Method for obtaining antibody molecules with high affinity

Abstract

Disclosed herein is a method for obtaining an antibody-producing cell that expresses an antibody molecule that exhibits a high binding affinity to an antigen, the method comprises: contacting a population of antibody-producing cells encompassing cells expressing an antibody molecule against the antigen on a cell surface with a first labeled form of the antigen to allow the antigen to bind to the antibody molecule on the cell surface, subsequently contacting the cell with (i) an unlabeled form of the antigen, (ii) a second labeled form of the antigen, or (iii) the unlabeled form of the antigen and the second labeled form of the antigen; and collecting the cells that remain bound to the first labeled form of the antigen, thereby obtaining a cell that expresses a high affinity antibody molecule against the antigen. The method of the invention allows cells expressing high affinity antibody molecules to be obtained from antibody-producing cell pools expressing antibody molecules having different affinity.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application 63 / 452,206, filed March 15, 2023, and U.S. Provisional Application 63 / 559,544, filed February 29, 2024, the contents of which are incorporated herein by reference. Background Technology

[0003] High affinity is a desired property 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 in isolating high-affinity antibody molecules.

[0004] Typically, monoclonal antibodies are obtained from mouse hybridomas, most commonly from the fusion of B lymphocytes from immunized mice with mouse myeloma cells. However, due to the throughput limitations of hybridoma cultures, the efficiency of isolating rare high-affinity antibodies using hybridoma technology is not high.

[0005] Another method for generating high-affinity antibody molecules involves using display techniques to generate lead antibody candidates from phage, yeast, or mammalian libraries. While DNA can be isolated directly from B cells expressing antibody molecules, DNA libraries are expressed in cellular expression systems (such as phage, yeast, or bacterial systems) and then “panned” or titrated to select for antibody molecules with high affinity. Display techniques can provide high-quality protein libraries, although the diversity offered is limited. Therefore, affinity maturation based on in vitro mutagenesis is often the next step in generating high-affinity antibody molecules derived from such libraries.

[0006] Therefore, there is a need for an efficient method to obtain large quantities of antibody molecules with the desired specificity and high binding affinity without having to perform several rounds of screening (such as "panning") or site-directed mutagenesis. Summary of the Invention

[0007] This document discloses methods for obtaining B cells and other antibody-producing cells expressing antibody molecules exhibiting high binding affinity to antigens, as well as mammalian host cells prepared using the disclosed methods. This disclosure is based on the observation that cells expressing antibody molecules with high affinity to antigens of interest can be directly selected and enriched from a population of antibody-producing cells with different affinities. Therefore, antibody molecules are directly selected from B cells and other antibody-producing cells prior to single-cell isolation and collection (e.g., using fluorescence-activated cell sorting (FACS)). This provides a simple 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.

[0008] On one hand, this disclosure relates to a method for obtaining antibody-producing cells, said antibody-producing cells expressing antibody molecules that exhibit high binding affinity to antigens, said method comprising:

[0009] (a) Contacting an antibody-producing cell population expressing antibody molecules against the antigen on the cell surface with a first labeled form of the antigen to allow the antigen to bind to the antibody molecules on the cell surface, wherein the first labeled form of the antigen is conjugated to a first detectable label;

[0010] (b) Wash the cells to remove unbound antigens;

[0011] (c) Expose the cells to the following:

[0012] (i) the unlabeled form of the antigen,

[0013] (ii) the second labeled form of the antigen, or

[0014] (iii) the unlabeled form of the antigen and the second labeled form of the antigen;

[0015] (d) Washing the cells to remove unbound antigens; and

[0016] (e) Collect cells that remain bound to the first labeled form of the antigen, thereby obtaining cells expressing high-affinity antibody molecules against the antigen.

[0017] In some embodiments of the method, the concentration of the first labeled form of the antigen is between 0.001 nM and 1 μM. In some embodiments of the method, the concentration of the first labeled form of the antigen is between 0.05 nM and 10 nM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.1 nM and 7.5 nM. In some embodiments, the concentration of the first labeled form of the antigen is about 0.2 nM. In some embodiments, the concentration of the first labeled form of the antigen is about 5 nM.

[0018] In some embodiments, the first detectable marker is a first fluorescent marker.

[0019] In some embodiments, the antigen is a protein in monomeric form. In some embodiments, the antigen is a protein in multimeric form (e.g., dimer, trimer, tetramer, pentamer, hexamer, etc., or mixtures thereof). In some embodiments, the antigen is a protein existing in both monomeric and multimeric forms.

[0020] In some embodiments, the first labeled form of the antigen is a monovalent form of the antigen. In such embodiments, the antigen may be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms, and the monovalent antigen is not multimeric. In some embodiments, the first labeled form of the antigen is a multivalent form of the antigen. In such embodiments, the antigen may 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 present in a multivalent form. In some embodiments, the first labeled form of the antigen is a mixture of monovalent and multivalent forms of the antigen. In embodiments employing a multivalent form of the antigen, the multivalent form of the antigen may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be bound to biotin conjugated to the antigen. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the multivalent molecule is a trimerized domain molecule, such as a trimer of a fold.

[0021] In some embodiments, the unlabeled form of the antigen that can be used for the tracking step is a monovalent form of the antigen, wherein 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 a multivalent form of the antigen, wherein 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 comprises a mixture of monovalent and multivalent forms of the antigen, wherein the antigen may be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In embodiments employing the multivalent form of the antigen, the multivalent form of the antigen may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be bound to biotin conjugated to the antigen. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the multivalent molecule is a trimerized domain molecule, such as a trimer of a fold.

[0022] In some embodiments, the second labeled form of the antigen that can be used for the tracking step is a monovalent form of the antigen, wherein 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 second labeled form of the antigen is a multivalent form, wherein 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 second labeled form of the antigen comprises a mixture of monovalent and multivalent forms, wherein the antigen may be a protein in monomeric form, a protein in multimeric form, or a protein in both monomeric and multimeric forms. In embodiments employing the multivalent form of the antigen, the multivalent form of the antigen may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be bound to biotin conjugated to the antigen. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment. In some embodiments, the multivalent molecule is a trimerized domain molecule, such as a trimer of a folded molecule. The antigen in its second labeled form 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.

[0023] In embodiments where the unlabeled form of the antigen is used in tracking, the unlabeled form may be a monovalent form of the antigen, a multivalent form of the antigen, or a mixture thereof, wherein the antigen may be a protein in monomeric form, a protein in polymeric form, or a protein in both monomeric and polymeric forms. In some embodiments, the unlabeled form of the antigen is the monovalent form of the antigen. In some embodiments, the molar ratio of the antigen in the unlabeled form is at least 2 to 150 times and at most, for example, 2500 times, the molar ratio of the antigen in the first labeled form of the antigen. For example, the molar ratio of the antigen in the unlabeled form is at least 2 to at least 120 times the molar ratio of the antigen in the first labeled form of the antigen, including 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, or 150 times, and at most, for example, 2500 times. In some embodiments, the concentration of the unlabeled form of the antigen in the tracking step is from 0.4 nM to 600 nM, depending on the concentration of the first labeled form of the antigen.

[0024] In embodiments where a second labeled form of the antigen is used in the tracking, the second labeled form of the antigen may be a monovalent form, a multivalent form, or a mixture thereof, a monomeric form, or a polymeric form, wherein the antigen may 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 a multivalent form of the antigen. In some embodiments, the molar ratio of the antigen in the second labeled form is at least 2 to 150 times and at most, for example, 2500 times, the molar ratio of the antigen in the first labeled form. The molar ratio of the antigen in the second labeled form is at least 2 to at least 120 times the molar ratio of the antigen in the first labeled form, including 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, or 150 times, at most, for example, 2500 times. In some embodiments, the concentration of the second labeled form of the antigen is from 15 nM to 600 nM, depending on the concentration of the first labeled form of the antigen.

[0025] In some embodiments, the tracking step and the washing following the tracking step are repeated at least once before collecting cells that remain bound to the first labeled form of the antigen.

[0026] In embodiments where the tracking uses both an unlabeled form of the antigen and a second labeled form of the antigen, the unlabeled form may be a monovalent, multivalent, or a mixture of monovalent and multivalent forms of the antigen, and the second labeled form of the antigen may be a monovalent, multivalent, or a mixture of monovalent and multivalent forms of the antigen. In some embodiments, tracking is performed using both the unlabeled monovalent form of the antigen and the second labeled form of the multivalent antigen. In some embodiments of combined tracking, the molar ratio of the unlabeled form of the antigen is at least 2 to 150 times and at most, for example, 2500 times, of the molar ratio of the first labeled form of the antigen. For example, at least 2 to at least 150 times, including 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, or 150 times and at most, for example, 2500 times; and the molar ratio of the second labeled form of the antigen is at least 2 to 150 times and at most, for example, 2500 times, of the molar ratio of the first labeled form of the antigen. For example, at least 2 to at least 150 times, including 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, or 150 times and at most, for example, 2500 times.

[0027] In some embodiments, the first detectable marker is a fluorescent marker, and fluorescently activated cell sorting is used to collect cells that retain binding to the first labeled form of the antigen. In some embodiments, the first detectable marker is a first fluorescent marker, the second detectable marker is a second fluorescent marker distinguished from the first fluorescent marker, and two-dimensional fluorescently activated cell sorting is used to collect cells that retain binding to the first labeled form of the antigen.

[0028] In some embodiments, the antibody-producing cells are mammalian cells or yeast (such as Saccharomyces cerevisiae or Pichia) that produce antibody molecules on their cell surface. In some embodiments, the antibody-producing mammalian cells are primary antibody-producing cells or immortalized cell lines that produce antibody molecules on their cell surface. In some embodiments, the primary antibody-producing cells are obtained from the spleen, lymph nodes, peripheral blood, and / or bone marrow of mammalian subjects (e.g., humans, rabbits, pigs, cattle, horses, and rodents). In some embodiments, the immortalized cell lines are selected from Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, and hybridoma cells that produce antibody molecules on their cell surface.

[0029] In some embodiments, the cells collected in step (e) are enriched with cells expressing high-affinity antibody molecules. In some embodiments, the KD of the high-affinity antibody molecule obtained is 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, such as KD, which is an antibody molecule with a KD of 0.1 pM to 25 nM. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., KD, which is an antibody molecule with a KD of 0.1 pM to 25 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 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 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 or without tracking. 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 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 or without tracking.

[0030] In some embodiments, the method further comprises isolating nucleic acids encoding antibodies from collected cells that retain binding to the first labeled form of the antigen.

