Methods for antibody characterization

Abstract

The present disclosure relates to methods for characterizing small amounts of immunogen-binding proteins, such as immunogen-binding antibodies, obtained directly from the supernatant of individually cultured B cells. These methods involve providing a preparation of B cells that avoid hybridoma formation and secrete immunogen-binding antibodies, and individually culturing the B cells under conditions suitable for the B cells to secrete the immunogen-binding antibodies into the culture supernatant. The culture supernatant containing the secreted antibodies is collected from the individually cultured B cells and subjected to one or more binding assays to characterize the immunogen-binding antibodies from the B cell preparation (e.g., for their binding affinity by exposing the cell culture supernatant to increasing concentrations of immunogen).

Description

[Technical Field]

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 420,965, filed October 31, 2022, is hereby claimed, the disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to a high-throughput method for characterizing antibody-target protein interactions using antibody-containing supernatants from individually cultured B cells. [Background technology]

[0003] Therapeutic monoclonal antibody (mAb) discovery typically begins with animal immunization, B cell isolation, and hybridoma formation and culture, resulting in the generation of large panels of mAbs per target. Identifying therapeutic candidates from these large panels involves evaluation of binding affinity, cross-reactivity, epitope binding, blocking activity, and antibody chain composition. Given the importance of identifying candidate antibodies with desired functional attributes, hybridoma formation and culture have been considered an essential component of the discovery process to ensure sufficient quantities of antibodies are available for full evaluation. However, due to the length of time involved in hybridoma generation, it can take more than 15 weeks to identify an initial candidate molecule. Given the high cost of developing therapeutic mAbs, the ability to identify quality leads more quickly and inexpensively would provide a significant advantage over current prior art methods. The present disclosure is directed to overcoming these and other deficiencies in the art. Summary of the Invention [Means for solving the problem]

[0004] A first aspect of the present disclosure is directed to a method for characterizing immunogen-binding antibodies from a preparation of B cells. The method includes providing a preparation of non-immortalized B cells, wherein the B cells of the preparation secrete immunogen-binding antibodies, and individually culturing the B cells of the preparation under conditions effective for the B cells to secrete the immunogen-binding antibodies into the culture supernatant. Culture supernatants containing the secreted antibodies are recovered from the individually cultured B cells, and each recovered supernatant is subjected to two or more different binding assays to characterize the immunogen-binding antibodies from the preparation of B cells.

[0005] Another aspect of the present disclosure is directed to a method for characterizing the binding affinity of antibodies from a preparation of B cells. The method includes providing a preparation of non-immortalized B cells, wherein the B cells of the preparation secrete immunogen-binding antibodies. The method further involves individually culturing the B cells of the preparation under conditions effective for the B cells to secrete the immunogen-binding antibodies into the culture supernatant. Culture supernatants containing the secreted antibodies are collected from the individually cultured B cells, and each culture supernatant is exposed to increasing concentrations of an immunogen, a fragment of the immunogen, or a homolog of the immunogen. The method further involves detecting association and dissociation between the secreted antibodies of the culture supernatant and the immunogen, a fragment thereof, or a homolog thereof at each of the increasing concentrations. The binding affinity of the secreted antibodies is characterized based on the detection.

[0006] The classical method for identifying antigen-specific antibodies involves harvesting the spleen and / or lymph nodes from immunized animals (e.g., mice), recovering B cells from the harvested tissue, and then immortalizing the recovered B cells due to challenges associated with ex vivo culture and B cell survival. Immortalization is typically achieved by fusing B cells with immortalized myeloma cells to generate hybridomas. The immortalized cells are plated into wells and cultured to produce antibody-enriched supernatants, which are used in various assays, such as antigen binding, affinity, blocking activity (e.g., receptor-ligand blocking), light chain determination, and epitope binning, to select lead antibodies possessing desired characteristics (see Figure 1A). After lead selection, sequencing is initiated to resolve the antibody heavy and light chain sequences required for binding and sequence transfer for recombinant production.

[0007] The main drawback of hybridoma technology is time. Protocols excluding immunization take approximately 4–6 months to identify a lead antibody candidate (see Pedrioli, A. and Oxenium, A., “Single B Cell Technologies for Monoclonal Antibody Discovery,” Trends Immunol. 42(12):1143–1158 (2021)). Additionally, this procedure can be low-throughput in terms of fusion efficiency and hybridoma formation, and some hybridomas are low-yield antibody producers.

[0008] To reduce the time and other drawbacks associated with hybridoma technology, advances have been made in defining primary B cell culture conditions that avoid immortalization and enable single B cell screening methodologies. Many of these screening approaches involve placing individual B cells in small wells or chambers, microcapillaries, or water-in-oil droplets containing detectable antigens (either in suspension or immobilized) to rapidly identify the binding specificity of B cell antibodies (see Pedrioli, A. and Oxenium, A., "Single B Cell Technologies for Monoclonal Antibody Discovery," Trends Immunol. 42(12):1143-1158 (2021) review article, pp. 1151-1153 and Figure 2). Once the desired B cells are identified, they are harvested and antibody coding sequences are obtained for recombinant production (see Figure 1B). These approaches are high-throughput to facilitate rapid identification of cells producing antigen-specific antibodies, but therefore could not accommodate the functional assays (e.g., binding affinity) required to identify potential lead antibodies (see ibid., p. 1153).

[0009] The process described herein was developed and implemented to overcome the above-mentioned deficiencies in the antibody discovery process. In this process, non-immortalized B cells (e.g., primary B cells isolated from immunized animals) are individually plated into wells. The B cells are cultured to allow secretion of antibodies into the culture supernatant, then harvested, and the secreted antibodies are screened for a number of desired characteristics, such as binding specificity, binding affinity, epitope binding, blocking activity, light chain composition, and other protein-based interactions, in a single experimental run (see Figures 1C and 2). A key difference between traditional hybridoma technology and the process described herein is that the B cells in the disclosed process are not immortalized prior to antibody characterization, thereby significantly reducing the time and resources involved in this process. The disclosed process also differs from current single B-cell screening strategies by facilitating the evaluation of two or more functional characteristics, such as binding affinity, thereby enabling candidate lead selection significantly earlier in the discovery process (i.e., before antibody sequencing and cell-based recombinant production). Additionally, because the B cell cultures in the methods described herein are separated from the screening process, no special techniques are required to recover the desired antibody-secreting cells from the screening assay.

[0010] As such, the methods described herein provide a substantial improvement to the antibody discovery process by identifying methods for assaying several functional endpoints using nominal amounts of antibody (e.g., antibody produced by individually cultured B cells). Because desired functional characteristics are identified without the need for hybridoma formation or antibody sequencing, the methods described herein significantly reduce the time and resources required to identify candidate lead antibodies worthy of further therapeutic development. [Brief explanation of the drawings]

