beads with cold monovalent tracking detected by flow cytometry. Four different IL13 antibody-coated beads were exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice with 100-fold (500 nM) unlabeled human IL13 for 45 minutes, with two washes in between. Similar experiments were performed using the same four different IL13 antibody-coated beads, exposing them to 0.2 nM A647-conjugated human IL13, washing, and then incubated twice with 100-fold (20 nM) unlabeled human IL13, yielding similar results (not shown). [Figure 6]This is a representative univariate histogram overlay of A647 staining of IL13 antibody-coated beads with fluorophore-labeled multivalent tracking detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice for 45 minutes with 4x (20 nM) biotinylated human IL13 pre-clustered with 5 nM SA-PE, with two washes in between. Similar experiments were performed by exposing four different IL13 antibody-coated beads to 0.2 nM A647-conjugated human IL13, washing, and then incubating twice with 100x (20 nM) biotinylated human IL13 pre-clustered with 5 nM SA-PE, yielding similar results (not shown). [Figure 7] This is a representative univariate histogram overlay of A647 staining of IL13 antibody-coated beads with combined tracking (cold monovalent antigen and hot polyvalent antigen). Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice for 45 minutes with 500 nM human IL13 and 20 nM biotinylated human IL13 pre-clustered with 5 nM SA-PE, with two washes during that time. [Figure 8]This is a representative A647 vs. PE dot plot (i.e., tracking map) of IL13 antibody-coated beads with fluorophore-labeled multivalent tracking detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice for 45 minutes with 4x (20 nM) biotinylated human IL13 pre-clustered with 5 nM SA-PE, with two washes during that time. Similar experiments were performed by exposing four different IL13 antibody-coated beads to 0.2 nM A647-conjugated human IL13, washing, and then incubating twice with 100x (20 nM) biotinylated human IL13 pre-clustered with 5 nM SA-PE, yielding similar results (not shown). [Figure 9] This is a representative A647 vs. PE dot plot (i.e., tracking map) of IL13 antibody-coated beads with combined tracking (cold monovalent and hot polyvalent) detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice for 45 minutes with 100x (500 nM) human IL13 and 4x (20 nM) biotinylated human IL13 pre-clustered with 5 nM SA-PE, with two washes during that time. [Figure 10] This is a representative A647 vs. PE dot plot (i.e., tracking map) of IL13 antibody-coated beads with fluorophore-labeled multivalent tracking detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 0.2 nM A647-conjugated human IL13, washed, and then incubated for 45 minutes with 100-fold (20 nM) biotinylated human IL13 pre-clustered with SA-PE, during which two washes were performed. [Figure 11]This is a representative A647 vs. PE dot plot (i.e., tracking map) of IL13 antibody-coated beads with combined tracking (cold monovalent and hot polyvalent) detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 0.2 nM A647-conjugated human IL13, washed, and then incubated for 45 minutes with 20 nM biotinylated human IL13 pre-clustered with 500 nM human IL13 and 5 nM SA-PE, during which two washes were performed. [Figure 12A] The results for splenocytes from a single mouse sorted according to the strategies described above are shown. A. No tracking. B. Hot multivalent tracking. C. Combined tracking (cold monovalent and hot multivalent). All cells were initially gated based on viability and IgG expression. The final sorting gate on the A647 vs PE dot plot is highlighted in yellow on the flow cytometry profile, in the upper panel. The gated cell populations were indexed and analyzed based on Biacore data, as shown in the lower part of the figure. The no-tracking sorting strategy (A) yielded antibodies with the shortest t1 / 2 values, while the tracking strategies enriched antibodies with longer t1 / 2 values ​​(B and C). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 13A] The results for antibodies from a representative mouse (#6018066), analyzed by Biacore, are shown below. A. No tracking. B. Hot multivalent tracking. C. Combined tracking (cold monovalent and hot multivalent). Antibodies isolated without tracking were mostly low affinity with short t1 / 2 (A). However, antibodies isolated using either of the two tracking strategies showed significant enrichment for sub-nanomoroa affinity antibodies with t1 / 2 exceeding 100 minutes (B and C). In addition to having improved affinity characteristics, antibodies from tracking conditions were also potent inhibitors in the IL13 inhibition assay (indicated by the color of the data points - yellow is 100% inhibition). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14-1] The antibodies from all mice analyzed by Biacore are shown. The trends observed in the individual mouse examples in Figures 13A–C were also consistently observed across all mice. Figures 14B, 14D, and 14F enlarge the view of the boxed inserts and focus on higher affinity antibodies. A and B: no tracking; C and D: hot multivalent tracking; E and F: cold monovalent tracking and hot multivalent tracking. Overall, enrichment of over 3x in longer t1 / 2 antibodies and over 2x in overall KD was observed. Additionally, high affinity antibodies from the cold monovalent + hot multivalent tracking strategies (E and F) also tended to be strong IL13 bioassay blockers (100–200 pM hIL13 was used in the bioassay). [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15-1] The dot plot overlay shows polystyrene microbeads separately coated with two monoclonal antibodies against the Zaire Ebola GP protein with different affinities: anti-Ebola GP-1 and anti-Ebola GP-2. The antibody-coated beads were further treated under either no-tracking conditions (A and C) or fluorophore-labeled multivalent tracking conditions (B and D). The green-shaded beads (t1 / 2 = 1155 min, coated with anti-Ebola GP-1) showed less PE staining and more A647 staining, which separated them from the purple-shaded beads (t1 / 2 = 3 min, coated with anti-Ebola GP-2) which showed more PE staining and less A647 staining. The beads coated with the two different antibodies can be separated from each other using the fluorophore-labeled multivalent tracking method based on their dissociation rate. [Figure 15-2] Same as above. [Modes for carrying out the invention]

[0026] Disclosed herein is a method for obtaining primary antibody-producing cells that express antibodies exhibiting high binding affinity to an antigen.

[0027] While the claimed subject matter is described in terms of a particular example, other examples, including those that do not provide all the benefits and features described herein, are also within the scope of this disclosure. Various structural, logical, and process step modifications may be made without departing from the scope of this disclosure.

[0028] The range of values ​​is disclosed herein. This range includes the lower and upper limits. Unless otherwise specified, this range includes, but is not limited to, all values ​​up to the minimum value (either the lower or upper limit), the lower limit, the upper limit, and all values ​​between the lower and upper limits.

[0029] The following description follows certain conventions regarding the use of terminology. In general, terms used herein are intended to be interpreted consistently with the meanings of those terms, as they are known to those skilled in the art. In making this disclosure, many conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the scope of the skill of the art, are used. These techniques are described, for example, in Molecular Cloning: a Laboratory Manual 4th edition, JFSambrook and DWRussell, ed. Cold Spring Harbor Laboratory Press 2012; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “Oligonucleotide Synthesis” (MJ Gait, ed., 1984); “Animal Cell “Current Protocols in Molecular Biology” (FMAusubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al. This is described in more detail in al., 2001). The contents of these references and other references, including standard protocols that are widely known and trusted by those skilled in the art, including manufacturer instructions, are incorporated herein by reference as part of this disclosure.

[0030] General explanation One aspect of the present disclosure relates to a method for obtaining antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen. A key feature of the method is an initial binding step that allows a first labeled form of the antigen to bind to an antibody molecule on the surface of an antibody-producing cell and form an antigen-antibody complex, followed by a “tracking” step in which the cell comes into contact with one of several forms of the antigen: (i) an unlabeled form of the antigen ("cold tracking"), (ii) a second labeled form of the antigen ("hot tracking"), or both the unlabeled and second labeled forms of the antigen ("combined tracking"). Cells that remain bound to the first labeled form of the antigen after tracking represent cells that express antibody molecules with high binding affinity.

[0031] Therefore, the method disclosed herein allows for the selection of cells expressing antibody molecules with different affinities, thereby enriching cells that express antibody molecules with high affinity.

[0032] As used herein, the term "antibody molecule" encompasses both full-length antibodies and their antigen-binding fragments (e.g., Fab, Fab', and F(ab')2).

[0033] The term "enrich" means increasing the frequency or percentage of a desired cell in a cell population, for example, increasing the percentage of antibody-producing cells expressing high-affinity antibody molecules within an antibody-producing cell population containing cells expressing antibody molecules of various affinities (e.g., high-affinity, medium-affinity, and low-affinity). Therefore, an antibody-producing cell population enriched with cells expressing high-affinity antibody molecules includes an antibody-producing cell population having a higher frequency and / or higher percentage of antibody-producing cells expressing high-affinity antibody molecules as a result of the enrichment process. In this context, the enrichment process is a process that includes antigen tracking, thereby selecting cells expressing high-affinity antibody molecules against a target antigen from a population of cells expressing antibody molecules of various affinities against the target antigen, and separating cells expressing high-affinity antibody molecules from cells expressing non-high-affinity antibody molecules.