[0031] In some embodiments, the method further comprises transfecting host cells with nucleic acids encoding an antibody heavy chain or a variable domain thereof and a nucleic acid encoding an antibody light chain or a variable domain thereof; and allowing the transfected cells to grow under conditions supporting antibody expression in the transfected cells. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells.

[0032] On the other hand, this disclosure relates to a mammalian host cell prepared by: isolating nucleic acids encoding antibodies from collected cells expressing high-affinity antibody molecules, and transfecting the host cell with nucleic acids encoding the antibody heavy chain or its variable domain and nucleic acids encoding the antibody light chain or its variable domain; and growing the transfected host cell under conditions supporting the expression of antibody molecules in the host cell. In some embodiments, the host cell is a CHO cell. Attached Figure Description

[0033] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or patent application publication with color drawings.

[0034] Figure 1 This is an illustration showing an overall view of the method used in this disclosure. Prior to undergoing a tracking step, beads or cells coated with antibodies of different affinities are exposed and bound to an antigen (i.e., a sorting agent or a first labeled form of the antigen) conjugated to A647 at a concentration of, for example, 5 nM or 0.2 nM. Three exemplary options for tracking are shown: A. Cold (unlabeled) monovalent tracking, wherein the concentration of the unlabeled monovalent antigen is at least 2 and at most 2500 times the concentration of the labeled sorted antigen; B. Fluorophore-labeled (or “hot”) multivalent tracking of pre-bound biotinylated antigen with streptavidin (SA)-phycoerythrin (PE) (where the concentration of the antigen is at least 4 and at most 2500 times the concentration of the labeled sorted antigen, and the ratio of biotinylated antigen to SA-PE is 4:1); or C. A combination of unlabeled monovalent antigen and hot multivalent agent, wherein the concentration of the unlabeled monovalent antigen is at least 2 and at most 2500 times the concentration of the labeled sorted antigen, and the hot multivalent agent uses pre-bound biotinylated antigen and SA-PE (where the concentration of the antigen is at least 2 and at most 2500 times the concentration of the labeled sorted antigen, and the ratio of biotinylated antigen to SA-PE is 4:1).

[0035] Figure 2This is an illustration showing an overall view of the method used in this disclosure, wherein human IL-13 is used as the antigen. Prior to undergoing the tracing step, beads coated with IL-13 antibodies of varying affinities are exposed and bound to A647-conjugated human IL-13 (i.e., the sorting agent) at concentrations such as 5 nM or 0.2 nM. Three exemplary options for tracing are shown: A. Cold (unlabeled) monovalent tracing, wherein the concentration of unlabeled human IL-13 is at least 4 times and at most 2500 times the concentration of the labeled sorting antigen; B. Fluorophore-labeled (thermal) multivalent tracing with streptavidin (SA)-phycoerythrin (PE) to fluorophoretically label pre-bound biotin-labeled human IL-13 (where the concentration of human IL-13 is at least 4 times and at most 2500 times the concentration of the labeled sorting antigen, and the biotin-labeled human IL-13 is...). 3) The ratio of human IL13 to SA-PE is 4:1; or C) A combination of unlabeled monovalent tracer human IL13 (at a concentration at least 4 times and at most 2500 times the concentration of labeled sorted antigen) and a thermal multivalent tracer, said thermal multivalent tracer using pre-bound biotin-labeled human IL13 and SA-PE (wherein the concentration of human IL13 is at least 4 times and at most 2500 times the concentration of labeled sorted antigen, and the ratio of biotin-labeled human IL13 to SA-PE is 4:1).

[0036] Figure 3 This is a representative univariate histogram stack of A647 staining on untraced IL13 antibody-coated beads, as detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies with different dissociation constants (as shown in the insert box) and exposed to 5 nM A647-conjugated human IL13, followed by two washes. Similar experiments were performed with 0.2 nM A647-conjugated human IL13, yielding similar results (not shown).

[0037] Figure 4 This is a representative univariate histogram stack of A647 staining on IL13 antibody-coated beads with cold monomer tracking, as detected by flow cytometry. Four different sets of beads were coated with IL13 antibodies with different dissociation constants (as shown in the insert box) and exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice with 4-fold (20 nM) unlabeled human IL13 for 45 minutes each time, with two washes in between, before A647 was detected by flow cytometry. A similar experiment was performed with the same four different sets of beads coated with IL13 antibody and exposed to 0.2 nM A647-conjugated human IL13, washed, and further incubated twice with 20 nM (i.e., 10-fold) unlabeled human IL13, yielding similar results (not shown).

[0038] Figure 5The above is a stacked univariate histogram of A647 staining of IL13 antibody-coated beads with cold monovalent tracking, as detected by flow cytometry. Beads coated with four different IL13 antibodies were exposed to 5 nM A647-conjugated human IL13, washed, and then further incubated twice with 100-fold (500 nM) unlabeled human IL13 for 45 minutes each time, with two washes in between. A similar experiment was performed in which the same beads coated with four different IL13 antibodies were exposed to 0.2 nM A647-conjugated human IL13, washed, and then further incubated twice with 100-fold (20 nM) unlabeled human IL13, producing similar results (not shown).

[0039] Figure 6 The above is a representative univariate histogram stack of A647 staining of multivalently tracked IL13 antibody-coated beads with fluorophore markers, as detected by flow cytometry. Four different groups of beads were coated with IL13 antibodies with different dissociation constants and exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice with 4-fold (20 nM) biotinylated human IL13 pre-aggregated with 5 nM SA-PE, for 45 minutes each time, with two washes in between. A similar experiment was performed in which beads coated with four different IL13 antibodies were exposed to 0.2 nM A647-conjugated human IL13, washed, and further incubated twice with 100-fold (20 nM) biotinylated human IL13 pre-aggregated with 5 nM SA-PE, yielding similar results (not shown).

[0040] Figure 7 This is a representative univariate histogram superposition of A647 staining on beads coated with IL13 antibody with combined tracking (cold monovalent antigen and hot multivalent antigen). Four different groups of beads were coated with IL13 antibody with different dissociation constants and exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice for 45 minutes each with 500 nM human IL13 pre-aggregated by 5 nM SA-PE and 20 nM biotinylated human IL13, with two washes in between.

[0041] Figure 8This is a representative A647 versus PE dot plot (i.e., tracking plot) of multivalently traced IL13 antibody-coated beads with fluorophore markers, detected by flow cytometry. Four different groups of beads coated with IL13 antibodies with different dissociation constants were exposed to 5 nM A647-conjugated human IL13, washed, and then further incubated twice with 4-fold (20 nM) biotinylated human IL13 pre-aggregated with 5 nM SA-PE, for 45 minutes each time, with two washes in between. A similar experiment was performed in which beads coated with four different IL13 antibodies were exposed to 0.2 nM A647-conjugated human IL13, washed, and further incubated twice with 100-fold (20 nM) biotinylated human IL13 pre-aggregated with 5 nM SA-PE, producing similar results (not shown).

[0042] Figure 9 This is a representative A647 versus PE dot plot (i.e., a tracking plot) of IL13 antibody-coated beads with combined tracking (cold monovalent and hot polyvalent) as detected by flow cytometry. Four different groups of beads coated with IL13 antibodies with different dissociation constants were exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice for 45 minutes each with 100-fold (500 nM) human IL13 pre-aggregated via 5 nM SA-PE and 4-fold (20 nM) biotinylated human IL13, with two washes in between.

[0043] Figure 10 This is a representative A647 dot plot (i.e., tracking plot) of multivalent tracing beads coated with IL13 antibody and fluorophore, as detected by flow cytometry. Four different groups of beads coated with IL13 antibodies with different dissociation constants were exposed to 0.2 nM A647-conjugated human IL13, washed, and then further incubated with 100-fold (20 nM) biotinylated human IL13 pre-aggregated via SA-PE for 45 minutes, with two washes in between.

[0044] Figure 11 This is a representative A647 versus PE dot plot (i.e., tracking plot) of IL13 antibody-coated beads with combined tracking (cold monovalent and hot polyvalent) as detected by flow cytometry. Four different groups of beads coated with IL13 antibodies with different dissociation constants were exposed to 0.2 nM A647-conjugated human IL13, washed, and further incubated with 500 nM human IL13 pre-aggregated via 5 nM SA-PE and 20 nM biotinylated human IL13 for 45 minutes, with two washes in between.

[0045] Figure 12A-C shows the results of spleen cells from a mouse sorted according to the strategy described above. A. No tracking. B. Hot multivalent tracking. C. Combined tracking (cold monovalent and hot multivalent). All cells were first gated based on viability and IgG expression. The final sorting gate of A647 relative to the PE dot plot is highlighted in yellow on the flow cytometry spectrogram (top). The gated cell population was index-sorted and analyzed based on Biacore data, as shown in the lower part of the figure. The no-tracking sorting strategy (A) produces cells with the shortest t 1 / 2 Antibodies with high t-values, while tracking strategies enrich antibodies with longer t-values. 1 / 2 Antibodies (B and C).

[0046] Figure 13A -C shows the antibody results from a representative mouse (number 6018066), where the antibodies were analyzed using Biacore. A. No tracking. B. Hot multivalent tracking. C. Combined tracking (cold monovalent and hot multivalent). Antibodies isolated in the no-tracking case mostly have short t... 1 / 2 The low affinity (A). However, antibodies isolated using either of the two tracking strategies showed t after 100 minutes. 1 / 2 Significant enrichment of sub-nanomolar affinity antibodies (B and C). In addition to having improved affinity properties, the antibodies from the trace condition were also effective inhibitors in the IL13 inhibition assay (as indicated by the color of the data points - yellow indicates 100% inhibition).

[0047] Figures 14A-F show antibodies from all mice analyzed using Biacore. Figure 13A The trend observed in individual mouse instances of -C was also consistently observed across all mice. Figures 14B, 14D, and 14F provide magnified views of the boxed chimeras and focus on antibodies with higher affinity. A and B: No tracking; C and D: Hot multivalent tracking; E and F: Cold monovalent heated multivalent tracking. Overall, longer t-values ​​were observed. 1 / 2 The antibody enrichment was greater than 3-fold, and the total K... D The enrichment was greater than 2-fold. In addition, high-affinity antibodies from cold monovalent + hot multivalent tracking strategies (E and F) also tend to be strong IL13 bioassay inhibitors (100-200 pM hIL13 for bioassay).