[0011] [Figure 1A]A schematic comparison of prior art antibody discovery and lead selection methods (FIGS. 1A and 1B) and the optimized antibody discovery and lead selection process disclosed herein (FIG. 1C) is shown. As shown in FIG. 1A, a standard hybridoma screening flow involves harvesting B cells from immunized mice and forming hybridoma cells. Media from the cultured hybridoma cells is screened for antibodies with desired immunogen binding, binding affinity, function, etc. This process, involving hybridoma formation, takes 15 weeks or more to complete. FIG. 1B shows an improvement of the process, involving antigen-binding screening performed on antibodies produced from individual B cells. Once a desired B-cell-produced antibody is identified, the antibody is sequenced and recombinantly produced to obtain sufficient quantities of the antibody to perform additional characterization, such as binding affinity and function. The process in FIG. 1B typically takes approximately 7 weeks to complete. In contrast, the process of the present disclosure, shown in FIG. 1C, involves obtaining a B-cell preparation, for example, by harvesting B cells from immunized mice and directly culturing the collected B cells without hybridoma or recombinant production. Although the concentration of secreted antibodies in the supernatant from cultured B cells is limited (due to the ultimate nature of primary B cells), the methods described herein allow for the evaluation of two or more binding properties (e.g., binding affinity) of the secreted antibodies in the supernatant. Candidate lead antibodies have been identified in as little as three weeks using this process. [Figure 1B]A schematic comparison of prior art antibody discovery and lead selection methods (FIGS. 1A and 1B) and the optimized antibody discovery and lead selection process disclosed herein (FIG. 1C) is shown. As shown in FIG. 1A, a standard hybridoma screening flow involves harvesting B cells from immunized mice and forming hybridoma cells. Media from the cultured hybridoma cells is screened for antibodies with desired immunogen binding, binding affinity, function, etc. This process, involving hybridoma formation, takes 15 weeks or more to complete. FIG. 1B shows an improvement of the process, involving antigen-binding screening performed on antibodies produced from individual B cells. Once a desired B-cell-produced antibody is identified, the antibody is sequenced and recombinantly produced to obtain sufficient quantities of the antibody to perform additional characterization, such as binding affinity and function. The process in FIG. 1B typically takes approximately 7 weeks to complete. In contrast, the process of the present disclosure, shown in FIG. 1C, involves obtaining a B-cell preparation, for example, by harvesting B cells from immunized mice and directly culturing the collected B cells without hybridoma or recombinant production. Although the concentration of secreted antibodies in the supernatant from cultured B cells is limited (due to the ultimate nature of primary B cells), the methods described herein allow for the evaluation of two or more binding properties (e.g., binding affinity) of the secreted antibodies in the supernatant. Candidate lead antibodies have been identified in as little as three weeks using this process. [Figure 1C]A schematic comparison of prior art antibody discovery and lead selection methods (FIGS. 1A and 1B) and the optimized antibody discovery and lead selection process disclosed herein (FIG. 1C) is shown. As shown in FIG. 1A, a standard hybridoma screening flow involves harvesting B cells from immunized mice and forming hybridoma cells. Media from the cultured hybridoma cells is screened for antibodies with desired immunogen binding, binding affinity, function, etc. This process, involving hybridoma formation, takes 15 weeks or more to complete. FIG. 1B shows an improvement of the process, involving antigen-binding screening performed on antibodies produced from individual B cells. Once a desired B-cell-produced antibody is identified, the antibody is sequenced and recombinantly produced to obtain sufficient quantities of the antibody to perform additional characterization, such as binding affinity and function. The process in FIG. 1B typically takes approximately 7 weeks to complete. In contrast, the process of the present disclosure, shown in FIG. 1C, involves obtaining a B-cell preparation, for example, by harvesting B cells from immunized mice and directly culturing the collected B cells without hybridoma or recombinant production. Although the concentration of secreted antibodies in the supernatant from cultured B cells is limited (due to the ultimate nature of primary B cells), the methods described herein allow for the evaluation of two or more binding properties (e.g., binding affinity) of the secreted antibodies in the supernatant. Candidate lead antibodies have been identified in as little as three weeks using this process. [Figure 2] This provides a schematic of how surface plasmon resonance (SPR) data from sequential characterization assays can be used to identify a lead immunogen-binding antibody derived directly from primary B cells in a single experiment (approximately 11 hours). In this example, the lead antibody exhibits no cross-reactive binding to off-target proteins, high affinity binding to the target protein, blocks receptor binding to the target protein, and possesses a kappa light chain. [Figure 3]1 is a graph showing the load signal response units (RU) of titrations of antibody samples (i.e., 500, 100, 50, and 25 ng / ml of antibody) loaded onto an HC30-M chip (Carterra® LSA) conjugated with anti-human Fc antibody (mAb1.35.1). The 500, 100, and 50 ng / mL samples were printed on the chip for a standard 10 minute period, and the 25 ng / mL sample was printed for 30 minutes to allow for capture of the small amounts of immunoglobulin present. [Figure 4] Figure 1 shows a series of SPR imaging (SPRi) sensorgrams showing binding affinity measurements obtained on a Carterra® LSA instrument using titrated antibody samples (500, 100, 50, and 25 ng / ml of antibody) printed on an HC30-M chip. The 25 ng / mL sample was printed over 30 minutes as described above. For affinity measurements, antibody target protein was injected sequentially at six concentrations (100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, and 0.41 nM). Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were generated using the Carterra® Kinetic software tool. Each sensorgram shows response units (RU; y-axis) over time in seconds (x-axis). [Figure 5] 1 is a collection of sensorgram readouts showing the results of an experiment performed according to the methods described herein and in Example 2, in which a panel of mAbs secreted from primary B cells were captured by a secondary antibody on an HC30-M chip. The panel of immobilized mAbs was sequentially evaluated for (i) binding to off-target proteins (column 3), (ii) binding affinity to the target protein (columns 7-12), (iii) blocking of receptor binding to the target (column 13), and finally light chain composition by binding to either an anti-kappa mAb (column 15) or an anti-lambda mAb (column 16). [Figure 6]The methods of the present application are shown to detect and differentiate cross-reactive binding activity of immunogen-binding antibodies recovered from B cell supernatants. SPRi sensorgrams obtained with a Carterra® LSA instrument show the binding response of captured antibody samples upon introduction of off-target proteins as described in Example 2, and the complete experimental data is shown in Figure 5. Binding to off-target proteins by a population of mAbs is readily identified through increased SPR signal (RU). [Figure 7] Representative binding affinity SPRi sensorgrams for several immunogen-binding antibodies recovered from primary B cell supernatants as described in Example 2 and the full experimental data shown in Figure 5 are shown. Five concentrations of target protein in a 1:3 dilution series starting from 100 nM were introduced to the HC30-M Carterra® chip containing the immunogen-bound antibody supernatant sample. Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were generated using the Carterra® Kinetic software tool. Each sensorgram shows response units (RU; y-axis) in seconds (x-axis) over time. The top panel of the sensorgrams shows very high affinity antibody candidates (<100 pM), while the middle and bottom panels show high- to medium-affinity antibody candidates as evidenced by discernible off-rates. [Figure 8] The methods of the present application are shown to detect and distinguish receptor-ligand (RL) blocking activity of representative immunogen-binding antibodies recovered from primary B cell supernatants as described in Example 2 and the full experimental data shown in Figure 5. The SPRi sensorgrams show the binding response (time = 0 to approximately 2600 seconds) after introduction of the target protein to an HC30-M chip containing the immunogen-binding antibody, followed by the binding response (time = approximately 2600 to 5000 seconds) when the target receptor protein is introduced. Antibodies that block receptor binding to the target protein show negligible binding (i.e., negligible increase in RU) when the receptor is introduced. Note that in this case, the receptor is significantly larger than the target protein, and therefore the binding signal is similarly large. [Figure 9]1 shows the determination of the light chain composition of immunogen-binding antibodies recovered from primary B cell supernatants using the methods of the present application. The center and right sensorgrams show the binding response for selected immunogen-antibody samples after introduction of an anti-kappa antibody (center sensorgram) or an anti-lambda antibody (right sensorgram). The left sensorgram is the response of a buffer-only control. [Figure 10] 1 is a collection of sensorgram readouts showing the results of a second experiment performed according to the methods described herein and in Example 3, in which a panel of mAbs (antibodies that bind IL-11) secreted from primary B cells were captured by a secondary antibody on an HC30-M chip. The panel of immobilized mAbs was sequentially evaluated for binding affinity to the target protein (IL-11) (columns 2-7) and ability to block binding of the receptor (IL-11R) to the IL-11 target (column 8). [Figure 11A] Representative binding affinity SPRi sensorgrams are shown for high-affinity (FIG. 11A), medium-affinity (FIG. 11B), and low-affinity (FIG. 11C) IL-11 binding antibodies recovered from primary B cell supernatants as described in Example 3 and the full experimental data shown in FIG. 10. Five concentrations of target protein in a 1:3 dilution series starting from 100 nM were introduced to the HC30-M Carterra® chip containing the immunogen-bound antibody supernatant sample. Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were generated using the Carterra® Kinetic software tool. [Figure 11B] Representative binding affinity SPRi sensorgrams are shown for high-affinity (FIG. 11A), medium-affinity (FIG. 11B), and low-affinity (FIG. 11C) IL-11 binding antibodies recovered from primary B cell supernatants as described in Example 3 and the full experimental data shown in FIG. 10. Five concentrations of target protein in a 1:3 dilution series starting from 100 nM were introduced to the HC30-M Carterra® chip containing the immunogen-bound antibody supernatant sample. Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were generated using the Carterra® Kinetic software tool. [Figure 11C]Representative binding affinity SPRi sensorgrams are shown for high-affinity (FIG. 11A), medium-affinity (FIG. 11B), and low-affinity (FIG. 11C) IL-11 binding antibodies recovered from primary B cell supernatants as described in Example 3 and the full experimental data shown in FIG. 10. Five concentrations of target protein in a 1:3 dilution series starting from 100 nM were introduced to the HC30-M Carterra® chip containing the immunogen-bound antibody supernatant sample. Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were generated using the Carterra® Kinetic software tool. [Figure 12] 1 is a table summarizing the affinity data and blocking activity of three candidate IL-11 antibodies determined using the methods disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0012] This disclosure describes the development of methods and assays that allow for early biochemical characterization and lead selection of binding proteins, such as antibodies, from samples containing nominal amounts of binding protein, such as about 1 ng to about 250 ng of total binding protein. The ability to generate larger quantities of binding protein without methods such as hybridoma formation and cell-based recombinant production of binding proteins before characterizing attributes such as binding affinity, blocking activity, cross-reactivity, epitope binding, and other properties of the binding protein saves significant amounts of time and resources and provides the functional information needed to identify suitable lead binding proteins for advancement.