[0034] The cell population obtained as a result of the disclosed method is enriched with antibody-producing cells that express antibody molecules having a high binding affinity to the antigen of interest. In other words, the enriched cell population (cells collected after tracking) contains a larger percentage of cells expressing antibody molecules that bind to the antigen of interest with high binding affinity compared to the cell population before or without tracking. In some embodiments, at least 40% of the collected cells express high-affinity antibody molecules, e.g., antibody molecules with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population having at least 50% of cells in the population expressing antibody molecules that bind to the antigen of interest with high binding affinity, e.g., antibody molecules with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population having at least 60% of cells in the population expressing antibody molecules that bind to the antigen of interest with high binding affinity, e.g., antibody molecules with a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population comprising at least 70% of the cells in the population expressing antibody molecules that bind to the target antigen with high binding affinity, for example, antibody molecules having a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population comprising at least 80% of the cells in the population expressing antibody molecules that bind to the target antigen with high binding affinity, for example, antibody molecules having a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population comprising at least 90% of the cells in the population expressing antibody molecules that bind to the target antigen with high binding affinity, for example, antibody molecules having a KD of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population comprising at least 95% of the cells in the population expressing antibody molecules that bind to the target antigen with high binding affinity, for example, antibody molecules having a KD of 0.1 pM to 25 nM. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in the cell population after tracking increases by at least 30% compared to the frequency in the cell population before tracking or without tracking.In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in a cell population after tracking increases by at least 40% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in a cell population after tracking increases by at least 50% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in a cell population after tracking increases by at least 75% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in a cell population after tracking increases by at least 100% compared to the frequency in the cell population before tracking or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., antibody molecules with a KD of 0.1 pM to 25 nM) in a follow-up cell population increases by at least 200% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in a follow-up cell population increases by at least 40% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in a follow-up cell population increases by at least 50% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in the cell population after tracking is increased by at least 75% compared to the frequency in the cell population before tracking or without tracking.In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in a follow-up cell population increases by at least 100% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 1 nM (e.g., antibody molecules with a KD of 0.1 pM to 1 nM) in a follow-up cell population increases by at least 200% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 0.1 nM (e.g., antibody molecules with a KD of 0.1 pM to 0.1 nM) in a follow-up cell population increases by at least 40% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 0.1 nM (e.g., antibody molecules with a KD of 0.1 pM to 0.1 nM) in a follow-up cell population increases by at least 50% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 0.1 nM (e.g., antibodies with a KD of 0.1 pM to 0.1 nM) in a follow-up cell population increases by at least 75% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 0.1 nM (e.g., antibody molecules with a KD of 0.1 pM to 0.1 nM) in a follow-up cell population increases by at least 100% compared to the frequency in the pre-follow-up or unfollowed cell population. In some embodiments, the frequency of cells expressing high-affinity antibody molecules with a KD of less than 0.1 nM (e.g., antibodies with a KD of 0.1 pM to 0.1 nM) in the cell population after tracking increases by at least 200% compared to the frequency in the cell population before tracking or without tracking.

[0035] In some embodiments, a method for obtaining antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen involves the following steps: (a) A population of antibody-producing cells expressing antibody molecules against an antigen on their cell surface is brought into contact with a first labeled form of the antigen, thereby enabling the antigen to bind to antibody molecules on the cell surface, wherein the first labeled form of the antigen is conjugated to a first detectable label. (b) Wash the cells to remove unbound antigens. (c) Contacting cells with (i) an unlabeled form of the antigen, (ii) a second labeled form of the antigen, or (iii) both an unlabeled form of the antigen and a second labeled form of the antigen. (d) Wash the cells to remove unbound antigens. (e) Collecting cells that remain bound to the first labeled form of the antigen, thereby obtaining a population of cells enriched with cells expressing a high-affinity antibody molecule.

[0036] Antibody molecules with high binding affinity "Binding affinity," as the term is known in the art, generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or a fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule to its binding partner is generally determined by the dissociation equilibrium constant (KD or K). D It can be represented by K. D There is an inverse relationship between the (molar) value and binding affinity, therefore K D A smaller value (M) indicates higher affinity. Therefore, "higher affinity" generally refers to antibody molecules that bind more strongly and / or more quickly to the antigen, and / or remain bound for a longer period. Generally, due to their strong binding interaction, a lower concentration (M) of the antigen is required to achieve the desired effect.

[0037] The term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody-binding fragment or molecular interaction. This value is also called the k off value.

[0038] The term "ka" (M-1 x sec -1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody, Ig, antibody-binding fragment or molecular interaction.

[0039] The term "KD" or "K D " (M) refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, or the equilibrium dissociation constant of an antibody, Ig, antibody-binding fragment, or molecular interaction. The equilibrium dissociation constant is obtained by dividing ka by kd.

[0040] Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present disclosure. The binding affinity obtained using a method is typically in the range of about 0.1 pM to about 25 nM when determined by surface plasmon resonance. In some embodiments, the binding affinity is less than about 10 nM when determined by surface plasmon resonance.

[0041] The term "high affinity" antibody molecule refers to those antibody molecules having a binding affinity of 25 nM or less, expressed as KD, e.g., having a value in the range of about 0.1 pM to 25 nM. For this purpose, high affinity antibody molecules are about 25 × 10 -9 M (25 nM) or less, about 10 × 10 -9 M (10 nM) or less, about 1 × 10 -9 M (1 nM) or less, about 1 × 10 -10 M (0.1 nM) or less, about 0.5 × 10 -10 M (0.05 nM) or less, about 0.05 × 10 -10It may have a measured KD of M (5 pM) or less, approximately 1 pM or less, or approximately 0.5 pM or less. A person skilled in the art would know that the KD value of the antibody molecule is nE -z as, or n × 10 -z It may be expressed numerically as either of the following, for example, 3.2E -12 is 3.2 × 10 -12It will be recognized that this is equivalent to and indicates a KD of 3.2 picomoles (pM). In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 25 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 20 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 15 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 10 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 5 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 1 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 0.5 nM. In some embodiments, the high-affinity antibody molecule has a measured KD in the range of about 0.1 pM to about 0.1 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 20 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 15 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 10 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 5 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 1 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 0.1 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 0.5 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 0.01 nM. In some embodiments, the high-affinity antibody molecule has a measured KD of less than about 0.001 nM (or 1 pM). In some embodiments, high-affinity antibody molecules have a measured KD of less than approximately 0.5 pM.

[0042] antibody-producing cells The terms “antibody-producing cells” and “antibody-expressing cells” refer to cells that express antibody molecules on their cell surface, i.e., the antibody molecules are bound to or anchored within the cell membrane. Cell surface expression of antibody molecules can occur naturally, for example, as a result of B cell activation, or as a result of recombinant technology and genetic engineering. Therefore, this term encompasses recombinant cells such as non-lymphoid cells engineered to express antibody molecules on their cell surface, including lymphocytes of antigen-dependent B cell lineages including memory B cells, immortalized cells and hybridoma cells, as well as yeast and mammalian cells. In some embodiments, immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, and mouse myeloma cells (e.g., NS0 and Sp2 / 0).

[0043] In some embodiments, the antibody-producing cells are primary antibody-producing cells. “Primary cells” refer to cells that have grown outside their natural environment, such as tissue cells isolated from mammals. In certain embodiments, primary antibody-producing cells are derived from tissues such as the spleen, lymph nodes, bone marrow, or peripheral blood. In some embodiments, the antibody-producing cells may be derived from primary antibody-producing cells. For example, primary antibody-producing cells may be fused to myeloma cells to create hybridomas, or otherwise immortalized, such as by infection with a virus (e.g., EBV), or distinguished by cell sorting techniques based on protein markers expressed by specific B cell types.

[0044] In some embodiments, antibody-producing cells are mammalian cells or yeast cells engineered to express antibody molecules on their cell surface. In relation to cells engineered to express antibody molecules, the cells may be engineered to express full-length immunoglobulin molecules or antigen-binding fragments. In some embodiments, antibody-producing cells are yeast cells (e.g., S. cerevisiae or Pichia) engineered to express antibody molecules on their cell surface. In some embodiments, antibody-producing cells are mammalian cells engineered to express antibody molecules on their cell surface. In some embodiments, mammalian cells are immortalized cells engineered to express antibody molecules on their cell surface, including, for example, CHO cells, HEK293 cells, and mouse myeloma cells (e.g., NS0 and Sp2 / 0).

[0045] Some of the methodologies described herein are also applicable to cell surface display platforms for various host cells, including mammalian cells such as yeast or CHO cells, which express antibody molecules on the cell surface to enable screening from an antibody gene library or a repertoire of antibody mature variants.

[0046] Yeast surface display (YSD) platforms have been described in antibody screening (Border and Wittrup, Nat Biotechnol. 1997; 15:553-7; Feldhaus MJ et al., Nat Biotechnol. 2003; 21:163-70; McMahon C et al., Nat Struct Mol Biol. 2018; 25:289-96) and are widely used. Displaying Fab regions on the yeast surface has been reported to increase antibody diversity and expand library size (Weaver-Feldhaus JM et al., FEBS Lett. 2004; 564:24-34; Rosowski S et al., Microb Cell Fact. 2018; 17:3; Sivelle C et al., MAbs. 2018; 10:720-9).

[0047] Mammalian cell surface display platforms for displaying full-length antibodies or Fab fragments on the surface of mammalian cells, including CHO cells, have been described, for example, by Zhou et al. MAbs. 2010; 2(5): 508-518; Nguyen et al., Protein Engineering, Design & Selection, 2018, vol. 31 no. 3, pp. 91-101). In addition, mammalian cells carrying a single antibody gene are preferred for use herein, and can be transfected with a gene encoding activation-inducible deaminase (AID) to initiate somatic hypermutation (SHM) by converting deoxycytidine (dC) to deoxyuracil (dU), thereby mutating the intracellular antibody gene during cell proliferation in cell culture. See, for example, Chen C. et al., Biotechnol Bioeng. 113, 39-51 (2016).