[0048] Figures 15A-D show a dot plot overlay of polystyrene microbeads coated with two monoclonal antibodies against Zaire Ebola GP protein with different affinities: anti-Ebola GP-1 and anti-Ebola GP-2. The antibody-coated beads were further treated with either no-tracking conditions (A and C) or fluorophore-labeled multivalent tracking conditions (B and D). Beads in green shading (t...) 1 / 2 =1155 minutes, coated with anti-Ebola GP-1) showed less PE staining and more A647 staining, which contrasted with the purple shading of the beads (t 1 / 2 =3 minutes, beads coated with anti-Ebola GP-2 were separated, the purple-shaded beads showing more PE staining and less A647 staining. Based on the dissociation rate, a fluorophore-labeled multivalent tracking method can be used to separate beads coated with two different antibodies from each other. Detailed Implementation

[0049] This article discloses a method for obtaining primary antibody-producing cells that express antibodies that exhibit high binding affinity to antigens.

[0050] Although the claimed subject matter will be described through certain examples, other examples, including those that do not provide all the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and methodological changes may be made without departing from the scope of this disclosure.

[0051] This document discloses a range of values. The range states a lower limit and an upper limit. Unless otherwise stated, the range includes the lower limit, the upper limit, and all values ​​between the lower limit and the upper limit, including but not limited to all values ​​up to the minimum value (lower limit or upper limit).

[0052] In the following description, the use of terminology will follow certain conventions. Generally, the terminology used herein is intended to be interpreted in accordance with the meanings known to those skilled in the art. In practicing this disclosure, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art, have been used. These techniques are described in detail in, for example, *Molecular Cloning: A Laboratory Manual*, 4th edition, edited by J.F. Sambrook and D.W. Russell, Cold Spring Harbor Laboratory Press, 2012; *Recombinant Antibodies for Immunotherapy*, edited by Melvyn Little, Cambridge University Press, 2009; "Oligonucleotide Synthesis" (edited by M.J. Gait, 1984); "Animal Cell Culture" (edited by R.R. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (edited by F.M. Mossubel et al., 1987 and regularly updated); and "PCR: The Polymerase Chain Reaction". "Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001). The contents of these references, and other references containing standard protocols (including manufacturer's instructions), which are widely known and relied upon by those skilled in the art, are hereby incorporated by reference as part of this disclosure.

[0053] General description

[0054] One aspect of this 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 this method involves an initial binding step that allows a first labeled form of the antigen to bind to antibody molecules on the surface of the antibody-producing cell, forming an antigen-antibody complex, followed by a “trace” step in which the cell comes into contact with one of several forms of the antigen: (i) an unlabeled form of the antigen (“cold trace”), (ii) a second labeled form of the antigen (“hot trace”), or both an unlabeled form and a second labeled form of the antigen (“combined trace”). Cells that remain bound to the first labeled form of the antigen after trace represent cells expressing antibody molecules with high binding affinity.

[0055] Therefore, the method disclosed herein allows for selection in cells expressing antibody molecules with different affinities to enrich cells expressing antibody molecules with high affinity.

[0056] 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).

[0057] The term "enrichment" refers to increasing the frequency or percentage of desired cells in a cell population. For example, within an antibody-producing cell population containing cells expressing antibody molecules with various affinities (e.g., high, intermediate, and low affinity), increasing the percentage of antibody-producing cells expressing high-affinity antibody molecules. Therefore, an antibody-producing cell population enriched in cells expressing high-affinity antibody molecules encompasses an antibody-producing cell population with a higher frequency and / or a higher percentage of cells expressing high-affinity antibody molecules due to the enrichment process. In this context, the enrichment process includes antigen tracking, thereby selecting cells expressing high-affinity antibody molecules for the antigen of interest from a cell population expressing antibody molecules with various affinities to the antigen of interest, and separating cells expressing high-affinity antibody molecules from cells expressing antibody molecules without high affinity.

[0058] The cell population obtained by the disclosed method is enriched with antibody-producing cells expressing antibody molecules with high binding affinity to the antigen of interest. In other words, the enriched cell population (collected after tracking) contains a greater percentage of cells expressing antibody molecules that bind to the antigen of interest with high binding affinity compared to a cell population before or without tracking. In some embodiments, at least 40% of the collected cells express high-affinity antibody molecules, for example, KD is an antibody molecule of 0.1 pM to 25 nM. In some embodiments, the enriched cell population may be a population in which at least 50% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the enriched cell population may be a population in which at least 60% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the enriched cell population may be a population in which at least 70% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the enriched cell population may be a population in which at least 80% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the enriched cell population may be a population in which at least 90% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the enriched cell population may be a population in which at least 95% of the cells express antibody molecules that bind to the antigen of interest with high binding affinity (e.g., KD is an antibody molecule of 0.1 pM to 25 nM). In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., KD is 0.1 pM to 25 nM antibody molecules) in a cell population after tracking increases by at least 30% compared to the frequency in a cell population before or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., KD is 0.1 pM to 25 nM antibody molecules) in a cell population after tracking increases by at least 40% compared to the frequency in a cell population before or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., KD is 0.1 pM to 25 nM antibody molecules) in a cell population after tracking increases by at least 50% compared to the frequency in a cell population before or without tracking. In some embodiments, the frequency of cells expressing high-affinity antibody molecules (e.g., KD is 0.1 pM to 25 nM antibody molecules) in a cell population after tracking increases by at least 75% compared to the frequency in a cell population before 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 a cell population before 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 200% compared to the frequency in a cell population before 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 cell population after tracking increases by at least 40% compared to the frequency in a cell population before 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 cell population after tracking increases by at least 50% compared to the frequency in a cell population before 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 cell population after tracking increases by at least 75% compared to the frequency in a cell population before 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 cell population after tracking increases by at least 100% compared to the frequency in a cell population before 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 cell population after tracking increases by at least 200% compared to the frequency in a cell population before or without tracking. 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 cell population after tracking increases by at least 40% compared to the frequency in a cell population before or without tracking. 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 cell population after tracking increases by at least 50% compared to the frequency in a cell population before or without tracking. 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 cell population after tracking increases by at least 75% compared to the frequency in a cell population before or without tracking.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 cell population after tracking increases by at least 100% compared to the frequency in a cell population before or without tracking. 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 cell population after tracking increases by at least 200% compared to the frequency in a cell population before or without tracking.

[0059] In some embodiments, the method for obtaining antibody-producing cells expressing antibody molecules that exhibit high binding affinity for antigens includes the following steps:

[0060] (a) Contacting an antibody-producing cell population expressing antibody molecules against the antigen on the cell surface with a first labeled form of the antigen to allow the antigen to bind to the antibody molecules on the cell surface, wherein the first labeled form of the antigen is conjugated to a first detectable label;

[0061] (b) Wash the cells to remove unbound antigens;

[0062] (c) Contact the cells with any of the following: (i) the unlabeled form of the antigen, (ii) the second labeled form of the antigen, or (iii) the unlabeled form of the antigen and the second labeled form of the antigen;

[0063] (d) Wash the cells to remove unbound antigens;

[0064] (e) Collect cells that remain bound to the first labeled form of the antigen, thereby obtaining a cell population enriched with cells expressing high-affinity antibody molecules.

[0065] Antibody molecules with high binding affinity

[0066] As is known in the art, the term "binding affinity" 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 fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise stated, 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., antibody and antigen). The affinity of a molecule for its binding partner can generally be determined by the dissociation equilibrium constant (KD or Kbinding affinity). D K is used to represent this. D There is an inverse relationship between the (molar) value and the binding affinity, therefore K DA lower M value indicates higher affinity. Therefore, "higher affinity" generally refers to antibody molecules that bind to the antigen more strongly and / or faster and / or remain bound for a longer period. Typically, due to their stronger binding interactions, a lower concentration (M) of antigen is required to achieve the desired effect.

[0067] The term "kd" (seconds⁻¹ or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction or the dissociation rate constant of interactions between antibodies, Ig, antibody-binding fragments, or molecules. This value is also known as k. off value.

[0068] The term "ka" (M-1x sec-1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction or the association rate constant of an interaction between an antibody, Ig, antibody-binding fragment, or molecule.

[0069] 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 the interaction between an antibody, Ig, antibody-binding fragment, or molecule. The equilibrium dissociation constant is obtained by dividing ka by kd.

[0070] Various methods for measuring binding affinity are known in the art, and any of these methods may be used for the purposes of this invention. Binding affinity obtained using these methods is typically in the range of about 0.1 pM to about 25 nM, as determined by surface plasmon resonance. In some embodiments, binding affinity is less than about 10 nM, as determined by surface plasmon resonance.

[0071] The term "high-affinity" antibody molecule refers to an antibody molecule with a binding affinity (expressed as KD) of 25 nM or lower (e.g., a value of about 0.1 pM to 25 nM). Therefore, the measured KD of a high-affinity antibody molecule can be approximately 25 x 10⁻⁶. -9 M (25nM) or lower, approximately 10 x 10 -9 M (10nM) or lower, approximately 1 x 10 -9 M (1nM) or lower, approximately 1 x 10⁻⁶ -10 M (0.1nM) or lower, approximately 0.5 x 10⁻⁶ -10 M (0.05nM) or lower, approximately 0.05 x 10⁻⁶ -10 M (5 pM) or lower, about 1 pM or lower, or about 0.5 pM or lower, as measured by surface plasmon resonance (e.g., BIACORE™ or solution affinity ELISA). Those skilled in the art will recognize that the KD value of an antibody molecule can be expressed numerically as nE. -z , or expressed as n x 10 -z For example, 3.2E-12 Equivalent to 3.2 x 10 -12 The KD is indicated to be 3.2 picomol (pM). In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 25 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 20 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 15 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 10 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 5 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 1 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 0.5 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is in the range of about 0.1 pM to about 0.1 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 20 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 15 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 10 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 5 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 1 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 0.1 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 0.5 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 0.01 nM. In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 0.001 nM (or 1 pM). In some embodiments, the measured KD of the high-affinity antibody molecule is less than about 0.5 pM.

[0072] Antibody-producing cells

[0073] The terms "antibody-producing cells" and "antibody-expressing cells" refer to cells that express antibody molecules on their cell surface (i.e., antibody molecules that bind to or anchor in 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 technologies and genetic engineering. Therefore, the terms encompass lymphocytes of antigen-dependent B cell lineages, including memory B cells, recombinant cells (such as non-lymphocytes engineered to express antibody molecules on their cell surface), including yeast and mammalian cells, such as immortalized cells and hybridoma cells. In some embodiments, immortalized cells include Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, and murine myeloma cells (e.g., NSO and Sp2 / O).