[0013] Accordingly, the present disclosure is directed to a method for characterizing binding proteins, such as antibodies, from a sample containing a nominal amount of the binding proteins. In a first aspect, the method involves characterizing immunogen-binding antibodies secreted from a preparation of B cells, wherein the B cell supernatant contains the nominal amount of secreted immunogen-binding antibodies. The method includes providing a preparation of non-immortalized B cells, wherein the B cells of the preparation secrete the immunogen-binding antibodies. The method further involves individually culturing the B cells of the preparation under conditions effective for the B cells to secrete the immunogen-binding antibodies into the culture supernatant. Culture supernatants containing the secreted antibodies are recovered from the individually cultured B cells, and each recovered supernatant is subjected to two or more different binding assays to characterize the immunogen-binding antibodies from the preparation of B cells.

[0014] According to this and all aspects of the present disclosure, binding assays suitable for functionally characterizing immunogen-binding antibodies from preparations of non-immortalized B cells include, but are not limited to, binding assays that characterize antibody binding affinity, antibody binding avidity, antibody cross-reactive binding (e.g., cross-reactive binding to the same immunogen in a different species or to a structurally similar immunogen), antibody blocking activity (e.g., blocking of immunogen binding to its cognate binding partner), immunogen binding conditions (e.g., optimal pH conditions for antibody-immunogen binding), antibody chain composition (e.g., antibody light chain composition), antibody epitope binding, antibody-antibody cross-competition, and any combination of the foregoing assays. Exemplary assays and reagents for performing the assays are disclosed below.

[0015] In any embodiment, the two or more binding assays used to characterize immunogen-binding antibodies are performed on a solid support (e.g., a biosensor chip). The solid support includes multiple reaction surfaces, each containing a capture reagent immobilized thereon. According to this embodiment, each of the recovered culture supernatants is contacted with the solid support under conditions effective for secreted antibodies from the recovered culture supernatants to bind to the immobilized capture reagents on the reaction surfaces, forming an array of capture antibodies. Thus, each reaction surface on the solid support contains immobilized antibodies from a different culture supernatant. The array of capture antibodies is exposed to a first binding analyte, and the presence or absence of an interaction between the first binding analyte and the capture antibody is detected to determine a first binding characteristic of the antibody. The method further involves repeating the exposing and detecting steps with a second binding analyte to determine a second binding characteristic of the antibody. The exposing and detecting steps may be repeated using a third, fourth, fifth, etc. binding detector to determine additional binding characteristics of the antibody. Preferably, the array of capture antibodies is exposed to binding analytes (i.e., first, second, third, etc. binding analytes) in a sequence that requires minimal or no washing steps between different binding analytes and does not require replenishment of the antibodies immobilized on the reaction surface. This allows for the determination of multiple functional properties of antibodies from a single supernatant sample in a single experimental run. Exemplary reaction sequences that achieve these goals are described in more detail herein.

[0016] According to this and all embodiments of the present disclosure, solid supports suitable for immobilizing immunogen-binding antibodies from non-immortalized B cell preparations for characterization can be formed of any porous or non-porous material, such as silica, glass, metal, plastic, or polymer. For example, the solid support can include materials such as metal, glass, ceramic, silica, polymeric materials (e.g., poly(methyl methacrylate) (PMMA), polystyrene, polycarbonate, and cycloolefin copolymer (COC)), or any combination of these materials. The solid support can comprise a solid surface or the surface of an optical fiber. If the solid support is not metallic, it can be coated with a metal, such as gold, platinum, silver, or metal nanoparticles, making it compatible with label-free real-time detection systems, such as surface plasmon resonance and biolayer interferometry platforms.

[0017] The surface of the solid support, which may be coated with gold, silver, or another sensor-compatible metal, may also be functionalized to promote or enhance attachment of immunogen-binding antibodies. Suitable materials for functionalizing the surface of the solid support include, but are not limited to, polymeric materials such as polycarboxylate hydrogels or carboxymethyldextran hydrogels. In any embodiment, the functionalized surface further comprises an immobilized capture reagent (e.g., streptavidin, protein A / G, one or more polynitrilotriacetic acid groups, capture antibody, or other binding moiety suitable for immobilizing immunogen-binding antibodies from the culture supernatant to the solid support surface).

[0018] Suitable solid supports for immobilizing antibodies from culture supernatants or other samples described herein are commercially available, see, for example, polycarboxylate and carboxymethyl dextran hydrogel sensor chips available from Carterra® Inc.

[0019] In any embodiment, the solid support surface comprises an immobilized capture reagent (e.g., streptavidin, protein A / G, one or more polynitrilotriacetic acid groups, a capture antibody suitable for immobilizing an immunogen-bound antibody to the solid support surface). In any embodiment, the immobilized capture reagent is an antibody. In any embodiment, the immobilized capture reagent is a polyclonal antibody reagent. In any embodiment, the immobilized capture reagent is a monoclonal antibody reagent. In any embodiment, the immobilized capture reagent is an antibody that binds to the constant portion of an antibody heavy or light chain (e.g., the Fc portion of an antibody heavy chain). In any embodiment, the immobilized capture reagent is an anti-Fc-specific antibody selected from an anti-IgG antibody, an anti-IgM antibody, an anti-IgD antibody, an anti-IgE antibody, or an anti-IgA antibody. In any embodiment, the antibody is an anti-human Fc-specific antibody, such as an anti-human IgG antibody, an anti-human IgM antibody, an anti-human IgD antibody, an anti-human IgE antibody, or an anti-human IgA antibody. Anti-Fc antibodies suitable for use in the methods of the present disclosure are readily known in the art and commercially available (see, for example, but not limited to, anti-Fc antibodies available from R&D Systems and SouthernBiotech). Selection of a suitable anti-Fc-specific antibody will vary depending on the heavy chain composition of the immunogen-binding antibody. Identification of a suitable capture antibody can be carried out using methods known in the art, and suitable antibodies include those with high binding affinity (e.g., less than 200 pM, less than 150 pM, less than 100 pM, or less than 50 pM).