[0048] Immunization and collection of primary antibody-producing cells Immunization of mammals, including humans and non-human animals, can be carried out by any method known in the art (see, for example, E. Harlow and D. Lane, Antibodies A Laboratory Manual, Cold Spring Harbor (1988); Malik and Lillehoj, Antibody techniques: Academic Press, 1994, CA). The antigen of interest is administered as a protein, protein fragment, protein fusion, or DNA plasmid that expresses the antigen polypeptide in vivo, containing the antigen gene of interest and expressing the antigen of interest using a host cell expression mechanism. It is understood that the immunized mammal may be a human who has been exposed to the antigen and expresses humoral immunity to the antigen of interest. The antigen may be administered directly to the mammal without an adjuvant, or with an adjuvant to assist in stimulating the immune response. Adjuvants known in the art include, but are not limited to, complete and incomplete Freund's adjuvants, the MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptide) (see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ). Without relying on any particular theory, adjuvants may contain factors that can prevent the rapid dispersion of polypeptides by sequestering antigens in a local depot and stimulate the host immune response.

[0049] Once an appropriate immune response has been achieved, antibody-producing cells are collected from the immunized animals.

[0050] Antibody-producing cells can be collected from different sources of immunized animals, including but not limited to the spleen, lymph nodes, bone marrow, and peripheral blood. In some embodiments, spleen cells are collected from immunized animals after immunization. In some embodiments, peripheral blood mononuclear cells (PBMCs) are collected from immunized animals.

[0051] In some embodiments of this method, the population of antibody-producing cells is antibody-producing B cells. In some embodiments, antibody-producing B cells can be obtained from immunized animals and isolated by FACS based on cell surface B cell markers. B cell markers are known in the art. For example, applicable B cell markers that can be detected through the use of FACS include, but are not limited to, IgG, IgM, IgE, IgA, IgD, CD1, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD38, CD40, CD78, CD80, CD138, CD319, TLR4, IL-6, PDL-2, CXCR3, CXCR4, CXCR5, CXCR6, IL-10, and TGFβ.

[0052] In some embodiments, after immunization, splenocytes are collected from the immunized animals. After removing red blood cells by lysis, IgG+ antigen-positive B cells are isolated and can be used as an antibody-producing cell population in this method.

[0053] In some embodiments, peripheral blood mononuclear cells (PBMCs) are collected from immunized animals known to have humoral immunity to the antigen of interest. Subsequently, IgG+ antigen-positive B cells can be isolated for use as primary antibody-producing cells in this method.

[0054] Collected primary antibody-producing cells, such as antibody-producing B cells, can be processed to enrich cells that express antibodies on their cell surface directed toward the antigen of interest. In some embodiments, the initial purification step is optionally performed to enrich the primary antibody-producing cells. Such purification includes affinity chromatography, also known as affinity purification. There are several types of affinity purification known in the art, such as affinity purification with ammonium sulfate precipitation, affinity purification with immobilized proteins A, G, A / G, or L, and affinity purification with immobilized antigens.

[0055] Target antigen The methods disclosed herein are available for use with any antigen of interest. The antigen of interest is any substance that elicits an immune response. In some embodiments, the antigen of interest is a soluble protein. In some embodiments, the antigen of interest is a transmembrane protein. The antigen of interest can be any substance to which an antibody can bind, including but not limited to peptides, proteins, or fragments thereof; carbohydrates; organic and inorganic molecules; receptors produced by animal cells, bacterial cells, and viruses; enzymes; agonists and antagonists of biological pathways; hormones; and cytokines. Exemplary antigens include, but are not limited to, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, angiotensin II, BAFF, CGRP, CXCL13, IP-10, PCSK9, NGF, Nav1.7, VEGF, EPO, EGF, and HRG. In some embodiments, the antigen of interest is a cytokine. In some embodiments, the antigen of interest is a growth factor. In some embodiments, the antigen of interest may be a tumor marker. In some embodiments, the antigen of interest is a viral protein. In some embodiments, the antigen of interest is a surface protein from a pathogen. In some embodiments, the antigen of interest is an interleukin (IL). In some embodiments, the antigen of interest is IL-13.

[0056] In some embodiments, the antigen is a protein that exists in monomeric form. Examples of proteins that exist in monomeric form include interleukin molecules such as IL-13. In some embodiments, the antigen is a protein that exists in multimeric form, including homomers and heteromers. In some embodiments, the antigen is a protein that exists in both monomeric and multimeric forms, in which case a mixture of protein monomers and multimers can be used in the manner described herein.

[0057] Whether the antigen is a protein existing in monomeric form, a protein existing in multimeric form, or a protein existing in a mixture thereof, the antigen may be used in the methods described herein in monovalent or polyvalent form. The terms “monovalent” and “polyvalent” refer to the number of units of the antigen presented and are used to distinguish them from the antigen itself being a protein in monomeric form, a multimeric form, or a mixture thereof. Thus, the monovalent form of an antigen refers to the single unit form of the antigen, and the antigen itself may be a protein in monomeric form, a multimeric form, or a mixture thereof. The polyvalent form of an antigen typically refers to multiple units of the antigen presented by a polyvalent molecule to which the antigen is bound or linked. The polyvalent molecule may be a dimer, trimer, tetramer, pentamer, hexamer, etc., or a combination thereof. In some embodiments, the polyvalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then conjugated to an antigen, thereby providing a polyvalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin multimer comprises a tetramer and may additionally comprise trimers and / or dimers. In some embodiments, the streptavidin multimer is conjugated with a fluorophore such as phycoerythrin. In some embodiments, the polyvalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen can be conjugated to provide a bivalent form of the antigen. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming molecule such as Fordon, to which an antigen can be conjugated to provide a trivalent form of the antigen.

[0058] Initial binding step and antigen labeling An initial binding or contact step is performed to select cells that express antibody molecules exhibiting the highest binding affinity to the target antigen. In this step, antibody-producing cells are brought into contact with a first labeled form of the antigen, allowing the antigen to bind to antibody molecules on the cell surface.

[0059] In some embodiments, the first labeled form of the antigen has a concentration of 0.001 nM to 1 μM. In some embodiments, the first labeled form of the antigen has a concentration of 0.01 nM to 100 nM. In some embodiments, the first labeled form of the antigen has a concentration of 0.05 nM to 10 nM. In some embodiments, the first labeled form of the antigen has concentrations of 0.05 nM to 9 nM, 0.05 nM to 8 nM, 0.05 nM to 7 nM, 0.05 nM to 6 nM, 0.05 nM to 5 nM, 0.05 nM to 4 nM, 0.05 nM to 3 nM, 0.05 nM to 2 nM, or 0.05 nM to 1 nM. In some embodiments, the first labeled form of the antigen has a concentration of 0.1 nM to 7.5 nM. In some embodiments, the first labeling form of the antigen is at a concentration of 0.1 nM to 7 nM, 0.1 nM to 6 nM, 0.1 nM to 5 nM, 0.1 nM to 4 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, or 0.1 nM to 1 nM. In some embodiments, the first labeling form of the antigen is at a concentration of 0.2 nM to 7.5 nM. In some embodiments, the first labeling form of the antigen is at concentrations of 0.2nM to 7nM, 0.2nM to 6nM, 0.2nM to 5nM, 0.2nM to 4nM, 0.2nM to 3nM, 0.2nM to 2nM, 0.2nM to 1nM, 0.3nM to 7nM, 0.3nM to 6nM, 0.3nM to 5nM, 0.3nM to 4nM, 0.3nM to 3nM, 0.3nM to 2nM, 0.3nM to 1nM, 0.5nM to 7nM, 0.5nM to 6nM, 0.5nM to 5nM, 0.5nM to 4nM, 0.5nM to 3nM, 0.5nM to 2nM, and 0.5nM to 1nM. In some embodiments, the first labeled form of the antigen is at a concentration of 1.0 nM to 10 nM, 1.0 nM to 9 nM, 1.0 nM to 8.0 nM, 1.0 nM to 7 nM, 1.0 nM to 6 nM, 1.0 nM to 5 nM, 1.0 nM to 4 nM, 1.0 nM to 3 nM, 1.0 nM to 2 nM, 2.0 nM to 10.0 nM, or 5.0 nM to 10.0 nM. In a specific embodiment, antibody-producing cells can be brought into contact with the first labeled form of the antigen, which is at a concentration of 0.2 nM. In another specific embodiment, antibody-producing cells can be brought into contact with the first labeled form of the antigen, which is at a concentration of 5.0 nM.In a specific embodiment, antibody-producing cells can be brought into contact with a first labeled form of the antigen, which is at a concentration of 7.5 nM. In another specific embodiment, antibody-producing cells can be brought into contact with a first labeled form of the antigen, which is at a concentration of 10 nM. In specific embodiments, antibody-producing cells can be brought into contact with a first labeled form of the antigen, the first labeled form of the antigen being at concentrations of 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 8.0 nM, 8.5 nM, 9.0 nM, 9.5 nM, or higher.

[0060] In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 5 to about 60 minutes. In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 30 minutes. In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 20 minutes. In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 40 minutes. In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 10 minutes. In some embodiments, contact between the antibody-producing cell and the first labeled form of the antigen occurs for about 50 minutes.