[0074] In some embodiments, antibody-producing cells are primary antibody-producing cells. "Primary cells" refers to cells that grow outside their natural environment, such as tissue cells isolated from mammals. In some embodiments, primary antibody-producing cells are tissue-derived, such as those derived from the spleen, lymph nodes, bone marrow, or peripheral blood. In some embodiments, antibody-producing cells may be derived from primary antibody-producing cells. For example, primary antibody-producing cells may be fused with myeloma cells to create hybridomas, or otherwise immortalized, such as through viral infection (e.g., EBV), or differentiated using cell sorting techniques based on protein markers expressed by specific B cell types.

[0075] In some embodiments, antibody-producing cells are mammalian cells or yeast cells engineered to express antibody molecules on their cell surface. In the context of 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., Saccharomyces cerevisiae or Pichia pastoris) 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, said immortalized cells including, for example, CHO cells, HEK293 cells, and murine myeloma cells (e.g., NSO and Sp2 / O).

[0076] Some of the methods described in this article are also applicable to cell surface display platforms for various host cells, including yeast or mammalian cells (such as CHO cells), which express antibody molecules on their cell surface to allow screening from antibody gene libraries or libraries of mature antibody variants.

[0077] Yeast surface display (YSD) platforms have been described and widely used for antibody screening (Border and Wittrup, *Nature Biotechnology* 1997; 15:553–7; Feldhaus MJ et al., *Nature Biotechnology* 2003; 21:163–70; McMahon C et al., *Nature Structural & Molecular Biology* 2018; 25:289–96). It has been reported that displaying Fab regions on yeast surfaces increases antibody diversity and expands library size (Weaver-Feldhaus JM et al., *FEBS Letters* 2004; 564:24–34; Rosowski S et al., *Microb Cell Fact* 2018; 17:3; Sivelle C et al., *Monoclonal Antibodies* 2018; 10:720–9).

[0078] 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, in: Zhou et al., Monoclonal Antibodies, 2010; 2(5):508–518; Nguyen et al., Protein Engineering, Design & Selection, 2018, Vol. 31, No. 3, pp. 91–101. Furthermore, suitable for this paper are mammalian cells carrying a single antibody gene that can be transfected with a gene encoding an activation-inducible deaminase (AID), which induces somatic hypermutation (SHM) by converting deoxycytidine (dC) to deoxyuracil (dU) to be a mutation of the antibody gene in the cell during cell proliferation in cell culture. See, for example, Chen C. et al., Biotechnol Bioeng., 113, 39–51 (2016).

[0079] Collection of immune and primary antibody-producing cells

[0080] Immunization of mammals, including humans and non-human animals, can be performed 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, California, CA). The antigen of interest is administered as a protein, protein fragment, protein fusion, or DNA plasmid containing the gene for the antigen of interest, and the antigen of interest is expressed using host cell expression mechanisms to express the antigenic peptide in vivo. It should be understood that the immunized mammal can be a human who has already been exposed to the antigen and has humoral immunity to the expression of the antigen of interest. The antigen can be administered directly to the mammal without adjuvant, or with adjuvant to help stimulate 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 (muramicyl dipeptide) (see O'Hagan, *Vaccine Adjuvants*, Human Press, 2000, New Jersey). Regardless of any specific theory, adjuvants can prevent rapid diffusion of peptides by isolating antigens in a local reservoir and can contain factors that can stimulate a host immune response.

[0081] Once an appropriate immune response is generated, antibody-producing cells are collected from the immunized animal.

[0082] Antibody-producing cells can be collected from various sources in immunized animals, including but not limited to spleen, lymph nodes, bone marrow, and peripheral blood. In some embodiments, spleen cells are collected from the immunized animal after immunization. In some embodiments, peripheral blood mononuclear cells (PBMCs) are collected from the immunized animal.

[0083] In some embodiments of the method, the antibody-producing cell population 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, suitable B cell markers that can be detected using 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.

[0084] In some embodiments, spleen cells are collected from the immunized animal after immunization. After removing red blood cells by lysis, IgG+ antigen-positive B cells can be isolated and used as the antibody-producing cell population in the method.

[0085] In some embodiments, peripheral blood mononuclear cells (PBMCs) are collected from immunized animals known to have humoral immunity to the antigen of interest. IgG+ antigen-positive B cells can then be isolated and used as primary antibody-generating cells in this method.

[0086] The collected primary antibody-producing cells (such as antibody-producing B cells) can be processed to enrich cells expressing antibodies against the antigen of interest on their cell surface. In some embodiments, an optional initial purification step is performed to enrich the primary antibody-producing cells. Such purification includes affinity chromatography, also known as affinity purification. Several types of affinity purification are known in the art, such as ammonium sulfate precipitation, affinity purification with immobilized proteins A, G, A / G, or L; and affinity purification with immobilized antigens.

[0087] Antigens of interest

[0088] The methods disclosed herein can be used 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 that can bind to an antibody, 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 can 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.

[0089] In some embodiments, the antigen is a protein in monomeric form. Examples of proteins present in monomeric form include interleukin molecules, such as IL-13. In some embodiments, the antigen is a protein in multimeric form, including homomers and heteromers. In some embodiments, the antigen is a protein in both monomeric and multimeric forms, in which case a mixture of protein monomers and multimers may be used in the methods described herein.

[0090] Regardless of whether the antigen is a protein present as a monomer, multimer, or mixture thereof, it can be used in the methods described herein in either monovalent or polyvalent form. The terms “monovalent” and “polyvalent” are used to refer to the number of units of antigen presented and are distinguished from the antigen itself, which is a protein present as a monomer, multimer, or mixture thereof. Thus, a monovalent form of antigen refers to a single-unit form of the antigen, wherein the antigen itself can be a protein present as a monomer, multimer, or mixture thereof. A polyvalent form of antigen refers to multiple antigen units typically presented by a polyvalent molecule bound to or linked to the antigen. The polyvalent molecule can be a dimer, trimer, tetramer, pentamer, hexamer, etc., or combinations thereof. 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 comprises a tetramer and may additionally comprise a trimer and / or a dimer. In some embodiments, the streptavidin multimer is conjugated to a fluorophore (such as phycoerythrin). In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen can be linked to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer, such as a trimer of a folded protein, to which an antigen can be linked to provide a trivalent form of the antigen.

[0091] Initial binding step and antigen labeling

[0092] To select cells expressing antibody molecules that exhibit the highest binding affinity for the antigen of interest, an initial binding or contact step is performed. In this step, the antibody-producing cell is first contacted with the antigen in a labeled form to allow the antigen to bind to antibody molecules on the cell surface.

[0093] In some embodiments, the concentration of the first labeled form of the antigen is between 0.001 nM and 1 μM. In some embodiments, the concentration of the first labeled form of the antigen is between 0.01 nM and 100 nM. In some embodiments, the concentration of the first labeled form of the antigen is 0.05 nM to 10 nM. In some embodiments, the concentration of the first labeled form of the antigen is 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 concentration of the first labeled form of the antigen is 0.1 nM to 7.5 nM. In some embodiments, the concentration of the first labeled form of the antigen is 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 concentration of the first labeled form of the antigen is 0.2 nM to 7.5 nM. In some embodiments, the concentration of the first labeled form of the antigen is 0.2 nM to 7 nM, 0.2 nM to 6 nM, 0.2 nM to 5 nM, 0.2 nM to 4 nM, 0.2 nM to 3 nM, 0.2 nM to 2 nM, 0.2 nM to 1 nM, 0.3 nM to 7 nM, 0.3 nM to 6 nM, 0.3 nM to 5 nM, 0.3 nM to 4 nM, 0.3 nM to 3 nM, 0.3 nM to 2 nM, 0.3 nM to 1 nM, 0.5 nM to 7 nM, 0.5 nM to 6 nM, 0.5 nM to 5 nM, 0.5 nM to 4 nM, 0.5 nM to 3 nM, 0.5 nM to 2 nM, or 0.5 nM to 1 nM. In some embodiments, the concentration of the first labeled form of the antigen is 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 may be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 0.2 nM. In a specific embodiment, antibody-producing cells may be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 5.0 nM. In a specific embodiment, antibody-producing cells may be contacted with the first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 7.5 nM. In a specific embodiment, antibody-producing cells may be contacted with a first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 10 nM.In a specific embodiment, antibody-producing cells may be contacted with a first labeled form of the antigen, wherein the concentration of the first labeled form of the antigen is 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.

[0094] In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 5 minutes to about 60 minutes. In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 30 minutes. In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 20 minutes. In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 40 minutes. In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 10 minutes. In some embodiments, contact between antibody-producing cells and the first labeled form of the antigen occurs from about 50 minutes.

[0095] In some embodiments, the first labeled form of the antigen is the monovalent form of the antigen. In some embodiments, the first labeled form of the antigen is the multivalent form of the antigen. In some embodiments, the first labeled form of the antigen is a mixture of the monovalent and multivalent forms of the antigen. Whether in monovalent, multivalent, or a mixture thereof, the antigen itself can be a protein that is a monomer, a polymer, or a mixture of monomers and polymers.

[0096] In some embodiments, the first labeled form of the antigen is an antigen conjugated with a first detectable label. The antigen can be labeled with small molecules, radioisotopes, enzymatic 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 routinely deployed detection assays known in the art.

[0097] In some embodiments, the detectable marker is an enzyme reporter gene. Enzyme markers are larger than biotin; however, they rarely disrupt antibody function. Commonly used enzyme markers are horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase. To use an enzyme-labeled antibody, the sample is incubated with an enzyme-specific substrate, which is catalyzed by the enzyme 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 react with diaminobenzidine to produce a brown product or with luminol to produce light. Conversely, AP can react with p-nitrophenyl phosphate (pNPP) to produce a yellow product detectable by spectrophotometry, or with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) to produce a purple precipitate.

[0098] In some embodiments, the detectable label is a fluorescent label. The fluorescent label is directly conjugated to the antibody, and detection does not require an enzyme / substrate or binding interaction. Therefore, the amount of detected fluorescence signal is proportional to the amount of the target protein in the sample. The fluorescent label can be covalently linked to the antibody via a primary amine or a thiol.

[0099] Removal of unbound antigens after initial binding

[0100] After incubating antibody-producing cells together with the first labeled form of the antigen (initial binding step), any unbound antigen can be removed from the antibody-producing cells.