[0020] In some embodiments, the immobilized antibody capture reagent is an anti-light chain-specific antibody, such as an anti-kappa chain antibody or an anti-lambda chain antibody. In some embodiments, the anti-light chain antibody is specific for a human antibody light chain, such as an anti-human kappa light chain or an anti-human lambda light chain. Anti-light chain antibodies suitable for use as immobilized capture reagents in the methods disclosed herein are readily known in the art and are commercially available (see, for example, but not limited to, anti-lambda and anti-kappa antibodies available from Abcam and R&D Systems). The selection of a suitable anti-light chain-specific antibody will vary depending on the light chain composition of the immunogen-binding antibody. Identification of a suitable anti-light chain capture antibody can be performed using methods known in the art, and suitable antibodies include those with high binding affinity (e.g., less than 200 pM, less than 150 pM, less than 100 pM, or less than 50 pM).

[0021] As described in more detail herein, culture supernatant containing immunogen-binding antibodies for analysis on a solid support is collected from individually cultured B cells. In some embodiments, the B cells are primary B cells. "Primary cells," as referred to herein, are terminal cells isolated directly from an in vitro or in vivo biological sample (e.g., tissue (e.g., spleen, lymph nodes), blood, plasma, serum, or bone). Importantly, the primary cells are terminal, non-immortalized cells. Thus, the amount of immunogen-binding antibodies produced by primary B cells and secreted into the culture supernatant is limited in concentration compared to the amount of antibodies available from more typically utilized hybridoma B cells or cell-based recombinant systems. Therefore, the use of a suitable antibody capture reagent (e.g., an anti-Fc-specific antibody) facilitates the capture of immunogen-binding antibodies on the solid support surface in sufficient quantities to detect the presence or absence of an interaction between the immobilized immunogen-binding antibodies and one or more binding analytes described herein.

[0022] According to this and all aspects of the disclosure, suitable B cells are antibody-secreting B cells, including any type of B cell that produces and secretes antibodies. Thus, a preparation of B cells can include plasmablasts (short-lived plasma cells), plasma cells (e.g., long-lived plasma cells), and germ cell (GC) B cells. In any embodiment, the preparation of B cells is a preparation of primary B cells that produce and secrete human antibodies. B cells that produce and secrete human antibodies include human B cells and non-human B cells that have been modified to produce human antibodies. In one embodiment, the B cells are derived from a transgenic animal (e.g., a transgenic mouse) that produces human B cells. Suitable transgenic mice whose B cells produce human antibodies include, but are not limited to, XenoMouse®, HuMab Mouse®, VelocImmune® mice (VelociMouse®), Harbor Mice®, OmniMouse®, Alloy mice, and Trianni mice. Other transgenic animals capable of producing human antibodies from B cells include, but are not limited to, transgenic chickens (e.g., OmniChicken®), transgenic rats (e.g., OmniRat®), transgenic llamas, transgenic rabbits, and transgenic cows (e.g., transchromosomal (TC) cows) (see, e.g., Brueggemann et al., "Human Antibody Production in Transgenic Animals," Arch. Immunol. Ther. Exp. 63:101-108 (2015)), which is incorporated herein by reference in its entirety). All forms of antibodies produced and secreted by B cells derived from transgenic animal models, including complete immunoglobulin molecules and smaller domains thereof (e.g., VhHs), are suitable for characterization according to the methods disclosed herein.

[0023] In another embodiment, the B cells produce and secrete non-human antibodies (e.g., primary B cells derived from a non-human animal that produces non-human antibodies). Suitable non-human B cell preparations include preparations of any mammalian B cells. Exemplary non-human mammalian B cells can be obtained from, for example, but not limited to, non-human primates, horses, pigs, cows, goats, sheep, llamas, camels, rabbits, dogs, cats, rats, guinea pigs, gerbils, and mice. In another embodiment, the B cells utilized in the methods described herein are derived from non-human animals such as birds (e.g., chickens and ducks), sharks, fish, or lampreys.

[0024] In some embodiments, the B cell preparation is isolated or obtained from a subject immunized with the immunogen of interest. The immunized subject can be any immunized animal, e.g., an immunized mammal. Suitable mammals for immunization include, but are not limited to, humans, non-human primates, horses, pigs, cows, goats, sheep, llamas, camels, rabbits, dogs, cats, rats, guinea pigs, gerbils, and mice. In other embodiments, the immunized subject is not a mammal. Suitable non-mammals for immunization include, but are not limited to, birds (e.g., chickens and ducks), sharks, fish, or lampreys.

[0025] In any embodiment, the non-human animal can be a natural animal or a transgenic animal, e.g., a transgenic non-human animal capable of producing human antibodies. Suitable techniques for immunizing non-human animals are known in the art. See, e.g., Coding, Monoclonal Antibodies: Principles and Practice, 3rd ed., Academic Press Limited, San Diego, CA, 1996, which is incorporated herein by reference in its entirety. For immunization of non-human animals, gene gun methods may also be used, as described, for example, in Barry et al., Biotechniques 16(4):616-8, 620 (1994); Tang et al., Nature 356(6365):152-4 (1992); Bergmann-Leitner and Leitner, Methods Mol Biol 1325:289-302 (2015); Aravindaram and Yang, Methods Mol Biol 542:167-178 (2009); Johnston and Tang, Methods Cell Biol 43 PtA:353-365 (1994); and Dileo et al., Human Gene Ther 14(1):79-87 (2003), which are incorporated herein by reference in their entireties. Alternatively, a non-human animal may be immunized by administering to the non-human animal cells expressing the antigen, or by administering antigen-loaded dendritic cells, tumor cell vaccines or immune cell-based vaccines.See, for example, Sabado et al., Cell Res 27(1):74-95 (2017), Bot et al., "Cancer Vaccines" in Plotkin's Vaccines. 7th ed., Editors: Plotkin et al., Elsevier Inc., 2018, and Lee and Dy, "The Current Status of Immunotherapy in Thoracic Malignancies" in Immune Checkpoint Inhibitors in Cancer. Editors: Ito and Emstoff, Elsevier Inc., 2019 (which are incorporated by reference in their entireties). In various cases, immunizing can be performed by microneedle delivery (see, e.g., Song et al., Clin Vaccine Immunol 17(9):1381-1389 (2010), which is incorporated herein by reference in its entirety); by virus-like particles (VLPs) (see, e.g., Temchura et al., Viruses 6(8):3334-3347 (2014), which is incorporated herein by reference in its entirety); or by any means known in the art. See, e.g., Shakya et al., Vaccine 33(33):4060-4064 (2015) and Cai et al., Vaccine 31(9):1353-1356 (2013), which are incorporated herein by reference in their entireties. Additional strategies for immunization and immunogen preparation, such as adding T cell epitopes to antigens, are also suitable for use and are described in Chen and Murawsky, Front Immunol 9:460 (2018), which is incorporated herein by reference in its entirety.

[0026] Thus, in any embodiment, the methods described herein can further include immunizing a subject with an immunogen of interest and recovering and isolating primary B cells from the immunized subject's spleen, lymph nodes, blood, and / or plasma using B cell isolation techniques known in the art (see, e.g., Moore et al. "Isolation of B-cells using Miltenyi MACS Bead Isolation Kits," PLoS One 14(3):e0213832 (2019), which is incorporated herein by reference in its entirety). The isolated B cells are then individually cultured, and the supernatant containing the secreted antibodies is subjected to the analyses described herein.