[0061] In some embodiments, the first labeling form of the antigen is the monovalent form of the antigen. In some embodiments, the first labeling form of the antigen is the polyvalent form of the antigen. In some embodiments, the first labeling form of the antigen is a mixture of the monovalent and polyvalent forms of the antigen. Whether in monovalent, polyvalent, or a mixture thereof, the antigen itself can be a protein that is a monomer, a polymer, or a mixture of monomers and polymers.

[0062] In some embodiments, the first labeling form of the antigen is an antigen conjugated with a first detectable label. The antigen can be labeled with small molecules, radioisotopes, enzyme proteins, and fluorescent dyes. In some embodiments, the detectable label is a small molecule. Detectable small molecule labels allow for easy labeling of proteins and can be used in many regularly introduced detection assays known in the art.

[0063] In some embodiments, the detectable label is an enzyme reporter. Enzyme labels are larger than biotin, but they rarely disrupt antibody function. Commonly used enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase. To use enzyme-labeled antibodies, the sample is incubated with an enzyme-catalyzed enzyme-specific substrate to produce a colored product (chromogenic assay) or light (chemiluminescence assay). Each enzyme has a set of substrates and detection methods that can be used. For example, HRP can be reacted with diaminobenzidine to produce a brown product, or with luminol to produce light. In contrast, AP can be reacted with p-nitrophenyl phosphate (pNPP) to produce a yellow product detectable by spectrophotometer, or with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitrobluetetrazolium (NBT) to produce a purple precipitate.

[0064] In some embodiments, the detectable label is a fluorescent label. The fluorescent label is directly conjugated to the antibody, and no enzyme / substrate or binding interaction is required for detection. Therefore, the amount of fluorescent signal detected is directly proportional to the amount of target protein in the sample. The fluorescent tag can be covalently bound to the antibody via a primary amine or thiol.

[0065] Removal of unbound antigen after initial binding After incubation (initial binding step) with the antibody-producing cells and the first labeled form of the antigen, any unbound antigen can be removed from the antibody-producing cells.

[0066] In some embodiments, unbound antigens are removed by washing. As is known in the Art, washing is a technique that uses a washing buffer to remove undesirable components. For the purposes of this disclosure, undesirable components include unbound antigens. In non-limiting examples, unbound antigens can be washed away from bound antigens by adding a washing buffer to a mixture of bound and unbound antigens, centrifuging the mixture, and removing the supernatant containing the washing buffer and unbound antigens.

[0067] Washing buffers are known in the art. Washing buffers and washing steps are used to remove unbound components and excess components. Washing buffers can be designed for specific techniques such as ELISA, immunoblotting, or immunohistochemistry. Some washing buffers are compatible with many different immunoassays. In some embodiments, the washing buffer is a phosphate-buffered saline (PBS) washing buffer. In some embodiments, the washing buffer is a Tris-buffered saline (TBS) washing buffer. In some embodiments, the washing buffer includes a washing agent. In some embodiments, the washing agent is Tween®-20.

[0068] The cells are washed with a wash buffer for a time allocated to remove unbound antigens. This allocated time is sufficient to remove the unbound antigens. In some embodiments, this allocated time can be about 10 to 60 minutes to remove the unbound antigens, and multiple washes totaling 10 to 60 minutes may be used, e.g., three washes of 10 minutes each, or one wash of 30 minutes; two to four washes of 5 to 15 minutes each, etc. In one embodiment, it may be used to wash the cells over a period including a single wash of about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or about 60 minutes in total. In some embodiments, washing the cells over a period of time may be used, including two washes, each lasting about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes. Additional wash intervals, essentially equivalent to those described herein, are intended.

[0069] After washing and aspirating the supernatant containing the washing buffer and unbound antigen, the pellet containing antigen-bound cells can be used in subsequent steps. In some embodiments, the pellet containing the bound antigen can be resuspended in a buffer and used in subsequent steps. In some embodiments, the buffer used to resuspend the pellet may be the same washing buffer. In some embodiments, the buffer used to resuspend the pellet may be a different buffer from the washing buffer.

[0070] Tracking Steps After antibody-producing cells are brought into contact with the first labeled form of the antigen (initial binding / contact step), and the unbound first labeled form of the antigen is removed, the antibody-producing cells either re-combine with the antigen or are "tracked" to selectively enrich cells expressing antibody molecules with high affinity for the antigen. This tracking can be performed using one of several forms of the antigen: (i) the unlabeled form of the antigen ("cold" tracking), (ii) the second labeled form of the antigen ("hot" tracking), or (iii) the unlabeled form and the second labeled form of the antigen (a combination of cold-tracked and hot-tracked antigens, also called "combined tracking"). This tracking allows the tracking antigen to bind to the antibody molecule initially bound by the first labeled form of the antigen, unless the antibody molecule has high affinity for the antigen and remains bound to the first labeled form of the antigen after tracking, thereby tracking the first labeled form of the antigen away from the antibody molecule.

[0071] In some embodiments, tracking is performed using an unlabeled form of the antigen (cold tracking). In some embodiments, the unlabeled form of the antigen is the monovalent form of the antigen, i.e., the antigen itself can be a protein in monomeric form, multimeric form, or a mixture of monomeric and multimeric forms, while the monovalent form of the antigen is the antigen itself without further secondary multimerization. In some embodiments, the unlabeled form of the antigen is the polyvalent form of the antigen, and the antigen itself can be a protein in monomeric form, multimeric form, or a mixture of monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is a mixture of the monovalent and polyvalent forms of the antigen. In embodiments where the polyvalent form of the antigen is used, such a form can be provided by a polyvalent molecule to which the antigen is bound or linked. In some embodiments, the polyvalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then linked to the antigen, thereby providing a polyvalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin polymer may include a trimer and / or dimer in addition to a tetramer. In some embodiments, the polyvalent molecule is a dimer of an immunoglobulin Fc fragment to which an antigen can be linked to provide a bivalent form of the antigen. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming molecule such as Fordon to which an antigen can be linked to provide a trivalent form of the antigen.

[0072] In some embodiments, tracking is performed using a second labeling form of the antigen (hot tracking). In such embodiments, after the initial binding step and the washing step, antibody-producing cells bound with the first labeling form of the antigen are tracked with the second labeling form of the antigen. The second labeling form of the antigen has a label that provides a detectable signal different from the label on the first labeling form of the antigen. Preferred selections of labels are described herein, insofar as the label on the second labeling form of the antigen is different from the label on the first labeling form of the antigen. Such labels include small molecules, radioisotopes, enzyme proteins, and fluorescent dyes. In some embodiments, the first label is AlexaFluor647 and the second label is phycoerythrin.

[0073] In some embodiments, the second labeled form of the antigen is the monovalent form of the antigen. In some embodiments, the second labeled form of the antigen is the polyvalent form of the antigen. In some embodiments, the second labeled form of the antigen includes a mixture of the monovalent and polyvalent forms of the antigen. In embodiments where the polyvalent form of the antigen is used, such a form can be provided by a polyvalent molecule to which the antigen is conjugated or linked. In some embodiments, the polyvalent molecule is a streptavidin multimer (e.g., a tetramer), which can be complexed with biotin and then linked to the antigen to provide a polyvalent (tetravalent) form of the antigen. In some embodiments, the streptavidin multimer may include trimers and / or dimers in addition to the tetramer. In some embodiments, the streptavidin multimer is conjugated with a fluorophore such as phycoerythrin. In some embodiments, the polyvalent molecule is a dimer of immunoglobulin Fc fragments to which the antigen is linked to provide a bivalent form of the antigen. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming molecule such as Foldon, to which an antigen can be linked to provide a trivalent form of the antigen.

[0074] The first and second label forms of the antigen may be the same-valence or different-valence forms of the antigen, but must have labels that emit different detectable signals. In some embodiments, the first label form of the antigen is the monovalent form of the antigen, and the second label form of the antigen is also the monovalent form. In some embodiments, the first label form of the antigen is the monovalent form of the antigen, while the second label form of the antigen is the polyvalent form.

[0075] In some embodiments, tracking is performed with an unlabeled form of the antigen (cold) and a second-labeled form of the antigen (hot), also referred to herein as combined tracking. In such embodiments, after the initial binding step and the washing step, antibody-producing cells are tracked with the unlabeled form of the antigen and the second-labeled form of the antigen. The two forms of tracking antigen: the unlabeled form of the antigen ("cold tracking antigen") and the second-labeled form of the antigen ("hot tracking antigen") can be brought into contact with the cells simultaneously or sequentially (for example, the cold tracking antigen may be added first, followed by the hot tracking antigen, or vice versa). In some embodiments, the unlabeled form of the antigen is monovalent. In some embodiments, the unlabeled form of the antigen is polyvalent. In some embodiments, the unlabeled form of the antigen is a mixture of the monovalent and polyvalent forms. In some embodiments, the second-labeled form of the antigen is monovalent. In some embodiments, the second-labeled form of the antigen is polyvalent. In some embodiments, the second-labeled form of the antigen includes a mixture of the monovalent and polyvalent forms. In embodiments where a polyvalent form of an antigen is used for combination tracking, such a form may be provided by a polyvalent molecule to which the antigen is bound or linked. In some embodiments, the polyvalent molecule is a streptavidin polymer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a polyvalent (e.g., tetravalent) form of the antigen. In some embodiments, the streptavidin polymer may include trimers and / or dimers in addition to tetramers. In some embodiments, the polyvalent molecule is a dimer of an immunoglobulin Fc fragment, to which the antigen can be linked to provide a bivalent form of the antigen. In some embodiments, the polyvalent molecule is a trimer of a trimer-forming molecule such as Fordon, to which the antigen can be linked to provide a trivalent form of the antigen.