[0101] In some embodiments, unbound antigens are removed by washing. As is known in the art, washing is a technique that uses a wash buffer to remove unwanted components. For the purposes of this disclosure, unwanted components include unbound antigens. In a non-limiting example, unbound antigens can be washed from bound antigens by adding a wash buffer to a mixture of bound and unbound antigens, centrifuging the mixture, and removing the supernatant containing the wash buffer and unbound antigens.

[0102] Wash buffers are known in the art. Wash buffers and washing steps are used to remove unbound and excess components. Wash buffers can be designed for specific techniques, such as ELISA, immunoblotting, or immunohistochemistry. Some wash buffers are compatible with a variety of different immunoassays. In some embodiments, the wash buffer is a phosphate-buffered saline (PBS)-based wash buffer. In some embodiments, the wash buffer is a Tris-buffered saline (TBS) wash buffer. In some embodiments, the wash buffer contains a detergent. In some embodiments, the detergent is Tween-20.

[0103] The cells are washed with a wash buffer for a specified time to remove unbound antigens. This specified time will be sufficient to remove unbound antigens. In some embodiments, this specified time may be from about 10 minutes to about 60 minutes to remove unbound antigens; multiple washes totaling 10 to 60 minutes may be used, such as three 10-minute washes or one 30-minute wash; 2 to 4 washes, each 5 to 15 minutes, etc. In one embodiment, the cells may be washed for a specified period of time, which includes a single (1) wash totaling 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 some embodiments, the cells may be washed for a period of time comprising two (2) washes, each for approximately 5 minutes, or approximately 10 minutes, or approximately 15 minutes, or approximately 20 minutes, or approximately 25 minutes, or approximately 30 minutes. Other wash intervals have been considered, which are substantially equivalent to the wash intervals described herein.

[0104] After washing and aspirating the supernatant containing the wash buffer and unbound antigen, the precipitate containing antigen-bound cells can be used in subsequent steps. In some embodiments, the precipitate containing bound antigen can be resuspended in a buffer and used in subsequent steps. In some embodiments, the buffer used to resuspend the precipitate can be the same wash buffer. In some embodiments, the buffer used to resuspend the precipitate can be a different buffer than the wash buffer.

[0105] Tracking steps

[0106] After antibody-producing cells have been contacted with the first labeled form of the antigen (initial binding / contact step), and once the unbound first labeled form of the antigen has been removed, the antibody-producing cells are contacted with the antigen again, or "traced," to selectively enrich cells expressing antibody molecules with high affinity for the antigen. This tracing can be performed using any of several antigenic forms: (i) the unlabeled form of the antigen ("cold" tracing), (ii) the second labeled form of the antigen ("hot" tracing); or (iii) both the unlabeled and second labeled forms of the antigen (a combination of cold-tracing and hot-tracing antigens, also known as "combined tracing"). Tracing allows the antigen to bind to antibody molecules initially bound by the first labeled form of the antigen, thereby tracing the first labeled form of the antigen off the antibody molecule unless the antibody molecule has high affinity for the antigen and remains bound to the first labeled form of the antigen after tracing.

[0107] In some embodiments, tracking is performed using the unlabeled form of the antigen (cold tracking). In some embodiments, the unlabeled form of the antigen is the monovalent form of the antigen—that is, while the antigen itself may be a protein in monomeric, multimeric, or a mixture of monomeric and multimeric forms, 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 multivalent form of the antigen, wherein the antigen itself may be a protein in monomeric, multimeric, or a mixture of monomeric and multimeric forms. In some embodiments, the unlabeled form of the antigen is a mixture of monovalent and multivalent forms of the antigen. In embodiments using the multivalent form of the antigen, such a form may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin polymer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, in addition to a tetramer, the streptavidin polymer may include a trimer and / or a dimer. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen may be linked to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimer, such as a trimer of a folded protein, to which an antigen may be linked to provide a trivalent form of the antigen.

[0108] In some embodiments, tracking is performed using a second labeled form of the antigen (thermal tracking). In such embodiments, antibody-producing cells bound to a first labeled form of the antigen are tracked using a second labeled form of the antigen after the initial binding and washing steps. The second labeled form of the antigen has a label that provides a detectable signal different from the label on the first labeled form of the antigen. Suitable selection of labels has been described herein, provided that the label on the second labeled form of the antigen differs from the label on the first labeled form of the antigen. Such labels include small molecules, radioisotopes, enzymatic proteins, and fluorescent dyes. In some embodiments, the first label is AlexaFluor647, and the second label is phycoerythrin.

[0109] In some embodiments, the second labeled form of the antigen is a monovalent form of the antigen. In some embodiments, the second labeled form of the antigen is a multivalent form of the antigen. In some embodiments, the second labeled form of the antigen comprises a mixture of monovalent and multivalent forms of the antigen. In embodiments using a multivalent form of the antigen, such a form may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin polymer (e.g., a tetramer), which may be complexed with biotin and then linked to the antigen, thereby providing a multivalent (tetravalent) form of the antigen. In some embodiments, in addition to a tetramer, the streptavidin polymer may include a trimer and / or a dimer. In some embodiments, the streptavidin polymer is conjugated to a fluorophore (such as phycoerythrin). In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which the antigen may be linked to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimerized molecule, such as a trimer of a folded protein, to which the antigen may be linked to provide a trivalent form of the antigen.

[0110] The first and second labeled forms of an antigen can be the same or different valence forms of the antigen, but they must have labels that emit detectable signals that are distinct from each other. In some embodiments, the first labeled form of the antigen is a monovalent form, and the second labeled form is also a monovalent form. In some embodiments, the first labeled form of the antigen is a monovalent form, while the second labeled form is a multivalent form.

[0111] In some embodiments, tracking is performed using 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, antibody-producing cells are tracked using both the unlabeled form of the antigen and the second labeled form of the antigen after an initial binding and washing step. The two forms of the antigen being tracked—the unlabeled form of the antigen (“cold-tracking antigen”) and the second labeled form of the antigen (“hot-tracking antigen”)—may be contacted with cells simultaneously or sequentially (e.g., the cold-tracking antigen is added first, followed by the hot-tracking antigen, or vice versa). In some embodiments, the unlabeled form of the antigen is a monovalent form. In some embodiments, the unlabeled form of the antigen is a multivalent form. In some embodiments, the unlabeled form of the antigen is a mixture of monovalent and multivalent forms. In some embodiments, the second labeled form of the antigen is a monovalent form. In some embodiments, the second labeled form of the antigen is a multivalent form. In some embodiments, the second labeled form of the antigen comprises a mixture of monovalent and multivalent forms. In embodiments where a multivalent form of the antigen is used in combined tracking, such a form may be provided by a multivalent molecule that binds to or is linked to the antigen. In some embodiments, the multivalent molecule is a streptavidin multimer (e.g., a tetramer), which may be complexed with biotin and then linked to an antigen, thereby providing a multivalent (e.g., tetravalent) form of the antigen. In some embodiments, in addition to a tetramer, the streptavidin multimer may include a trimer and / or a dimer. In some embodiments, the multivalent molecule is a dimer of an immunoglobulin Fc fragment, to which an antigen may be linked to provide a divalent form of the antigen. In some embodiments, the multivalent molecule is a trimerized molecule, such as a trimer of a folded protein, to which an antigen may be linked to provide a trivalent form of the antigen.

[0112] For any form of tracer, the concentration of the tracer antigen used is excessive compared to the concentration of the first labeled form of the antigen, regardless of the form of the antigen used for tracer (i.e., unlabeled form, second labeled form, or both). In embodiments using the unlabeled form of the antigen (in cold tracer or combined tracer), the molar ratio of the unlabeled antigen is at least twice the molar ratio of the first labeled form of the antigen, i.e., twice to at most, for example, 2500 times. In some embodiments, the molar ratio of the unlabeled antigen is twice the molar ratio of the first labeled form of the antigen. In some embodiments, the molar ratio of the unlabeled antigen is three times the molar ratio of the first labeled form of the antigen. In some embodiments, the molar ratio of the unlabeled antigen is four times the molar ratio of the first labeled form of the antigen. In some embodiments, the molar ratio of the unlabeled antigen is five times the molar ratio of the first labeled form of the antigen. In some embodiments, the molar ratio of the unlabeled antigen is six times the molar ratio of the first labeled form of the antigen. In some embodiments, the molar ratio of the unlabeled antigen is 7 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 8 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 9 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 10 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 15 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 20 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 25 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 30 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the unlabeled antigen is 35 times the molar ratio of the first labeled antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 40 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 45 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 50 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 60 times that of the first labeled form of the antigen.In some embodiments, the molar ratio of the antigen in its unlabeled form is 70 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 80 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 90 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 100 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 110 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 120 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 130 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 140 times that of the first labeled form of the antigen. In some embodiments, the molar ratio of the antigen in its unlabeled form is 150 times that of the first labeled form of the antigen. In some embodiments, the concentration of the unlabeled form of the antigen may be 0.4 nM to 1 μM or 10 nM to 600 nM, depending on the concentration of the first labeled form of the antigen. In some embodiments, the concentration of the unlabeled form of the antigen may be 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, depending on the concentration of the first labeled form of the antigen. In embodiments using a second labeled form of the antigen (in thermal or combined tracking), the molar ratio of the antigen in the second labeled form is at least twice that of the antigen in the first labeled form, i.e., twice to at most, for example, 2500 times. In some embodiments, the molar ratio of the antigen in the second labeled form is twice that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is three times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is four times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is five times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is six times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is seven times that of the antigen in the first labeled form.In some embodiments, the molar ratio of the antigen in the second labeled form is 8 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 9 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 10 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 15 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 20 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 25 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 30 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 35 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 40 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 45 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 50 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 60 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 70 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 80 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 90 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 100 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 110 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 120 times that of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 130 times that of the molar ratio of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 140 times that of the molar ratio of the antigen in the first labeled form. In some embodiments, the molar ratio of the antigen in the second labeled form is 150 times that of the molar ratio of the antigen in the first labeled form.In some embodiments, the concentration of the second labeled form of the antigen may be 0.4 nM to 1 mM or 10 nM to 600 nM, depending on the concentration of the first labeled form of the antigen. In some embodiments, the concentration of the second labeled form of the antigen may be 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, depending on the concentration of the first labeled form of the antigen.

[0113] In some embodiments of combined tracking, the cold-tracking antigen and the hot-tracking antigen may be at the same concentration or at different concentrations.