[0027] In another embodiment, the B cell preparation is isolated or obtained from a subject with an autoimmune condition, and the subject's B cells produce antibodies that bind to the autoimmunogen. Suitable subjects include mammalian and non-mammalian subjects. Exemplary mammalian subjects include, but are not limited to, humans, non-human primates, horses, pigs, cows, goats, sheep, llamas, camels, rabbits, dogs, cats, rats, guinea pigs, gerbils, and mice.

[0028] In another embodiment, the B cell preparation is isolated or obtained from a subject who has or previously had a viral or bacterial infection, and the subject's B cells produce antibodies that bind to the viral or bacterial immunogen. Suitable subjects include mammalian and non-mammalian subjects. Exemplary mammalian subjects include, but are not limited to, humans, non-human primates, horses, pigs, cows, goats, sheep, llamas, camels, rabbits, dogs, cats, rats, guinea pigs, gerbils, and mice.

[0029] In any embodiment, the preparation of B cells is isolated or obtained from an in vitro immune organoid model, and the B cells of this model are exposed to an immunogen for antibody production.The in vitro immune organoid model can comprise immune cells from any mammal and / or non-mammal, including but not limited to: human, non-human primate, horse, pig, cow, goat, sheep, llama, camel, rabbit, dog, cat, rat, guinea pig, gerbil, mouse, bird, shark, fish, lamprey or any combination thereof.

[0030] Once isolated or obtained, primary B cells that produce and secrete immunogen-binding antibodies are cultured individually, i.e., a single primary B cell is placed in a cell culture well to seed the culture. The primary B cells are cultured and expanded using standard cell culture methods appropriate for B cell culture; see, e.g., Weitkamp et al., "Generation of Recombinant Human Monoclonal Antibodies to Rotavirus from Single Antigen-specific B Cells Selected with Fluorescent Virus-like Particles," J. Immunol. Meth. 275:223-37 (2003) and Lagerkvist et al., "Single, Antigen-Specific B Cells Used to Generate Fab Fragments using C40-mediated Amplification of Direct PCR Cloning," BioTechniques 18:862-69 (1995), which are incorporated herein by reference in their entireties.

[0031] In any embodiment, the primary B cell culture is maintained at least until the antibody concentration in the culture supernatant reaches about 20 ng / mL to about 100 ng / mL or more. A sample of the supernatant is then contacted with the reaction surface of a solid support. In any embodiment, about 50 μL to about 250 μL of the supernatant sample is contacted with the solid support, and this supernatant sample contains an antibody concentration of about 20 ng / mL to about 100 ng / mL or is diluted to contain this antibody concentration. Therefore, the total amount of immunogen-bound antibody that contacts the reaction surface of the solid support is at least about 1 ng to about 25 ng, more preferably at least about 3 ng to about 20 ng. In any embodiment, the total amount of immunogen-bound antibody contacted with the reaction surface of the solid support for analysis is at least about 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 11 ng, 12 ng, 13 ng, 14 ng, 15 ng, 16 ng, 17 ng, 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, or 25 ng.

[0032] In any embodiment, about 150 μL to about 250 μL of supernatant sample is contacted with the solid support, and the supernatant sample contains or is diluted to contain an antibody concentration of about 20 ng / mL to about 100 ng / mL. In any embodiment, the supernatant sample contacted with the solid support contains an antibody concentration of about or at least 20 ng / mL, about or at least 25 ng / mL, about or at least 30 ng / mL, about or at least 35 ng / mL, about or at least 40 ng / mL, about or at least 45 ng / mL, about or at least 50 ng / mL, about or at least 55 ng / mL, about or at least 60 ng / mL, about or at least 65 ng / mL, about or at least 70 ng / mL, about or at least 75 ng / mL, about or at least 80 ng / mL, about or at least 85 ng / mL, about or at least 90 ng / mL, about or at least 95 ng / mL, or about 100 ng / mL.

[0033] In any embodiment, a sample of the collected culture supernatant is contacted with a solid support under conditions effective to capture a sufficient amount of secreted antibody onto the solid support surface. In any embodiment, this contacting can involve repeatedly flowing the supernatant sample over a reaction surface of a solid support, for example, using continuous-flow microspotting technology. Using this approach, the supernatant sample is periodically flowed over the reaction surface under conditions suitable for binding of the antibody in the supernatant to a capture reagent (e.g., an anti-Fc antibody) immobilized on the solid support surface. In any embodiment, the flow of the antibody-containing culture supernatant is performed in a bidirectional manner to maximize exposure of the antibody in the supernatant to the solid support surface. In any embodiment, the flow of the supernatant over the surface continues for a duration of at least 10 minutes. In any embodiment, the flow continues for a duration of about 10 to about 40 minutes, for example, at least or about 15 minutes, at least or about 20 minutes, at least or about 25 minutes, at least or about 30 minutes, at least or about 35 minutes, or at least or about 40 minutes. In some embodiments, the circulation continues for a duration of about 40 minutes. In some embodiments, the circulation continues for a duration of more than 30 minutes. In some embodiments, the circulation continues for a duration of 30-40 minutes.

[0034] Suitable spotting devices and methods for immobilizing secreted antibodies onto a solid support surface are known in the art, see, e.g., U.S. Pat. No. 10,300,450 to Gale et al., U.S. Pat. No. 8,210,119 to Gale et al., and U.S. Pat. No. 9,682,372 to Gale, which are incorporated herein by reference in their entireties.

[0035] According to the methods disclosed herein, the solid support is suitable for high-throughput analysis and therefore includes multiple reaction surfaces suitable for antibody immobilization. Thus, in any embodiment, the method allows for the analysis of 96 to 384 different supernatant samples. In any embodiment, the solid support includes 96 reaction surfaces, each suitable for capturing antibodies from a different B cell culture supernatant. In any embodiment, the solid support includes more than 96 reaction surfaces, each suitable for capturing antibodies from a different B cell culture supernatant. In any embodiment, the solid support includes 192 reaction surfaces, each suitable for capturing antibodies from a different B cell culture supernatant. In any embodiment, the solid support includes 288 reaction surfaces, each suitable for capturing antibodies from a different B cell culture supernatant. In any embodiment, the solid support includes 384 reaction surfaces, each suitable for capturing antibodies from a different B cell culture supernatant.

[0036] Immobilization of antibodies from B cell culture supernatants onto a solid support forms an array of capture antibodies suitable for analyzing and detecting the presence or absence of an interaction between a first binding analyte and the capture antibody to determine a first binding property of the capture antibody. The first binding analyte can be any analyte suitable for use in a binding assay to determine one or more characteristics of the antibodies in the culture supernatant. Suitable binding analytes include biomolecules, i.e., any molecule produced by a living organism, including, but not limited to, proteins, peptides, nucleic acid molecules (e.g., deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, hybrid DNA-RNA molecules), lipids, and carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides). According to the methods described herein, suitable binding analytes include (i) biomolecules produced in a living organism (e.g., cells) and isolated for use, and (ii) recombinantly or synthetically produced biomolecules.

[0037] In any embodiment, the first binding analyte is a biomolecule suitable for characterizing the binding cross-reactivity of the capture antibody. The term "cross-reactivity," as used herein, refers to the binding of an antibody to an immunogen other than the immunogen against which the antibody was raised. Cross-reactivity encompasses antibodies that bind to homologous immunogens from different species. For example, an antibody raised against a human immunogen may exhibit cross-reactive binding to a corresponding immunogen from a different species (e.g., mouse or monkey). Cross-reactivity also encompasses antibodies that bind to proteins (or other biomolecules) that are structurally similar (e.g., primary, secondary, tertiary, or quaternary structure) to the immunogen against which the antibody was raised. For example, an antibody raised against a human immunogen may exhibit cross-reactive binding to a structurally similar protein within the same protein superfamily as the immunogen of interest.