[0076] In any form of tracking, the tracking antigen concentration used is excessive compared to the concentration of the first labeled form of the antigen, regardless of the form of the antigen used for tracking (i.e., unlabeled form, second labeled form, or both unlabeled and second labeled forms). In embodiments where the unlabeled form of the antigen is used (in cold tracking or combined tracking), the antigen in the unlabeled form is at least twice as high as the antigen in the first labeled form, i.e., a molar ratio of 2 to, for example, 2500 times. In some embodiments, the antigen in the unlabeled form is twice as high as the antigen in the first labeled form. In some embodiments, the antigen in the unlabeled form is three times as high as the antigen in the first labeled form. In some embodiments, the antigen in the unlabeled form is four times as high as the antigen in the first labeled form. In some embodiments, the antigen in the unlabeled form is five times as high as the antigen in the first labeled form. In some embodiments, the antigen in the unlabeled form is six times as high as the antigen in the first labeled form. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 7:1 to 10:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 8:1 to 10:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 9:1 to 10:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 15:1 to 10:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 20:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 25:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 30:1. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 35:1. In some embodiments, the unlabeled form of the antigen is present in a 40-fold molar ratio to the first labeled form of the antigen.In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 45 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 50 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 60 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 70 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 80 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 90 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 100 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 110 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 120 times that of the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 130-fold relative to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 140-fold relative to the first labeled form of the antigen. In some embodiments, the unlabeled form of the antigen is present in a molar ratio of 150-fold relative to the first labeled form of the antigen. In some embodiments, depending on the concentration of the first labeled form of the antigen, the unlabeled form of the antigen may be at a concentration of 0.4 nM to 1 μM or 10 to 600 nM. In some embodiments, depending on the concentration of the first labeled form of the antigen, the unlabeled form of the antigen may be at concentrations of 10 nM to 600 nM, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 65 nM, 10 nM to 50 nM, 10 nM to 40 nM, 10 nM to 30 nM, 10 nM to 25 nM, or 10 nM to 20 nM. In embodiments where a second labeled form of the antigen is used (in hot tracking or combined tracking), the antigen in the second labeled form is at least twice as much as the antigen in the first labeled form, i.e., a molar ratio of 2 to, for example, 2500 times.In some embodiments, the antigen in the second labeling form is in a molar ratio of 2:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 3:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 4:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 5:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 6:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 7:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 8:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 9:1 to 1:1 relative to the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 15 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 20 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 25 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 30 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 35 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 40 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 45 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 50 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 60 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a 70-fold molar ratio to the antigen in the first labeling form.In some embodiments, the antigen in the second labeling form is in a molar ratio of 80 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 90 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 100 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 110 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 120 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 130 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 140 times that of the antigen in the first labeling form. In some embodiments, the antigen in the second labeling form is in a molar ratio of 150 times that of the antigen in the first labeling form. In some embodiments, depending on the concentration of the first labeled form of the antigen, the second labeled form of the antigen may be at a concentration of 0.4 nM to 1 mM or 10 nM to 600 nM. In some embodiments, depending on the concentration of the first labeled form of the antigen, the second labeled form of the antigen may be at a concentration of 10 nM to 600 nM, 10 nM to 500 nM, 10 nM to 400 nM, 10 nM to 300 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 75 nM, 10 nM to 65 nM, 10 nM to 50 nM, 10 nM to 40 nM, 10 nM to 30 nM, 10 nM to 25 nM, or 10 nM to 20 nM.

[0077] In some embodiments where combined tracking is performed, the cold tracking antigen and the hot tracking antigen may be at the same or different concentrations.

[0078] In some embodiments of combined tracking, cells are sequentially contacted with a cold tracking antigen and a hot tracking antigen, and in some such embodiments, a washing step may be included between incubations with the two tracking antigens. In such embodiments, cells are washed with a washing buffer for a sufficient allocated time to remove unbound antigens. In some embodiments, washing cells over a period including one wash for a total of about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or about 60 minutes. In some embodiments, washing cells over a period including two washes, each for about 5 minutes, or about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes. Additional washing intervals are contemplated, which are essentially equivalent to those described herein.

[0079] The tracking is performed for a period of time sufficient to allow the tracking antigen to bind to the antibody. In some embodiments, the tracking is performed for a period of time of about 5 to about 60 minutes in the unlabeled form of the antigen. In some embodiments, the tracking is performed for about 50 minutes in the unlabeled form of the antigen. In some embodiments, the tracking is performed for about 45 minutes in the unlabeled form of the antigen. In some embodiments, the tracking is performed for about 40 minutes in the unlabeled form of the antigen. In some embodiments, the tracking is performed for a period of time of about 30 minutes. In some embodiments, the tracking is performed for about 20 minutes in the unlabeled form of the antigen. In some embodiments, the tracking is performed for about 10 minutes in the unlabeled form of the antigen.

[0080] In some embodiments, tracking is performed for a period of time of about 5 to 60 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for about 50 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for about 45 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for about 40 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for a period of time of about 30 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for about 20 minutes with the second labeled form of the antigen. In some embodiments, tracking is performed for about 10 minutes with the second labeled form of the antigen.

[0081] In some embodiments of combined tracking, tracking is performed with the unlabeled form of the antigen for a period of about 5 to about 60 minutes (e.g., 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes), followed by contact with a second labeled form of the antigen for a period of about 5 to about 60 minutes (e.g., 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes). The contact times for the unlabeled form of the antigen and the second labeled form of the antigen can be the same or different. As a non-limiting example, the unlabeled form of the antigen may be in contact with a primary antibody-producing cell bound to the first labeled form of the antigen for about 30 minutes, while the second labeled form of the antigen may then be in contact with the cell for about 45 minutes, and vice versa. In some embodiments of combined tracking, tracking is performed simultaneously with the unlabeled form of the antigen and the second labeled form of the antigen for about 5 to about 60 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes.

[0082] In some embodiments, tracking is performed two or more times. In some embodiments, cold tracking is performed two or more times. In some embodiments, hot tracking is performed two or more times. In some embodiments, cold tracking is performed once, followed by two or more hot trackings. In some embodiments, cold tracking is performed two or more times, followed by one hot tracking. In some embodiments, cold tracking is performed two or more times, followed by two or more hot trackings. In some embodiments, combined tracking is performed two or more times. In some embodiments, a washing step is included after each tracking step. In some embodiments, two or more washing steps, for example, two washes or three washes, are included after each tracking step.

[0083] Removal of unbound antigens after follow-up After the follow-up step (for example, antibody-producing cells bound to the first labeled form of the antigen are followed by the unlabeled form of the antigen, the second labeled form of the antigen, or both the unlabeled and second labeled forms of the antigen), the unbound antigen is removed again.

[0084] In some embodiments, unbound antigens are removed by washing.

[0085] In some embodiments, cells that remain bound to the first labeled form of the antigen are collected, and tracking and washing are repeated two or more times before a population of cells enriched with cells expressing high-affinity antibodies is obtained.

[0086] Fluorescence-activated cell sorting (FACS) Flow cytometry is a common analytical cell biology technique that uses light to count and profile cells in heterogeneous fluid mixtures. Flow cytometry is a particularly powerful method because it allows researchers to quickly, accurately, and easily collect data relevant to many parameters from heterogeneous fluid mixtures containing living cells. Fluorescence-activated cell sorting (FACS) is a derivative of flow cytometry that adds superior functionality. Using FACS, researchers can physically sort heterogeneous cell mixtures into different populations.

[0087] Two-dimensional (2D) FACS is cell sorting based on two different fluorescent labels. Two-dimensional FACS provides more selective results than FACS based on a single parameter. Two-dimensional FACS can be used in embodiments of this disclosure that use hot tracking. In some embodiments, two-dimensional FACS is used to collect cells that remain bound to the first labeled form of the antigen, where the first detectable label is a first fluorescent label and the second detectable label is a second fluorescent label different from the first fluorescent label. In a specific embodiment, the first detectable label is A647 and the second detectable label is phycoerythrin.

[0088] Obtain a population of cells enriched with cells expressing high-affinity antibody molecules. Antibody-producing cells that remain bound to the primary labeling form of the antigen after tracking are collected. This collection of antibody-producing cells allows for obtaining a population of cells enriched with cells expressing high-affinity antibody molecules.

[0089] A population of cells enriched with cells expressing high-affinity antibody molecules can be sorted or isolated into single cells. In some embodiments, fluorescence-activated cell sorting (FACS) is used to sort and select single antibody-producing cells. Protocols for single-cell isolation by flow cytometry are well known (Huang, J. et al, 2013, see above). Single antibody-producing cells may be sorted and collected by alternative methods known in the art, including but not limited to manual single-cell picking, limited dilution, B-cell panning of adsorbed antigens, microfluidics, laser-capture microdissection, and gel bead emulsion (GEM), all of which are well known in the art. For example, see Rolink et al., J Exp Med (1996) 183:187-194; Lightwood, D. et al, J. Immunol. Methods (2006) 316(1-2):133-43; Gross et al., Int. J. Mol. Sci. (2015) 16:16897-16919; and Zheng et al., Nature Communications (2017) 8:14049. Gel bead emulsions (GEMs) are also commercially available (for example, the 10X Chromium System from 10x Genomics (Pleasanton, CA)).