[0114] In some embodiments of combined tracing, cells are sequentially contacted with cold tracing antigens and hot tracing antigens; and in some such embodiments, a washing step may be included between incubation with both tracing antigens. In such embodiments, the cells are washed with a washing buffer for a period sufficient to remove unbound antigens. In some embodiments, the cells may be washed for a period of time comprising a single (1) 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, the cells may be washed for a period of time comprising two (2) washes, each for about 5 minutes, or each for about 10 minutes, or each for about 15 minutes, or each for about 20 minutes, or each for about 25 minutes, or each for about 30 minutes. Other washing intervals have been considered, which are substantially equivalent to the washing intervals described herein.

[0115] The tracking is performed for a period of time sufficient to allow for tracking of antigen-antibody binding. In some embodiments, tracking is performed for approximately 5 minutes to approximately 60 minutes using the unlabeled form of the antigen. In some embodiments, tracking is performed for approximately 50 minutes using the unlabeled form of the antigen. In some embodiments, tracking is performed for approximately 45 minutes using the unlabeled form of the antigen. In some embodiments, tracking is performed for approximately 40 minutes using the unlabeled form of the antigen. In some embodiments, tracking is performed for approximately 30 minutes. In some embodiments, tracking is performed for approximately 20 minutes using the unlabeled form of the antigen. In some embodiments, tracking is performed for approximately 10 minutes using the unlabeled form of the antigen.

[0116] In some embodiments, tracking is performed for approximately 5 to approximately 60 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 50 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 45 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 40 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 30 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 20 minutes using a second biolabeled form of the antigen. In some embodiments, tracking is performed for approximately 10 minutes using a second biolabeled form of the antigen.

[0117] In some embodiments of combined tracing, the antigen is traced in its unlabeled form for approximately 5 to approximately 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 approximately 5 to approximately 60 minutes (e.g., 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes). The contact time lengths for the unlabeled and second labeled forms of the antigen can be the same or different. As a non-limiting example, the unlabeled form of the antigen may be contacted with a primary antibody that binds to the first labeled form of the antigen for approximately 30 minutes, while the second labeled form of the antigen may subsequently be contacted with the cells for approximately 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 approximately 5 minutes to approximately 60 minutes, for example, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes.

[0118] In some embodiments, tracking is performed more than once. In some embodiments, cold tracking is performed more than once. In some embodiments, hot tracking is performed more than once. In some embodiments, cold tracking is performed once, followed by hot tracking more than once. In some embodiments, cold tracking is performed more than once, followed by hot tracking once. In some embodiments, cold tracking is performed more than once, followed by hot tracking more than once. In some embodiments, combined tracking is performed more than once. In some embodiments, a washing step is included after each tracking step. In some embodiments, more than one washing step is included after each tracking step, such as two or three washes.

[0119] Removal of unbound antigens after tracking

[0120] After the tracing step (e.g., in which antibody-producing cells bound to the first labeled form of the antigen are traced using 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.

[0121] In some embodiments, unbound antigens are removed by washing.

[0122] In some embodiments, before collecting cells that remain bound to the first labeled form of the antigen, the tracing and washing are repeated more than once, thereby obtaining a cell population enriched with cells expressing high-affinity antibodies.

[0123] Fluorescence-activated cell sorting (FACS)

[0124] Flow cytometry is a popular analytical cell biology technique that uses light to count and characterize cells in a heterogeneous fluid mixture. Flow cytometry is a particularly powerful method because it allows researchers to rapidly, accurately, and easily collect data related to many parameters from a heterogeneous fluid mixture containing live cells. Fluorescence-activated cell sorting (FACS) is a derivative of flow cytometry that adds a degree of specialization. Using FACS, researchers can physically sort heterogeneous mixtures of cells into different populations.

[0125] Two-dimensional (2D) FACS sorts cells 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 utilize thermal tracing. In some embodiments where the first detectable label is a first fluorescent label and the second detectable label is a second fluorescent label distinct from the first fluorescent label, two-dimensional FACS is used to collect cells that retain binding to the first labeled form of the antigen. In a specific embodiment, the first detectable label is A647 and the second detectable label is phycoerythrin.

[0126] Obtain a cell population enriched with cells expressing high-affinity antibody molecules.

[0127] Collect and track antibody-producing cells that retain their first labeled binding to the antigen. This collection of antibody-producing cells allows for the acquisition of a cell population enriched with cells expressing high-affinity antibody molecules.

[0128] The resulting cell population enriched with cells expressing high-affinity antibody molecules can be sorted or separated into individual cells. In some embodiments, fluorescence-activated cell sorting (FACS) is used to sort and select individual antibody-producing cells. Protocols for separating individual cells by flow cytometry are well known (Huang, J. et al., 2013, ibid.). Individual antibody-producing cells can be sorted and collected by alternative methods known in the art, including but not limited to manual single-cell picking, limiting dilution, B-cell panning with adsorbed antigens, microfluidics, laser capture microdissection, and gel bead emulsion (GEM), all of which are well known in the art. See, for example, Rolink et al., *Journal of Experimental Medicine* (1996) 183:187-194; Lightwood, D. et al., *Journal of Immunological Methods* (2006) 316(1-2):133-43; Gross et al., *International Journal of Molecular Sciences* (2015) 16:16897-16919; and Zheng et al., *Nature Communications* (2017) 8:14049. Gel bead emulsions (GEMs) are commercially available (e.g., the 10X Chromium system from 10x Genomics, Pleasanton, CA).

[0129] Once collected, individual antibody-producing cells can be propagated using standard cell culture techniques for subsequent DNA preparation. Alternatively, the antibody gene can be amplified directly from individual antibody-producing cells and subsequently cloned into a DNA vector.

[0130] Antibodies are produced from nucleic acids obtained from antibody-producing cells that express high-affinity antibody molecules.

[0131] Nucleic acids encoding antibodies or fragments thereof can be isolated from antibody-producing cells obtained using the methods described herein.

[0132] In some embodiments, genes or nucleic acids encoding the variable heavy and light chains of immunoglobulins (i.e., VH and VL, where VL can be a Vκ or Vλ chain) can be recovered from nucleic acids isolated from antibody-producing cells using an RT-PCR protocol. These RT-PCR protocols are well-known and routine techniques, as described, for example, by the following: Wang et al., *Journal of Immunological Methods* (2000) 244:217-225 and described herein.

[0133] In some embodiments, the nucleic acid encodes a fragment of the antibody, such as a variable domain, a constant domain, or a combination thereof. In some embodiments, the nucleic acid isolated from antibody-producing cells encodes a variable domain of the antibody. In some embodiments, the nucleic acid encodes an antibody heavy chain or a fragment thereof (e.g., a variable domain of the antibody heavy chain). In other embodiments, the nucleic acid encodes an antibody light chain or a fragment thereof (e.g., a variable domain of the antibody light chain).

[0134] Once recovered, the antibody-encoding gene or nucleic acid can be cloned into IgG heavy and light chain expression vectors and expressed by transfection of host cells. For example, the antibody-encoding gene or nucleic acid can be inserted into a reproducible vector for further cloning (DNA amplification) or expression in cells (stable or transient). Many vectors, especially expression vectors, are available or can be engineered to contain the appropriate regulatory elements required to regulate the expression of the antibody-encoding gene or nucleic acid.

[0135] In the context of this disclosure, the expression vector can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (a nucleic acid sequence containing a suitable set of expression control elements) as described herein. Examples of such vectors include derivatives of SV40, bacterial plasmids, bacteriophage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage 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, linear expression elements (as described in, for example, Sykes and Johnston, Nature Biotechnology (1997) 12:355-59), compact nucleic acid vectors (as described in, for example, US 6,077,835), or plasmid vectors such as pBR322 or pUC 19 / 18. Such nucleic acid vectors and their uses are well known in the art. See, for example, US 5,589,466 and US 5,973,972. In some embodiments, the expression vector can be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, those containing constitutive or inducible promoters such as yeast α-factor, alcohol oxidase, and PGH. See F. Ausubel et al., eds., *Current Protocols in Molecular Biology*, Greene Publishing and Wiley InterScience New York (1987); and Grant et al., *Methods in Enzymol*, 153, 516-544 (1987).

[0136] In some embodiments, the vector comprises a nucleic acid molecule (or gene) encoding the heavy chain of an antibody and a nucleic acid molecule (or gene) encoding the light chain of an antibody, wherein the antibody is produced by antibody-producing cells obtained by the methods of this disclosure.

[0137] Suitable host cells for expressing antibody molecules include, but are not limited to, cells derived from prokaryotic or eukaryotic (typically mammalian) sources. In some embodiments, the host cell is a bacterial or yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cell may be, for example, Chinese hamster ovary cells (CHO), such as CHO K1, DXB-11CHO, Veggie-CHO cells; COS (e.g., COS-7); stem cells; retinal cells; Vero cells; CV1 cells; kidney cells, such as HEK293, 293EBNA, MSR 293, MDCK, aHaK, BHK21 cells; HeLa cells; HepG2 cells; WI38; MRC 5; Colo25; HB 8065; HL-60; Jurkat 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 above-mentioned cells. In one specific embodiment, the host cell is a CHO cell. In another specific embodiment, the host cell is a CHO K1 cell.

[0138] It should be understood that the full-length antibody nucleic acid sequence or gene can subsequently be cloned into one or more suitable vectors. Alternatively, the Fab region of the isolated antibody can be cloned into one or more vectors that correspond to a constant region of any isotype. Therefore, any constant region can be used to construct isolated antibodies, including IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, and IgE heavy chain constant regions, or chimeric heavy chain constant regions. Depending on the intended use of the antibody, such constant regions can be obtained from any human or animal species. Furthermore, the antibody variable region or Fab region can be cloned into a suitable vector for protein expression in other formats, such as ScFv, bifunctional antibodies, etc.

[0139] In some embodiments, host cells comprising one or more nucleic acids encoding antibodies are cultured under conditions expressing full-length antibodies, and antibodies can then be generated and isolated for further use. In some embodiments, the host cells comprise nucleic acids encoding variable domains of antibodies, and the cells are cultured under conditions expressing variable domains. In other embodiments, the host cells comprise nucleic acids encoding variable heavy chain (VH) domains of antibodies, and the cells are cultured under conditions expressing VH domains. In yet another embodiment, the host cells comprise nucleic acids encoding variable light chain (VL) domains of antibodies, and the cells are cultured under conditions expressing VL domains. In a specific embodiment, the host cells comprise nucleic acids encoding both VH and VL domains of antibodies, and the cells are cultured under conditions expressing both VH and VL domains.