[0038] Exemplary biomolecules suitable for characterizing the cross-reactive binding activity of an antibody include, but are not limited to, proteins or peptides that are homologous (i.e., similar in sequence or structure) to the target immunogen to which the antibody binds. In any embodiment, the protein or peptide is from the same immunogen but a different species; for example, a human antibody that specifically binds to a human immunogen may be evaluated for cross-reactive binding to a cynomolgus monkey homolog of that human immunogen. In another embodiment, the protein or peptide belongs to the same or related protein family as the immunogen and / or shares an amino acid sequence or structure with the target immunogen.

[0039] In another embodiment, the binding analyte is a biomolecule suitable for characterizing the binding affinity or binding avidity of the capture antibody. The term "binding affinity," as used herein, refers to the strength of the interaction between two molecules (e.g., an antibody and its antigenic epitope). This interaction may include hydrogen bonding, ionic bonding, van der Waals interactions, and electrostatic interactions. Binding affinity is typically measured by the equilibrium dissociation constant (K D The dissociation constant is measured and reported as the rate at which the antibody-immunogen complex dissociates (k off) is the rate at which antibody-immunogen complexes form (k on ) and expressed as molar concentration (M). D and affinity are inversely related. D The lower the value, the higher the binding affinity of the antibody for its antigen.

[0040] The term "binding avidity" describes a measure of the overall or cumulative strength of a protein-protein complex (i.e., the total strength of all non-covalent interactions between an antibody and its antigen). Binding avidity is determined by three parameters: (i) the binding affinity of the antibody-antigen complex, (ii) the valency of the antibody, and (iii) the structural arrangement of the antibody and its antigen in the complex.

[0041] Suitable binding analytes for assessing the binding affinity and binding avidity of a capture antibody include the antibody's immunogen or a fragment of the immunogen that contains the epitope to which the antibody binds. If cross-reactive binding to a homologous immunogen is desired, for example, cross-reactive binding of a human immunogen to a cynomolgus monkey homolog is often desired, then binding affinity and / or avidity to the homologous immunogen or a fragment thereof may also be assessed.

[0042] In another embodiment, the binding analyte comprises one or more biomolecules suitable for characterizing the blocking activity of an antibody. The term "blocking activity" of an antibody, as used herein, refers to the antibody's ability to block or disrupt the binding of an immunogen to its cognate binding partner. For example, if the immunogen is a receptor ligand, an antibody has blocking activity if it blocks or disrupts the interaction between the receptor ligand immunogen and its cognate receptor. Suitable biomolecules for testing antibody blocking activity include an immunogen or a fragment thereof and the immunogen's binding partner. To test the blocking activity of an immobilized capture antibody, an array of capture antibodies is contacted with an immunogen or a fragment thereof under conditions suitable for binding between the immunogen and the antibody. Once an antibody-immunogen complex is formed, the immunogen binding partner is introduced. If the immunogen binding partner binds to the antibody-binding immunogen, the antibody does not have blocking activity. Conversely, if the immunogen binding partner does not bind to the antibody-binding immunogen, the antibody possesses blocking activity.

[0043] In another embodiment, the binding analyte is one or more biomolecules suitable for characterizing immunogen binding of the capture antibody. In one embodiment, the binding analyte comprises a combination of an immunogen and a second immunogen-binding antibody. First, an array of capture antibodies is contacted with the immunogen, allowing binding between the capture antibody and the immunogen to occur. Subsequently, a second immunogen-binding antibody is introduced. If the second immunogen-binding antibody cannot bind to the immunogen (i.e., is complexed with the capture antibody), the capture antibody and the second immunogen-binding antibody can be "binned" together to bind to the same region of the immunogen. This type of assay is often referred to as an antibody-antibody cross-competition assay.

[0044] In another embodiment, the one or more biomolecules suitable for characterizing the immunogen binding of the capture antibody comprise fragments of the immunogen. According to this embodiment, an array of capture antibodies is sequentially exposed to a series of immunogen fragments covering the length of the immunogen. By detecting the binding of one or more immunogen fragments to the immobilized capture antibody, the epitope region of the immunogen to which the capture antibody binds can be identified.

[0045] In another embodiment, antibody-immunogen binding conditions can also be evaluated using an array of capture antibodies. For example, if the desired property of an antibody is that it binds to a target immunogen under selective pH conditions, the immunogen can be sequentially contacted with the antibody array, with each introduction gradually increasing or decreasing the pH of the buffer containing the immunogen. By detecting the immunogen binding to the capture antibody under various conditions, it is possible to select antibodies with the desired binding properties.

[0046] In another embodiment, the bound analyte is one or more biomolecules suitable for characterizing the chain composition of the capture antibody. For example, exemplary biomolecules for characterizing the light chain composition of the capture antibody include anti-kappa chain antibodies and / or anti-lambda chain antibodies. Light chain antibodies (i.e., anti-kappa chain antibodies and anti-lambda chain antibodies) suitable for use in the methods described herein are known in the art and commercially available (e.g., mouse and rabbit anti-human anti-kappa and anti-lambda antibodies are available, for example, but not limited to, from Abcam, Invitrogen, and R&D Systems). Other exemplary biomolecules for characterizing chain composition include, but are not limited to, Fc-specific antibodies, such as anti-IgG antibodies, anti-IgA antibodies, anti-IgE antibodies, anti-IgM antibodies, and anti-IgD antibodies. Fc-specific antibodies (e.g., anti-human Fc antibodies suitable for use in the methods described herein) are known in the art and commercially available.

[0047] In any embodiment of the methods disclosed herein, the steps of exposing the array of capture antibodies to a first binding analyte and detecting the presence or absence of an interaction between the first binding analyte and the capture antibody to determine a first binding property of the antibody are repeated one or more times. For example, if the first binding analyte is an immunogen or a fragment thereof, and the binding affinity of the capture antibody is to be characterized, the capture antibody can be repeatedly exposed to increasing concentrations of the immunogen or fragment thereof, and the K can be determined using detection of the binding association and dissociation rates at each concentration of immunogen. D A value is calculated and provides a measure of binding affinity.

[0048] In other embodiments, the exposing and detecting steps are repeated at least twice with different binding analytes (e.g., a first binding analyte and a second binding analyte) to determine at least two different binding properties of the antibody in a single assay run (i.e., the same capture antibody sample). In another embodiment, the exposing and detecting steps are repeated at least three times with different binding analytes (e.g., a first, second, and third binding analyte) to determine at least three different binding properties of the antibody in a single assay run. In yet another embodiment, the exposing and detecting steps are repeated at least four times with four different binding analytes to determine at least four different binding properties of the antibody in a single assay run. Preferably, the exposing and detecting steps are performed in an order that minimizes or eliminates wash steps and / or antibody regeneration steps between the introduction of different binding analytes.

[0049] In any embodiment, the exposing and detecting steps are repeated two or more times in an order that allows for characterization of two or more binding properties of the capture antibody sequentially (i.e., in a single assay run using a single capture antibody sample). Importantly, as demonstrated in the Examples herein, repeating the exposing and detecting steps with a second, and optionally a third, fourth, or additional binding analyte to determine additional binding properties of the antibody can be performed on a single supernatant sample without the need to provide additional supernatant samples. Because nominal amounts of antibody are present in supernatants from individually cultured primary B cells, the ability to assess multiple binding properties from a single antibody sample is critical for obtaining sufficient characterization data to select a subset of potential lead antibodies from an antibody campaign.

[0050] Thus, in one embodiment, the exposing and detecting steps are repeated in a sequence that allows for characterization of at least cross-reactive binding activity, binding affinity, receptor-ligand blocking activity, and light chain composition (in that order). Figure 2 shows an overview of this method and the sensorgram data generated from it, as further described in Example 2 herein. As shown, the combined evaluation of these parameters can be used to identify lead candidate antibodies with desired properties in less than one day (approximately 11 hours) and from a series of primary B cell culture supernatant samples.