[0090] At the time of collection, single-antibody-producing cells may be grown using a common cell culture technique for subsequent DNA preparation. Alternatively, the antibody gene may be amplified directly from the single-antibody-producing cells and then cloned into a DNA vector.

[0091] The process of generating antibodies from nucleic acids obtained from antibody-producing cells that express high-affinity antibody molecules. Nucleic acids encoding antibodies or fragments thereof can be isolated from antibody-producing cells obtained using the methods described herein.

[0092] In some embodiments, genes or nucleic acids encoding immunoglobulin variable heavy and variable light chains (i.e., VH and VL, where VL may be a Vκ or Vλ chain) can be recovered using RT-PCR protocols with nucleic acids isolated from antibody-producing cells. These RT-PCR protocols are well-known and conventional techniques, as described, for example, in Wang et al., J.Immunol.Methods(2000)244:217-225 and herein.

[0093] In some embodiments, the nucleic acid encodes a fragment of an antibody, such as a variable domain, a constant domain, or a combination thereof. In certain embodiments, the nucleic acid isolated from antibody-producing cells encodes the variable domain of an antibody. In some embodiments, the nucleic acid encodes an antibody heavy chain or a fragment thereof (e.g., the variable domain of an antibody heavy chain). In other embodiments, the nucleic acid encodes an antibody light chain or a fragment thereof (e.g., the variable domain of an antibody light chain).

[0094] Upon recovery, the antibody-encoding gene or nucleic acid can be cloned into IgG heavy and light chain expression vectors and expressed via transfection into host cells. For example, the antibody-encoding gene or nucleic acid can be inserted into a replicable vector for further cloning (DNA amplification) or expression in cells (stable or transient). Many vectors, particularly expression vectors, are available or can be engineered to include appropriate regulatory elements necessary to modulate the expression of antibodies encoding genes or nucleic acids.

[0095] Expression vectors in connection with this disclosure may be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a preferred set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the nucleic acid molecule is contained in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., described in Sykes and Johnston, Nat Biotech (1997) 12:355-59), a compressed nucleic acid vector (e.g., described in US6,077,835), or a plasmid vector such as pBR322 or pUC 19 / 18. Such nucleic acid vectors and their uses are well known in the art. See, for example, US5,589,466 and US5,973,972. In certain embodiments, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in yeast systems may be used. Suitable vectors include, for example, those containing constitutive or inductive promoters such as yeast alpha factor, alcohol oxidase, and PGH. See F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987); and Grant et al., Methods in Enzymol 153, 516-544 (1987).

[0096] In a particular embodiment, the vector comprises a nucleic acid molecule (or gene) encoding the heavy chain of the antibody and a nucleic acid molecule (or gene) encoding the light chain of the antibody, and the antibody is produced by an antibody-producing cell obtained by the method of the present disclosure.

[0097] Suitable host cells for antibody molecule expression include, but are not limited to, cells of prokaryotic or eukaryotic (generally mammalian) origin. In some embodiments, the host cell is a bacterial cell or a yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cells may be, for example, Chinese hamster ovary cells (CHO), e.g., CHO K1, DXB-11 CHO, Veggie-CHO cells; COS (e.g., COS-7); stem cells; retinal cells; Vero cells; CV1 cells; kidney cells, e.g., HEK293, 293 EBNA, MSR 293, MDCK, aHaK, BHK21 cells; HeLa cells; HepG2 cells; WI38, MRC 5; Colo25; HB 8065; HL-60; Jurkat cells or Daudi cells; A431 (epidermal) cells; CV-1, U937, 3T3, or L cells; C127 cells, SP2 / 0, NS-0, or MMT cells; tumor cells; and cell lines derived from any of the aforementioned cells. In a particular embodiment, the host cells are CHO cells. In a specific embodiment, the host cells are CHO K1 cells.

[0098] It will be understood that the full-length antibody nucleic acid sequence or gene can then be cloned into a suitable vector or vectors. Alternatively, the Fab region of an isolated antibody can be cloned into a vector or vector along the constant region of any isotype. Thus, any constant region can be used to construct isolated antibodies containing IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, and IgE heavy chain constant regions, or chimeric heavy chain constant regions. Such constant regions can be obtained from any human or animal species, depending on the intended use of the antibody. Furthermore, the antibody variable region or Fab region can be cloned into a suitable vector for protein expression in other forms, such as scFv, diabodies, etc.

[0099] In some embodiments, host cells containing one or more nucleic acids encoding an antibody are cultured under conditions that express a full-length antibody, and the antibody can then be produced and isolated for further use. In certain embodiments, the host cells contain nucleic acids encoding the variable domain of the antibody, and the cells are cultured under conditions that express the variable domain. In other embodiments, the host cells contain nucleic acids encoding the variable heavy chain (VH) domain of the antibody, and the cells are cultured under conditions that express the VH domain. In yet another embodiment, the host cells contain nucleic acids encoding the variable light chain (VL) domain of the antibody, and the cells are cultured under conditions that express the VL domain. In specific embodiments, the host cells contain nucleic acids encoding the VH domain and nucleic acids encoding the VL domain of the antibody, and the cells are cultured under conditions that express the VH and VL domains. [Examples]

[0100] Example 1. Bead preparation As a proof of concept, polystyrene microbeads were used instead of B cells. The polystyrene microbeads used were the same size as B cells. Polystyrene microbeads conjugated with a polyclonal anti-hFc capture antibody (Spherotech, Lake Forest, IL) were coated with four monoclonal antibodies against human IL13 protein with varying affinities: anti-IL13-1, anti-IL13-2, anti-IL13-3, and anti-IL13-4. Dissociation of the binding between these four antibodies and hIL13 was observed. 1 / 2 As shown in Table 1, the time intervals are 1.3 minutes for anti-IL13-1, 10 minutes for anti-IL13-2, 102 minutes for anti-IL13-3, and 1155 minutes for anti-IL13-4. [Table 1]

[0101] Polystyrene beads were incubated overnight at 4°C with four anti-human IL13 antibodies (anti-IL13-1, anti-IL13-2, anti-IL13-3, and anti-IL13-4). The following day, the beads were washed with PBS to remove unbound antibodies. The conjugated beads were then incubated with A647 conjugate hIL13 at either 5 nM or 0.2 nM for 30 minutes. A staining profile using 5 nM was preferred, but concentrations ranging from 0.2 nM to 5 nM were also tested and successful. Unbound A647 conjugate hIL13 was removed by two washes using staining buffer, and the mAb-coated beads were ready for use in tracking experiments: no tracking, monovalent cold tracking, fluorophore-labeled polyvalent tracking, and combined tracking with monomeric cold antigen and fluorophore-labeled polyvalent antigen.

[0102] Example 2: Proof-of-concept separation of polystyrene beads in FACS experiments based on antigen binding and dissociation rates To demonstrate the ability to isolate B cells in FACS-based experiments based on antigen binding and dissociation rates, fluorophore-labeled antigens were used, followed by variable tracking steps: 1) no tracking, 2) monovalent cold tracking, 3) fluorophore-labeled ("hot") multivalent tracking, and 4) combined tracking of monovalent cold and hot multivalent antigens. A schematic diagram illustrating the overall picture of the three exemplary tracking methods used is shown in Figure 2.

[0103] No tracking For samples without tracking, no further steps were required; however, the cells were pelleted by centrifugation, resuspended in PBS, and analyzed by flow cytometry. The results can be seen in Figure 3, which is a representative univariate histogram overlay of A647 staining for IL13 antibody-coated beads without tracking, detected by flow cytometry.

[0104] Monovalent cold tracking For monovalent cold tracking experiments, hIL13 mAb-coated beads were incubated with 20 nM or 500 nM unlabeled hIL13 for 45 minutes, followed by two washes with PBS. A second incubation period of another 45 minutes was performed with unlabeled hIL13. After the second incubation, the beads were pelletized by centrifugation, resuspended in PBS, and analyzed by flow cytometry.

[0105] The results are shown in Figures 4 and 5, which are representative univariate histogram overlays of A647 staining of IL13-coated beads detected by flow cytometry. Figure 4 presents the results of a monovalent cold tracking experiment in which four different groups of beads were coated with IL13 antibodies having different dissociation constants, exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice with 4x (20 nM) unlabeled human IL13 for 45 minutes each, with two washes during that time, after which A647 was detected by flow cytometry. Figure 5 presents the results of a monovalent tracking experiment in which four different IL13 antibody-coated beads were exposed to 5 nM A647-conjugated human IL13, washed, and then incubated twice with 100x (500 nM) unlabeled human IL13 for 45 minutes each, with two washes during that time. 1 / 2 Beads coated with two IL13 mAbs (anti-IL13-3 and anti-IL13-4) exhibited the same staining level with and without tracking; on the other hand, shorter t 1 / 2 Beads coated with the other two mAbs (anti-IL13-1 and anti-IL13-2) showed weaker staining under cold tracking conditions and longer t than samples without cold tracking. 1 / 2 This shows better separation from beads coated with mAb.