[0140] Example

[0141] Example 1. Preparation of beads

[0142] As a proof-of-concept, polystyrene microbeads were used to replace B cells. The polystyrene microbeads used were the same size as the B cells. The polystyrene microbeads, conjugated with a polyclonal anti-hFc capture antibody (Spherotech, Lake Forest, IL, Illinois), were coated with four monoclonal antibodies against human IL13 protein with different affinities: anti-IL13-1, anti-IL13-2, anti-IL13-3, and anti-IL13-4. Table 1 shows the dissociation t-cell ratios of these four antibodies binding to hIL13. 1 / 2 The time to treatment is 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.

[0143] Table 1

[0144] Anti-hIL13 antibody Ka(1 / Ms) Kd(1 / s) KD(M) t1 / 2 (minutes) Anti-IL13-1 1.42E+06 8.89E-03 6.26E-09 1.3 Anti-IL13-2 1.25E+06 1.19E-03 9.53E-10 10 Anti-IL13-3 6.27E+05 1.13E-04 1.81E-10 102 Anti-IL13-4 2.03E+06 1.00E-05* 4.93E-12 1155

[0145] 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 next day, the beads were washed with PBS to remove unbound antibodies. The conjugated beads were then incubated with A647-conjugated hIL13 at 5 nM or 0.2 nM for 30 minutes. A staining profile at 5 nM was preferred; however, concentrations in the range of 0.2 nM to 5 nM were also tested successfully. Unbound A647-conjugated hIL13 was removed by two washes using staining buffer, and the mAb-coated beads were ready for tracking experiments: no tracking, monovalent cold tracking, fluorophore-labeled multivalent tracking, and combined tracking with a monomeric cold antigen and a fluorophore-labeled multivalent antigen.

[0146] Example 2: Proof of Concept: Separation of Polystyrene Beads in FACS Experiments Based on Antigen Binding and Dissociation Rates

[0147] To demonstrate the ability to isolate B cells in FACS-based experiments based on antigen binding 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 with monovalent cold antigens and hot multivalent antigens. Figure 2 A schematic representation of the overall view showing the three exemplary tracking methods used is shown.

[0148] Untracked

[0149] In untracked samples, no additional steps are required, but cells are resuspended in PBS after centrifugation and analyzed by flow cytometry. Results can be obtained from... Figure 3 The image shows a stack of representative univariate histograms of A647 staining on untracked IL13 antibody-coated beads, as detected by flow cytometry.

[0150] Unit price cold tracking

[0151] For the monovalent cold tracking assay, beads coated with hIL13 mAb were incubated with 20 nM or 500 nM unlabeled hIL13 for 45 minutes, followed by two washes with PBS. A second incubation of 45 minutes with unlabeled hIL13 was then performed. After the second incubation, the beads were centrifuged, resuspended in PBS, and analyzed by flow cytometry.

[0152] The results are shown in Figure 4 and Figure 5 In the figure, the graph is a stack of representative univariate histograms of A647 staining of IL13-coated beads as detected by flow cytometry. Figure 4 The results of a monovalent cold-tracking experiment are shown, in which four different groups of beads were coated with IL13 antibodies with different dissociation constants and exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice with 4 times (20 nM) unlabeled human IL13 for 45 minutes each time, with two washes in between, and then A647 was detected by flow cytometry. Figure 5 The results of a monovalent tracking experiment are shown, in which beads coated with four different IL13 antibodies were exposed to 5 nM A647-conjugated human IL13, washed, and further incubated twice with 100-fold (500 nM) unlabeled human IL13 for 45 minutes each time, with two washes in between. Beads coated with these antibodies exhibited a longer dissociation time. 1 / 2 Both types of IL13 mAb beads (anti-IL13-3 and anti-IL13-4) showed the same staining levels with and without tracking; while beads coated with shorter t... 1 / 2 Beads containing the other two mAbs (anti-IL13-1 and anti-IL13-2) showed weaker staining and longer t-coating with the coated material under cold-tracking conditions compared to samples without cold-tracking. 1 / 2 mAb beads are better separated.

[0153] Multivalent tracking of fluorophores

[0154] For fluorophore-labeled multivalent tracking assays, beads coated with hIL13 mAb were incubated with 20 nM biotin-hIL13 pre-bound to 5 nM phycoerythrin (PE)-streptavidin (SA) for 45 minutes, followed by two PBS washes. A second incubation was then performed with the 20 nM biotin-hIL13 pre-bound to 5 nM PE-SA for 45 minutes. After the second incubation, the beads were centrifuged, resuspended in PBS, and analyzed by flow cytometry.

[0155] The results of the multivalent tracking experiment with fluorophore labels are shown in the A647 histogram overlay ( Figure 6 ) and A647 overlay relative to PE2 color dot map ( Figure 8 and Figure 10 Of the two. In the case of hIL13 with A647 labeling applied at 5 nM (e.g. Figure 6 As shown), when only A647 staining levels were observed, all mAb-coated beads subjected to multivalent fluorophore tracking showed the same difference in A647 staining levels as their corresponding mAb-coated beads subjected to monomeric cold tracking as described above; however, when PE staining levels were observed, beads coated with the longest t... 1 / 2 (t 1 / 2 =1155 minutes, beads coated with mAb containing anti-IL13-4 showed minimal PE staining, compared to those coated with anti-IL13-3 (t 1 / 2 The beads coated with anti-IL13-1 and anti-IL13-2 (t = 102 minutes) showed a greater PE staining distance, which was significantly different from the beads coated with anti-IL13-1 and anti-IL13-2, which had shorter t values. 1 / 2 (1.3 minutes and 10 minutes), such as Figure 8 and Figure 10 As shown. Adding a second color to this trace helps to further enhance the effect of coating two longer t... 1 / 2 The beads are separated from each other.

[0156] Combination of monovalent cold tracking and fluorophore-labeled multivalent tracking

[0157] For the combined experiment of monovalent cold tracking and fluorophore-labeled multivalent tracking, beads coated with hIL13 mAb were incubated with the above two tracking agents, namely, with 500 nM unlabeled hIL13 and 20 nM biotin-hIL13 pre-bound with 5 nM PE-streptavidin for 45 minutes, followed by two PBS washes, and then a second incubation with 500 nM unlabeled hIL13 and 20 nM biotin-hIL13 pre-bound with 5 nM PE-streptavidin for another 45 minutes.

[0158] For the combination of monovalent cold tracking and fluorophore-labeled multivalent tracking experiments, the results are again shown in the A647 histogram overlay ( Figure 7 ) and shown in the A647 v PE 2 color dot overlay ( Figure 8 and Figure 10 Of the two, in the case of hIL13 with A647 labeling applied at 0.2 nM, the coating with the longest t 1 / 2 (t 1 / 2 The beads coated with mAb (=1155 oz) were clearly prominent and separated from the remaining mAb-coated beads in the A647 v PE dot plot overlay. The beads coated with anti-IL13-3 (t) 1 / 2 =102 minutes), anti-IL13-2 (t 1 / 2 =10 minutes) and anti-IL13-1 (t 1 / 2 =1.3 minutes) of beads not only with the longest t 1 / 2 The mAb (anti-IL13-4) beads are separated and are closer to each other in the dot plot. Adding a second color to this trace helps to further separate the two longer t-coatings. 1 / 2 The beads are separated from each other.

[0159] Therefore, B cells expressing surface-anchored antibodies with fast or slow dissociation constants can be isolated by labeling B cells using various tracking conditions of this disclosure and incorporating them into sorting strategies to enrich high-affinity antibodies.

[0160] Example 3: Isolation of B cells in FACS experiments based on antigen binding and dissociation rates

[0161] During the standard B-cell sorting workflow, spleen cells from immunized mice are stained with fluorescently labeled antigens, washed, and then sorted by flow cytometry. Sorting gating is primarily based on selection of IgG and antigen-double-positive populations, thus allowing the isolation of antigen-specific B cells expressing antibodies with a range of affinities. This method has previously been modified to identify higher-affinity antibodies by utilizing monomeric sorting reagents and by specifically sorting cells with the highest antigen-specific + / IgG+ ratio (“diagonal sorting”).

[0162] In this example, the B cell sorting process is further improved by adding a tracking step designed to remove B cells expressing lower-affinity antibodies from those expressing high-affinity antigen-specific antibodies. In this improved workflow, the tracking step is performed in the presence or absence of additional unlabeled (“cold”) antigens polymerized and differentially labeled antigens. Human IL13 labeled with AlexaFluor647 (A647) is incubated with mouse spleen cells for 30 minutes, followed by two washes to remove unbound sorting reagents and to ensure only B cells bound to labeled human IL13 are present. The labeled B cells are further subjected to the tracking step using one of two methods: multivalent tracking or a combination of (cold monovalent tracking and hot multivalent) tracking. Multivalent tracking consists of pre-polymerized biotin-hIL13 in a 4:1 ratio with streptavidin-PE, resulting in a mixture of tetramers, trimers, dimers, and some monomers. The combined tracking consists of “cold” (unlabeled) monovalent hIL3 in combination with multivalent tracking antigens. These tracking methods greatly improve the efficiency of the isolation process for ultra-high affinity mAbs by reducing the total number of antibodies that need to be cloned, expressed, and screened.

[0163] A high-affinity antibody against the soluble cytokine target IL13 was isolated. Genetically modified mice with an unrearranged human immunoglobulin variable region gene segment at the endogenous mouse immunoglobulin locus were generated using the method described in U.S. Patent No. 8,502,018. The genetically modified mice were immunized with human IL13 in the form of an mFc dimer protein and fused with an antibody targeting antigen-presenting cells. Following the tracking strategy disclosed herein, a total of 58 t cells were identified during B cell isolation. 1 / 2 Antibodies with a duration exceeding 100 minutes. Of all antibodies isolated using this method, over 50% exhibit nanomolar or better affinity. Among them, 47 antibodies possess ultra-high affinity, with affinity less than 1 x 10⁻⁶. -10 (M). This contrasts sharply with the 20% of antibodies exhibiting sub-nanomolar affinity, where only 7 ultra-high affinity antibodies were generated using conventional sorting strategies without any "tracking" steps. Of the 47 ultra-high affinity antibodies, 39 were also potent inhibitors of IL13 in bioassays. Therefore, the disclosed B-cell sorting strategy represents a significant improvement over current methods for applications requiring high-affinity antibodies to achieve therapeutic efficacy.

[0164] As described, after sorting B cells from IL13-immunized mice, variable domains of both the heavy and light chains were cloned into a fully human IgG1 expression construct and transiently expressed in CHO cells. Target-specific binding of the antibody containing the supernatant was determined by Luminex or SPARCL assays. All IL13 binders across all sorting strategies were moved to Biacore for bioassay screening, and the results are shown in Table 2. The numbers shown in Table 2 refer to the number of mice used for a specific sorting strategy, where each mouse could be used across multiple sorting strategies. Therefore, the total number of mice sorted was 13.