[0051] According to this embodiment, the first binding analyte introduced to the array of antibodies secreted from immobilized primary B cells comprises one or more biomolecules suitable for characterizing the binding cross-reactivity of the capture antibody. As described above, exemplary first binding analytes for this purpose include one or more biomolecules homologous to the immunogen. The homologous biomolecule may be a protein, nucleic acid molecule, carbohydrate, or lipid structurally similar to the immunogen (e.g., a protein from the same or related protein family or a protein homolog from a different species), or it may be a protein, nucleic acid molecule, carbohydrate, or lipid structurally dissimilar to the immunogen. According to this embodiment, one or more different binding analytes suitable for assessing the binding cross-reactivity of the capture antibody may be sequentially contacted with the immobilized antibody. To the extent that undesired cross-reactive binding is detected, the immobilized antibody bound to the cross-reactive antigen is eliminated as a potential lead candidate, and subsequent assays to determine binding characteristics are therefore irrelevant to this antibody.

[0052] The exposing and detecting steps are then repeated with increasing concentrations of a second binding analyte suitable for measuring the binding affinity of the capture antibody. As noted above, this second binding analyte may comprise an immunogen, a fragment of an immunogen, or a homolog of an immunogen.

[0053] After exposing the antibody array with an immunogen or a fragment thereof, the antibody of interest on the array should bind to the immunogen. To test the blocking activity of the capture antibody, the exposing and detecting steps are repeated with a third binding analyte containing the immunogen's binding partner (e.g., the immunogen's receptor or ligand). If receptor or ligand binding to the antibody-bound immunogen is detected, the immobilized antibody is not a blocking antibody. If receptor or ligand binding to the antibody-bound immunogen is not detected, the immobilized antibody is a blocking antibody. If low levels of receptor or ligand binding to the antibody-bound immunogen are detected, the immobilized antibody can be characterized as a partially blocking antibody.

[0054] Alternatively, the third binding analyte may comprise a second immunogen-binding antibody to perform an antibody-antibody competition assay. As noted above, if the second immunogen-binding antibody (from the second analyte assay) cannot bind to the antibody-immunogen present on the array, the immobilized antibody is likely to bind to the same region of the immunogen as the second immunogen-binding antibody. Thus, the immobilized antibody and the second immunogen-bound antibody will have the same epitope bin.

[0055] After exposing the antibody array to the third binding analyte, the immobilized antibodies may be complexed with an immunogen, with an immunogen and an immunogen-binding partner, or with an immunogen and a second immunogen-binding antibody. Despite the complexed nature of the immobilized antibodies, the exposing and detecting steps may be repeated at least one more time with a fourth binding analyte, which contains one or more biomolecules suitable for characterizing the light chain composition of the captured antibodies. For example, the exposing and detecting steps may be repeated with additional anti-lambda chain antibodies, anti-kappa chain antibodies, and combinations thereof. Binding of one of these antibodies to the immobilized antibody or antibody complex identifies the light chain identity of the immobilized antibody.

[0056] Another aspect of the present disclosure is directed to a method for characterizing the binding affinity of antibodies from a preparation of B cells. The method includes providing a preparation of non-immortalized B cells, wherein the B cells of the preparation secrete immunogen-binding antibodies. The method further involves individually culturing the B cells of the preparation under conditions effective for the B cells to secrete the immunogen-binding antibodies into the culture supernatant. Culture supernatants containing the secreted antibodies are collected from the individually cultured B cells, and each culture supernatant is exposed to increasing concentrations of an immunogen, a fragment of the immunogen, or a homolog of the immunogen. The method further involves detecting association and dissociation between the secreted antibodies of the culture supernatant and the immunogen, a fragment thereof, or a homolog thereof at each of the increasing concentrations, and characterizing the binding affinity of the secreted antibodies based on the detection.

[0057] According to this aspect of the disclosure, the method may further include providing a solid support comprising a plurality of reaction surfaces, each reaction surface comprising a capture reagent immobilized thereon as described above. Each of the recovered culture supernatants is contacted with the solid support under conditions effective for secreted antibodies from one of the recovered culture supernatants to bind to the capture reagent immobilized on one of the reaction surfaces, forming an array of captured antibodies on the solid support. Exposing the culture supernatants to increasing concentrations of an immunogen, a fragment of the immunogen, or a homolog of the immunogen, and detecting association and dissociation between the antibodies in the culture supernatant and the immunogen, a fragment thereof, or a homolog thereof is carried out on the solid support.

[0058] In any embodiment, the method may optionally further involve contacting the array of capture antibodies with a biomolecule that is homologous to the immunogen and determining the presence or absence of an interaction between the homologous biomolecule and the capture antibody to identify the binding cross-reactivity of the capture antibody. As described above, a homologous biomolecule is any biomolecule (e.g., a protein, nucleic acid molecule, lipid, or carbohydrate) that is structurally similar to the immunogen. In a preferred embodiment, the cross-reactivity of the capture antibody is determined before determining the binding affinity. This allows certain immunogen-binding antibodies to be excluded from further analysis if the tested cross-reactivity is undesirable. In another embodiment, the cross-reactivity of the capture antibody is determined after the binding affinity. This approach may include a washing step to remove bound immunogen from the array of capture antibodies.

[0059] The method may optionally further include, after assessing binding affinity to the immunogen, contacting the array of capture antibodies with one or more additional binding analytes and determining the presence or absence of an interaction between the one or more additional binding analytes and the capture antibodies to characterize one or more additional characteristics of the capture antibodies. In any embodiment, the capture antibodies are bound to the immunogen (antibody-immunogen complex) at the time the one or more additional binding analytes are introduced to the solid support reaction surface.

[0060] In any embodiment, the one or more additional binding analytes are binding partners of an immunogen, and the blocking activity of the capture antibody is characterized as described above. In any embodiment, the one or more additional binding analytes are agents that bind to antibody light chains, and the light chain composition of the capture antibody is characterized as described above. In any embodiment, the one or more additional binding analytes comprise another immunogen-binding antibody, and the epitope binning of the capture antibody is characterized as described above. In any embodiment, the one or more additional binding analytes comprise one or more immunogen fragments, and the epitope mapping of the capture antibody is characterized as described above.

[0061] Methods for analyzing binding affinity, binding kinetics, cross-reactivity, and other binding interactions are known in the art (see, e.g., Ernst et al., Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009), incorporated herein by reference in its entirety), and are suitable for use in carrying out the methods described herein. These methods include, but are not limited to, solid phase binding assays (e.g., ELISA assays), immunoprecipitation, flow cytometry, fluorescence activated cell sorting (FACS), surface plasmon resonance (SPR), surface plasmon resonance imaging (SPRi) such as Carterra® LSA (Salt Lake City, UT) and Biacore™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion assays (e.g., KinExA®), BioLayer interferometry such as Octet™ (Sartorius, Fremont, CA), MicroScale Thermophoresis (MST) such as NanoTemper Monolith (NanoTemper Technologies GmbH, Munich Germany), and isothermal titration calorimetry (ITC) such as Microcal ITC200 (Malvern Panalytical, Malvern UK).