[0106] Fluorophore-labeled multivalent tracking For fluorophore-labeled multivalent tracking experiments, hIL13 mAb-coated beads were incubated for 45 minutes with 20 nM biotin-hIL13 pre-conjugated with 5 nM phycoerythrin (PE)-streptavidin (SA). Subsequently, after two washes with PBS, a second incubation was performed for another 45 minutes with 20 nM biotin-hIL13 pre-conjugated with 5 nM PE-SA. After the second incubation, the beads were pelletized by centrifugation, resuspended in PBS, and analyzed by flow cytometry.

[0107] The results of the fluorophore-labeled multivalent tracking experiment are shown in both the A647 histogram overlay (Figure 6) and the A647 vs. PE two-color dot plot overlay (Figures 8 and 10). When A647-labeled hIL13 was applied at 5 nM (shown in Figure 6), looking only at the A647 staining level, all mAb-coated beads that underwent fluorophore-labeled multivalent tracking showed the same difference in A647 staining level as their corresponding mAb-coated beads that underwent monomeric cold tracking shown above; however, looking at the PE staining level, the longest t 1 / 2 (t 1 / 2 Beads coated with mAb having resistance to IL13-4 (=1155 min) are resistant to IL13-3 (t 1 / 2 The lowest PE staining is observed when the coated beads are further away from PE staining (=102 mins), which is shown in Figures 8 and 10, with shorter t 1 / 2 These are clearly separated from the beads coated with anti-IL13-1 and anti-IL13-2, which are two mAbs with 1.3 min and 10 min respectively. The addition of a second color in this trace is two longer t 1 / 2 This helped to further separate the coated beads from each other.

[0108] A combination of monovalent cold tracking and fluorophore-labeled multivalent tracking. For experiments combining monovalent cold tracking and fluorophore-labeled polyvalent tracking, hIL13 mAb coated beads were incubated for 45 minutes with the combination of the two tracking agents described above, namely 500 nM unlabeled hIL13 and 20 nM biotin-hIL13 pre-conjugated with 5 nM PE-streptavidin. Subsequently, after two washes with PBS, a second incubation was performed for another 45 minutes with 500 nM unlabeled hIL13 and 20 nM biotin-hIL13 pre-conjugated with 5 nM PE-streptavidin.

[0109] For experiments combining monovalent cold tracking and fluorophore-labeled multivalent tracking, the results are again shown in the A647 histogram overlay (Figure 7) and the A647 v PE 2-color dot plot overlay (Figures 8 and 10). When A647-labeled hIL13 was applied at 0.2 nM, the longest t 1 / 2 (t 1 / 2 Beads coated with mAb (=1155 min) were clearly noticeable and separated from the remaining mAb-coated beads in the A647 v PE dot plot overlay. 1 / 2 =102 min), anti-IL13-2 (t 1 / 2 =10 minutes), and anti-IL13-1(t 1 / 2 The beads coated in 1.3 minutes had the longest t 1 / 2 Not only are the beads coated with mAb (anti-IL13-4) separated, but they are also close to each other in the dot plot. The addition of a second color in this trace is two longer t 1 / 2 It helps to further separate the coated beads from each other.

[0110] Therefore, B cells expressing surface-anchored antibodies with fast or slow dissociation constants can be isolated by labeling the B cells using the various tracking conditions of this disclosure and incorporating them into a sorting strategy for enriching high-affinity antibodies.

[0111] Example 3: Separation of B cells in FACS experiments based on antigen binding and dissociation rate In a standard B cell sorting workflow, splenocytes from immunized mice are stained with fluorescently labeled antigens, washed, and then sorted into single cells using flow cytometry. Sorting gating is primarily based on the selection of IgG and antigen-double positive populations, thus allowing the isolation of antigen-specific B cells expressing antibodies with varying affinities. This process has been previously refined to identify higher-affinity antibodies by utilizing monomer sorting reagents and specifically sorting cells with the highest antigen-specific + / IgG+ ratio ("diagonal sorting").

[0112] In this embodiment, the B cell sorting process was further refined by adding a tracking step designed to remove B cells expressing lower affinity antibodies from B cells expressing high affinity antigen-specific antibodies. In this improved workflow, the tracking step was performed in the presence of multimerized and differentially labeled antigens, with or without additional unlabeled ("cold") antigens. AlexaFluor647 (A647)-labeled human IL13 was incubated with mouse splenocytes for 30 minutes, followed by two washes to remove unbound sorting reagents, presenting only B cells bound to labeled human IL13. Labeled B cells were further subjected to a tracking step using one of two methods: multivalent tracking or combined (cold monovalent tracking and hot multivalent) tracking. Multivalent tracking consisted of biotin-hIL13 pre-clustered with streptavidin-PE in a 4:1 ratio, resulting in a mixture of tetramers, trimers, dimers, and several monomers. The combined tracking consisted of "cold" (unlabeled) monovalent hIL3 combined with a multivalent tracking antigen. These tracking approaches significantly improved the efficiency of the isolation process for ultra-high affinity mAbs by reducing the total number of antibodies that needed to be cloned, expressed, and screened.

[0113] A very high-affinity antibody against the soluble cytokine target IL13 was isolated. Genetically modified mice possessing a human immunoglobulin variable region gene segment that was not rearranged at the endogenous mouse immunoglobulin locus were generated by the method described in U.S. Patent No. 8,502,018. The genetically modified mice were immunized with a fusion of human IL13 in the form of an mFc dimer protein and an antibody targeting antigen-presenting cells. When the tracking strategy disclosed herein was implemented, B cell isolation was observed for longer than 100 minutes. 1 / 2 A total of 58 antibodies possessing this characteristic were identified. More than 50% of all antibodies isolated using this method had nanomolar or better affinity. Of these, 47 antibodies had ultra-high affinity, 1 × 10⁻⁶. -10 The antibodies exhibited affinity less than (M). This is in stark contrast to 20% of antibodies with sub-nanomolecular affinity identified using conventional sorting strategies without any "follow-up" steps, resulting in only 7 ultra-high affinity antibodies. Of the 47 ultra-high affinity antibodies, 39 were also potent inhibitors of IL13 in bioassays. Thus, the disclosed B-cell sorting strategy offers a significant improvement over current methods for those applications where high affinity antibodies are required to achieve therapeutic efficacy.

[0114] As described, after selecting B cells from IL13-immunized mice, variable domains for both the heavy and light chains were cloned into a fully human IgG1 expression construct and transiently expressed in CHO cells. Antibodies containing the supernatant were assayed for target-specific engagement using Luminex or SPARCL assays. All IL13 conjugates across all selection strategies were moved to Biacore and Bioassay screening, and the results are shown in Table 2. The numbers shown in Table 2 refer to the number of mice used in a particular selection strategy, and each mouse can be used across multiple selection strategies. Therefore, the total number of mice selected overall was 13. [Table 2]

[0115] Antibodies from representative mice were analyzed using Biacore, and the results are shown in Figures 13A-C. Under conditions without tracking (Figure 13A), most of the isolated antibodies were short t 1 / 2 It showed low affinity. However, antibodies isolated using either of the two tracking strategies (Figure 13B and Figure 13C) showed better results than 100 minutes. 1 / 2 This shows a significant enrichment of sub-nanomolecular affinity antibodies. In addition to having improved affinity properties, these antibodies were also potent inhibitors in the IL13 inhibition assay. This is shown in Figures 13B-C, where yellow indicates 100% inhibition, indicated by the color of the data points.

[0116] Table 3 shows the antibodies analyzed by Biacore from all experimental mice. The trends observed in all mice are consistent with the trends observed in the individual mouse examples in Figures 13A-C. Figures 14B, 14D, and 14F, in enlarged view, focus on antibodies with higher affinity. Along with Figures 14A-F, Table 3 shows the longer t 1 / 2 More than 3-fold enrichment in antibodies, and overall K D This indicates that a more than twofold enrichment was observed. Additionally, as seen in Figures 14E and 14F, high affinity antibodies from the cold monovalent + multivalent tracking strategy also tend to be strong IL13 bioassay blockers (using 100–200 pM hIL13 in the bioassay).

[0117] Overall, the cold + multimer tracking strategy is more effective over longer periods. 1 / 2 Antibodies containing [the specified characteristic] were enriched more than 3-fold (2.3% to 7.4%). Although IL13 was used in these examples, the tracking strategies presented herein can be implemented for other targets requiring ultra-high affinity antibodies. [Table 3]

[0118] Example 4. Proof of Concept: Separation of antibody-coated beads based on the dissociation rate between antibody and multimeric antigen in FACS experiments. Objective: To demonstrate the ability to isolate antibody-expressing cells based on the dissociation rate between antibodies and multimeric antigens, specifically Ebola GP trimer proteins, using a fluorophore-labeled multivalent tracking method in FACS-based experiments.

[0119] In this proof-of-concept experiment, polystyrene microbeads were used instead of antibody-expressing cells. As described in Example 1, the beads were separately coated with two monoclonal antibodies against the Zaire Ebola GP protein with different affinities: anti-Ebola GP-1 and anti-Ebola GP-2. Dissociation of the binding between these two antibodies 1 / 2 As shown in Table 4, the time required for the anti-Ebola GP-1 strain was 1155 minutes, and for the amti-Ebola GP-2 strain it was 3 minutes. [Table 4]

[0120] Next, antibody-coated beads were incubated with either 5 nM or 0.2 nM A647 conjugate Ebola GP trimer protein for 30 minutes. Unbound A647 conjugate Ebola GP trimer protein was removed by two washes using staining buffer, and the antibody-coated beads were further applied under either no-tracking or fluorophore-labeled multivalent tracking conditions. The samples were subsequently analyzed by flow cytometry. The results are shown in the A647 vs. PE two-color dot plot overlay in Figure 15.