[0165] Table 2

[0166] Sorting strategy Sorted mice B cells collected Cloned antibodies IL13 adhesive (%) Untracked 12 1496 637 519(81％) Multimer tracking 6 675 199 157(79％) Cold + Polymer Tracking 13 1907 775 700(90％) total 13 4078 1611 1376(85％)

[0167] Antibodies from representative mice were analyzed using Biacore, and the results are shown in... Figure 13A -C. Under no-tracking conditions ( Figure 13A Most of the isolated antibodies have short-term T-cell activity. 1 / 2 The low affinity. However, antibodies isolated using either of the two tracking strategies ( Figure 13B and Figure 13C The text appears to be a fragmented collection of phrases and sentences, seemingly from different sources, and lacks coherence. A direct translation 1 / 2 Significant enrichment of sub-nanomolar affinity antibodies. In addition to their improved affinity properties, these antibodies were also potent inhibitors in IL13 inhibition assays. This is in Figure 13B As can be seen in -C, it is specified by the color of the data points, where yellow represents 100% suppression.

[0168] Antibodies from all experimental mice analyzed by Biacore are shown in Table 3. Trends observed in all mice are similar to those observed in... Figure 13A The trend observed was consistent in individual mouse examples of -C. Enlarged views in Figures 14B, 14D, and 14F focus on antibodies with higher affinity. Together with Figures 14A-F, Table 3 shows the observed longer t... 1 / 2 The antibody enrichment was greater than three-fold, and the total K... D The enrichment was greater than twofold. In addition, as seen in Figures 14E and 14F, high-affinity antibodies from the cold monovalent + multivalent tracking strategy also tended to be strong IL13 bioassay inhibitors (100-200 pM hIL13 for bioassay).

[0169] Overall, the cold + multimer tracking strategy enrichment has a longer t 1 / 2 The antibody concentrations were more than three times higher (2.3% to 7.4%). Even though IL13 was used in these examples, the tracking strategy presented in this paper can be implemented for other targets that require ultra-high affinity antibodies.

[0170] Table 3

[0171]

[0172] Example 4. Proof of Concept: Separation of antibody-coated beads in FACS experiments based on the dissociation rate between the antibody and the multimeric antigen.

[0173] Objective: To demonstrate the ability of antibody-expressing cells to be separated based on the dissociation rate between the antibody and the multimeric antigen Ebola GP trimer protein using a fluorophore-labeled multivalent tracking method in FACS-based experiments.

[0174] In this proof-of-concept experiment, polystyrene microbeads were used instead of antibody-expressing cells. As described in Example 1, the beads were coated with two monoclonal antibodies with different affinities against the Zaire Ebola GP protein: anti-Ebola GP-1 and anti-Ebola GP-2. The dissociation t of the binding between the two antibodies was... 1 / 2 The time was 1155 minutes for anti-Ebola GP-1 and 3 minutes for anti-Ebola GP-2, as shown in Table 4.

[0175] Table 4

[0176] Anti-Ebola antibody KD(M) t1 / 2 (minutes) Anti-Ebola GP-1 2.28E-10 1155 Anti-Ebola GP-1 5.23E-08 3

[0177] The antibody-coated beads were then incubated together with A647-conjugated Ebola GP trimer protein at 5 nM or 0.2 nM for 30 minutes. Unbound A647-conjugated 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 then analyzed by flow cytometry. The results are shown in Figure 15 in the A647 vs. PE 2 dot plot overlay.

[0178] For untraceable samples, no additional steps are required. The samples are centrifuged to precipitate and resuspended in PBS, then analyzed by flow cytometry. As shown in Figure 15A, for beads incubated with 5 nM of Ebola GP trimer protein conjugated with A647, the A647 MFI of beads coated with anti-Ebola GP-1 (green shading) is 6,335, and the A647 MFI of beads coated with anti-Ebola GP-2 (purple shading) is 3,781. Although their staining is different, the overlay shows that the two populations overlap significantly and are therefore difficult to separate in A647 staining (Y-axis). Figure 15C shows similar results for beads incubated with 0.2 nM of Ebola GP trimer protein. The A647 MFI of beads coated with anti-Ebola GP-1 (green shading) is 1,466, and the A647 MFI of beads coated with anti-Ebola GP-2 (purple shading) is 908. The two bead groups are heavily overlapped and inseparable during superposition.

[0179] In beads using a fluorophore-labeled multivalent tracking method, after removing unbound A647-conjugated Ebola GP trimer protein, antibody-coated beads were incubated for 45 minutes with 20 nM biotin-labeled Ebola GP trimer protein pre-bound to 5 nM phycoerythrin (PE)-streptavidin (SA). This was followed by two PBS washes, and then a second incubation of 20 nM biotin-labeled Ebola GP trimer protein pre-bound to 5 nM PE-SA for 45 minutes. After the second incubation, the beads were centrifuged, resuspended in PBS, and analyzed by flow cytometry.

[0180] The results of the fluorophore-labeled multivalent tracking experiments are shown in the A647 vs. PE 2-color dot plot overlays in Figures 15B and 15D. In Figure 15B, with A647-labeled Ebola GP trimer protein applied at 5 nM, when only A647 staining levels are observed, both the beads coated with anti-Ebola GP-1 and anti-Ebola GP-2, subjected to fluorophore-labeled multivalent tracking, show similar differential A647 staining levels to their corresponding untracked antibody-coated beads, as shown in Figure 15A; however, when PE staining levels are also considered in the dot plots, the groups coated with both antibodies are far apart. Green-shaded beads (t...) 1 / 2 =1155 minutes, coated with anti-Ebola GP-1) showed less PE staining and more A647 staining, which contrasted with the purple shading of the beads (t 1 / 2 =3 minutes, coated with anti-Ebola GP-2) is separated, the purple-shaded beads have more PE staining and less A647 staining. Adding a second color to this trace helps to separate beads with different t 1 / 2The beads can be separated from each other. In the case of trimer antigen binding, beads coated with two different antibodies can be separated from each other based on the dissociation rate using a fluorophore-labeled multivalent tracking method.

Claims

1. A method for obtaining antibody-producing cells, said antibody-producing cells expressing antibody molecules exhibiting high binding affinity to antigens, said method comprising: (a) Contacting an antibody-producing cell population comprising cells expressing antibody molecules against the antigen on the cell surface with a first labeled form of the antigen to allow the antigen to bind to the 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) Expose the cells to the following: (i) the unlabeled form of the antigen, (ii) the second labeled form of the antigen, or (iii) the unlabeled form of the antigen and the second labeled form of the antigen; (d) Wash the cells to remove unbound antigens; as well as (e) Collect cells that remain bound to the first labeled form of the antigen, thereby obtaining cells expressing high-affinity antibody molecules against the antigen.

2. The method of claim 1, wherein the concentration of the first labeled form of the antigen is between 0.001 nM and 1 μM.

3. The method of claim 1, wherein the concentration of the first labeled form of the antigen is between 0.1 nM and 7.5 nM.

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

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 polymeric form.

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

8. The method according to any one of claims 5 to 7, wherein the first labeled 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 a multivalent form of the antigen.

11. The method of claim 10, wherein the multivalent form of the antigen is provided by a multivalent molecule that binds to or is linked to the antigen.

12. The method of claim 11, wherein the multivalent molecule is selected from streptavidin multimers, such as tetramers, dimers of immunoglobulin Fc fragments, or trimerized molecules, such as trimers of folded molecules.

13. The method according to any one of claims 5 to 12, wherein the second labeled 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 labeled form of the antigen is a multivalent form of the antigen.

15. The method of claim 14, wherein the multivalent form of the antigen is provided by a multivalent molecule that binds to the antigen.

16. The method of claim 15, wherein the multivalent molecule is a streptavidin multimer (e.g., a tetramer), a dimer or trimer of an immunoglobulin Fc fragment, such as a trimer of a fold.

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

18. The method of claim 17, wherein the second detectable marker is a second fluorescent marker.

19. The method of claim 8, wherein in step (c), the cell is contacted with the unlabeled form of the antigen.

20. The method of 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 molar ratio of the antigen in the unlabeled form is at least 2 to 4 times the molar ratio of the antigen in the first labeled form used in step (a).

22. The method of claim 8, wherein in step (c), the cell is contacted with the second labeled form of the antigen.

23. The method of claim 22, wherein the second labeled form of the antigen is a multivalent form of the antigen.

24. The method according to claim 22 or 23, wherein the molar ratio of the antigen in the second labeled form is at least 2 to 4 times the molar ratio of the antigen in the first labeled form used in step (a).

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

26. The method of claim 8, wherein in step (c), the cell is contacted with the unlabeled form of the antigen and the second labeled form of the antigen.

27. The method of claim 26, wherein the unlabeled form is the monovalent form of the antigen, and the second labeled form of the antigen is the multivalent form of the antigen.

28. The method of 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 multivalent form of the antigen.

29. The method of claim 26, wherein the antigen is a multimeric protein, the unlabeled form is a monovalent form of the antigen, and the second labeled form of the antigen is a multivalent form of the antigen.

30. The method of claim 26, wherein the antigen is a protein existing in both monomeric and multimeric forms, the unlabeled form is the monovalent form of the antigen, and the second labeled form of the antigen is the multivalent form of the antigen.

31. The method according to any one of claims 26 to 30, wherein the cell is simultaneously in contact with both the unlabeled form of the antigen and the second labeled form of the antigen.

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

33. The method according to any one of the preceding claims, wherein the first detectable label is a fluorescent label, and wherein fluorescently activated cell sorting is used to collect cells that retain binding to the first labeled form of the antigen.

34. The method according to any one of claims 19 to 32, wherein the first detectable marker is a first fluorescent marker, and the second detectable marker is a second fluorescent marker distinguished from the first fluorescent marker, wherein two-dimensional fluorescence-activated cell sorting is used to collect cells that retain binding to the first labeled form of the antigen.

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

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

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

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

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

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

41. The method of claim 40, further comprising transfecting a host cell with a nucleic acid encoding an antibody heavy chain or a variable domain thereof and a nucleic acid encoding an antibody light chain or a variable domain thereof; and growing the transfected host cell under conditions supporting antibody expression in the host cell.

42. The method according to claim 41, wherein the host cell is a Chinese hamster ovary (CHO) cell.

43. A mammalian host cell prepared by the method according to claim 41 or 42.

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