[0121] For samples without tracking, no further steps were required. Samples were pelletized by centrifugation, resuspended in PBS, and analyzed by flow cytometry. As shown in Figure 15A, for beads incubated with 5 nM A647-conjugated Ebola GP trimer protein, the A647 MFI for anti-Ebola GP-1 coated beads (shaded in green) was 6,335, and for anti-Ebola GP-2 coated beads (shaded in purple) was 3,781. Although they stain differently, the overlay showed that the two populations overlapped considerably and were therefore difficult to separate with A647 staining (Y axis). Figure 15C showed similar results for beads incubated with 0.2 nM Ebola GP trimer protein. The A647 MFI for anti-Ebola GP-1 coated beads (shaded in green) is 1,466, and the A647 MFI for anti-Ebola GP-2 coated beads (shaded in purple) is 908. The two bead populations overlap significantly and are inseparable in the overlay.

[0122] In beads treated with a fluorophore-labeled multivalent tracking method, unbound A647-conjugated Ebola GP trimer protein was removed. The antibody-coated beads were then incubated for 45 minutes with 20 nM biotin-labeled Ebola GP trimer protein pre-conjugated with 5 nM phycoerythrin (PE)-streptavidin (SA). Subsequently, after two washes with PBS, a second incubation was performed for another 45 minutes with 20 nM biotin-labeled Ebola GP trimer protein pre-conjugated with 5 nM PE-SA. After the second incubation, the beads were pelletized by centrifugation, resuspended in PBS, and analyzed by flow cytometry.

[0123] The results of the fluorophore-labeled multivalent tracking experiment are shown in the A647 vs. PE two-color dot plot overlays in Figures 15B and 15D. In Figure 15B, when A647-labeled Ebola GP trimer protein was applied at 5 nM, both the anti-Ebola GP-1 and anti-Ebola GP-2 coated beads that underwent fluorophore-labeled multivalent tracking showed similar differences in A647 staining levels to their corresponding antibody-coated beads without tracking, as shown in Figure 15A; however, when PE staining levels are also considered in the dot plot, the two antibody-coated populations are separated from each other. (Beads shaded in green (t)) 1 / 2 (Coated with anti-Ebola GP-1 for 1155 minutes) Less PE staining, more A647 staining, this is a purple shaded bead (t 1 / 2 (=3 minutes, coated with anti-Ebola GP-2) is separated. The addition of a second color in this tracking indicates a different t 1 / 2 This helped to separate beads having one another. Beads coated with two different antibodies can be separated from each other using a fluorophore-labeled multivalent tracking method based on the dissociation rate in the case of trimer antigen binding.

Claims

1. A method for obtaining antibody-producing cells that express antibody molecules exhibiting high binding affinity to an antigen, (a) Contacting a population of antibody-producing cells, including cells expressing antibody molecules against the antigen on their cell surface, with a first labeled form of the antigen, thereby enabling the antigen to bind to the antibody molecules on the cell surface, wherein the antigen in the first labeled form is conjugated to a first detectable label. (b) Washing the cells to remove unbound antigens, (c) The cells (i) Unlabeled form of the antigen, (ii) A second labeling form of the antigen, or (iii) Contacting the unlabeled form of the antigen with the second labeled form of the antigen, (d) Washing the cells to remove unbound antigens, (e) A method comprising collecting cells that remain bound to the first labeled form of the antigen, thereby obtaining cells that express antibody molecules with high affinity for the antigen.

2. The method according to claim 1, wherein the first labeling form of the antigen has a concentration of 0.001 nM to 1 μM.

3. The method according to claim 1, wherein the first labeling form of the antigen has a concentration of 0.1 to 7.5 nM.

4. The method according to any one of claims 1 to 3, wherein the first detectable label is a first fluorescent label.

5. The method according to any one of claims 1 to 4, wherein the antigen is a protein in monomeric form.

6. The method according to any one of claims 1 to 4, wherein the antigen is a protein in a multimeric form.

7. The method according to any one of claims 1 to 4, wherein the antigen is a protein that exists in both monomeric and polymeric forms.

8. The method according to any one of claims 5 to 7, wherein the first labeling form of the antigen is the monovalent form of the antigen.

9. The method according to any one of claims 5 to 8, wherein the unlabeled form of the antigen is the monovalent form of the antigen.

10. The method according to any one of claims 5 to 8, wherein the unlabeled form of the antigen is the polyvalent form of the antigen.

11. The method according to claim 10, wherein the polyvalent form of the antigen is provided by a polyvalent molecule to which the antigen is bound or linked.

12. The method according to claim 11, wherein the polyvalent molecule is selected from streptavidin macromers such as tetramers, dimers of immunoglobulin Fc fragments, or trimers of trimer-forming molecules such as Fordon.

13. The method according to any one of claims 5 to 12, wherein the second labeling form of the antigen is the monovalent form of the antigen.

14. The method according to any one of claims 7 to 12, wherein the second labeling form of the antigen is the polyvalent form of the antigen.

15. The method according to claim 14, wherein the polyvalent form of the antigen is provided by a polyvalent molecule to which the antigen is bound.

16. The method according to claim 15, wherein the polyvalent molecule is a streptavidin macromer (e.g., a tetramer), a dimer of an immunoglobulin Fc fragment, or a trimer of a trimer-forming molecule such as Fordon.

17. The method according to any one of claims 14 to 16, wherein the polyvalent form of the antigen is labeled with a second detectable label.

18. The method according to claim 17, wherein the second detectable label is a second fluorescent label.

19. The method according to claim 8, wherein in step (c), the cells are brought into contact with the unlabeled form of the antigen.

20. The method according to claim 19, wherein the unlabeled form of the antigen is the monovalent form of the antigen.

21. The method according to claim 19 or 20, wherein the antigen in the unlabeled form is in a molar ratio of at least 2 to 4 times that of the first labeled form of the antigen used in step (a).

22. The method according to claim 8, wherein in step (c), the cells are brought into contact with the second labeled form of the antigen.

23. The method according to claim 22, wherein the second labeling form of the antigen is the polyvalent form of the antigen.

24. The method according to claim 22 or 23, wherein the antigen in the second labeling form is at least 2 to 4 times more molarly than the antigen used in step (a) in the first labeling form.

25. The method according to any one of claims 19 to 24, wherein the contacting in step (c) and the washing in step (d) are repeated at least once before the collection of cells in step (e).

26. The method according to claim 8, wherein in step (c), the cells are brought into contact with the unlabeled form of the antigen and the second labeled form of the antigen.

27. The method according to claim 26, wherein the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a polyvalent form of the antigen.

28. The method according to claim 26, wherein the antigen is a monomeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a polyvalent form of the antigen.

29. The method according to claim 26, wherein the antigen is a polymeric protein, the unlabeled form is the monovalent form of the antigen, and the second labeled form of the antigen is the polyvalent form of the antigen.

30. The method according to claim 26, wherein the antigen is a protein that exists in both monomeric and polymeric forms, the unlabeled form is the monovalent form of the antigen, and the second labeled form of the antigen is the polyvalent form of the antigen.

31. The method according to any one of claims 26 to 30, wherein the cells are brought into contact with the unlabeled form of the antigen and the second labeled form of the antigen simultaneously.

32. The method according to any one of claims 26 to 31, wherein the unlabeled form of the antigen is in a molar ratio of at least 2 to 4 times the first labeled form of the antigen used in step (a).

33. The method according to any one of the prior claims, wherein the first detectable label is a fluorescent label, and cells that remain bound to the first labeled form of the antigen are collected using fluorescence-activated cell sorting.

34. The method according to any one of claims 19 to 32, wherein the first detectable label is a first fluorescent label, and the second detectable label is a second fluorescent label different from the first fluorescent label, and cells remaining bound to the first labeled form of the antigen are collected using two-dimensional fluorescence-activated cell sorting.

35. The method according to any one of the prior claims, wherein the antibody-producing cell is a primary antibody-producing cell that produces antibody molecules on its cell surface, yeast, or immortalized mammalian cell.

36. The method according to claim 35, wherein the primary antibody-producing cells are obtained from the spleen, lymph nodes, peripheral blood, and / or bone marrow.

37. The method according to claim 35, wherein the immortalized mammalian cells that produce antibody molecules are selected from Chinese hamster ovary (CHO) cells and hybridoma cells.

38. The method according to any one of the prior claims, wherein the high affinity is in the range of about 0.1 pM to about 25 nM (KD).

39. The method according to claim 38, wherein the high affinity is less than about 10 nM (KD).

40. The method according to any one of the prior claims, further comprising isolating nucleic acids encoding antibodies from the cells collected in step (e).

41. The method according to claim 40, further comprising transfecting host cells with nucleic acids encoding an antibody heavy chain or its variable domain, and an antibody light chain or its variable domain, and growing the transfected host cells under conditions that support antibody expression by the host cells.

42. The method according to claim 41, wherein the host cells are Chinese hamster ovary (CHO) cells.

43. Mammalian host cells prepared by the method described in claim 41 or 42.

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