[0062] In some embodiments, detecting the presence or absence of binding interactions of immunogen-binding antibodies derived from primary B cells is performed using surface plasmon resonance (SPR). SPR technology is reviewed, for example, in Hahnfeld et al., "Determination of Kinetic Data Using SPR Biosensors," Methods Mol. Med. 94:299-320 (2004) and Nguyen et al., "Surface Plasmon Resonance: A Versatile Technique for Biosensor Applications," Sensors (Basel) 15(5):10481-510 (2015), which are incorporated herein by reference in their entireties. In a typical SPR experiment, antibodies derived from B cells are immobilized on an SPR-active gold-coated glass slide in a flow cell, and a sample containing one of the binding analytes described herein is introduced and flowed across the surface. When polychromatic light of a given wavelength is shone on a gold surface at a specific angle (angle of incidence), a portion of the light energy excites electrons on the surface. This angle of incidence is strongly affected by the refractive index of the material bound on or near the gold surface. Thus, when a binding interaction occurs between an immobilized antibody and a potential binding analyte, the refractive index increases, causing a change in the angle of incidence. The change in the angle of incidence can be measured to generate a response curve in real time, from which the kinetics of binding can be estimated.

[0063] In an optional embodiment, binding interactions of immunogen-binding antibodies derived from B cells are performed using SPR imaging (SPRi) or SPR microscopy. SPRi follows the same general principles as traditional SPR, but the information measured and detection method are slightly different, allowing for a higher throughput method of studying binding interactions. In particular, a polarized light beam (as opposed to polychromatic light) is directed onto a thin gold film, and a charge-coupled device (CCD) camera is utilized to capture high-resolution images of the binding region.

[0064] As demonstrated in the Examples herein, the Carterra® LSA instrument, which uses high-throughput surface plasmon resonance imaging (HT-SPRi) to measure kinetic interactions of biomolecules, is a particularly suitable platform for performing the methods described herein in high throughput. Other instruments that utilize SPR detection in a high-throughput format are also suitable for detecting and measuring the kinetic interactions described herein, including, for example, Biacore T200 and Biacore 8K instruments (Cytiva, Marlborough, MA).

[0065] An alternative, preferred detection method that can be used in the methods described herein involves detection based on biolayer interferometry (BLI). This technique is described, for example, in Wilson et al., Biochemistry and Molecular Biology Education, 38:400-407 (2010) and Dysinger et al., J. Immunol. Methods, 379:30-41 (2012), which are incorporated herein by reference in their entireties. BLI is an optical technique that measures macromolecular interactions by analyzing the interference pattern of white light reflected from the surface of a biosensor chip. In a typical BLI experiment, antibodies derived from B cells are immobilized on a biosensor chip, and the chip is introduced into a solution well containing one of the binding analytes described herein. Binding of the analyte to the immobilized antibody on the biosensor chip causes a shift in the interference pattern, which is measured in real time. Sartorius' Octet® BLI label-free detection system is suitable for performing the methods described herein in high throughput.

[0066] As demonstrated in the Examples herein, multiple binding properties of an immunogenic binding protein can be accurately characterized from a B cell supernatant sample containing a nominal antibody concentration. Therefore, the methods described herein are suitable for characterizing binding proteins in other samples containing minimal concentrations of binding protein. Accordingly, another aspect of the present disclosure is directed to a method for characterizing binding proteins in a sample containing 250 ng or less of binding protein. In any embodiment, characterizing binding proteins according to the methods described herein can be performed using samples containing about 1 ng to about 250 ng of binding protein, samples containing 1 ng to about 100 ng of binding protein, and samples containing 1 ng to about 25 ng of binding protein. The method involves contacting the samples with a solid support comprising multiple reaction surfaces, each reaction surface comprising a capture reagent immobilized thereon. The contacting of the sample with the solid support is performed under conditions effective for binding proteins in the sample to bind to the immobilized capture reagent on the reaction surface, thereby forming an array of captured binding proteins on the solid support. The method further involves subjecting the array of captured binding proteins to two or more different binding assays to characterize the binding proteins in the sample.

[0067] Suitable solid supports and capture reagents for immobilizing binding proteins from a sample to form an array of captured binding proteins are described above. As illustrated in the Examples herein, contacting a sample containing a low amount of binding protein with a solid support reaction surface is carried out by flowing the sample over the reaction surface of the solid support and repeating or circulating the flow over the reaction surface of the solid support for at least 15 minutes. In any embodiment, the flow of the sample containing binding proteins is carried out in a bidirectional manner to maximize exposure of the binding proteins in the sample to the capture reagent on the solid support surface. In any embodiment, the flow of the sample over the surface continues for a duration of at least 10 minutes. In any embodiment, the flow continues for a duration of about 10 to about 40 minutes, e.g., at least or about 15 minutes, at least or about 20 minutes, at least or about 25 minutes, at least or about 30 minutes, at least or about 35 minutes, or at least or about 40 minutes. In any embodiment, the circulating flow continues for a duration of about 40 minutes. In any embodiment, the circulating flow continues for a duration of more than 30 minutes. In any embodiment, this circulation continues for a duration of 30 to 40 minutes.

[0068] As described above, the concentration of the binding protein in the sample is about 1 ng to about 25 ng. More preferably, the concentration of the binding protein in the sample is about 3 ng to about 20 ng. To maximize the immobilization of the binding protein in the sample to the solid support, a sample having a sample volume of about 50 μL to about 250 μL containing about 20 ng / mL to about 100 ng / mL of binding protein is contacted with the reaction surface of the solid support. In any embodiment, about 150 μL to about 250 μL of sample is contacted with the solid support, and this sample contains a binding protein concentration of about 20 ng / mL to about 100 ng / mL or is diluted to contain this binding protein concentration. In any embodiment, the sample contacted with the solid support has a sample volume of about 150 μL to about 250 μL and a total amount of binding protein comprising or consisting of about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 18 ng, about 19 ng, about 20 ng, about 21 ng, about 22 ng, about 23 ng, about 24 ng, or about 25 ng.

[0069] According to this aspect of the disclosure, suitable binding proteins that can be characterized by the described methods include antibodies, i.e., intact immunoglobulin molecules, immunoglobulin domains (e.g., VhH domains, unibody) and fragments thereof (Fab, Fab', F(ab')2, antibody derivatives (e.g., scFv, diabodies, tribodies, minibodies, etc.), and synthetic binders such as minibinders.

[0070] According to this aspect of the disclosure, characterizing the binding proteins in a sample by subjecting the array of captured binding proteins to two or more different binding assays is carried out as disclosed above, i.e., exposing the array of captured binding proteins to a first binding analyte and detecting the presence or absence of an interaction between the first binding analyte and the captured binding proteins to determine a first binding property of the binding proteins. This process of exposure to and detection by a second, and optionally a third, fourth, fifth binding analyte is repeated sequentially to determine additional binding properties of the binding proteins.

[0071] Importantly, as demonstrated in the examples herein, repeating the steps of exposing to a second, and optionally a third, fourth, or additional binding analyte and detecting with this binding analyte to determine additional binding properties of the antibody can be performed on a single sample without the need to provide additional supernatant samples. Preferably, the array of capture antibodies is exposed to the binding analytes (i.e., the first, second, third, etc. binding analytes) in the order described above, with minimal or no washing steps required between different binding analytes and without the need to replenish the binding proteins on the reaction surface. This allows for the determination of multiple functional properties of binding proteins from a single supernatant sample in a single experimental run. Exemplary reaction sequences that achieve these goals are described in more detail herein.

[0072] Suitable binding assays for functionally characterizing binding proteins from a sample include, but are not limited to, binding assays that characterize antibody binding affinity, antibody binding avidity, antibody cross-reactive binding (e.g., cross-reactive binding to the same immunogen in different species or to structurally similar immunogens), antibody blocking activity (e.g., blocking of immunogen binding to its cognate binding partner), immunogen binding conditions (e.g., optimal pH conditions for antibody-immunogen binding), antibody chain composition (e.g., antibody light chain composition), antibody epitope binding, antibody-antibody cross-competition, and any combination of the foregoing assays. Exemplary assays and reagents for performing the assays are disclosed above.

[0073] The invention having been described, the following examples are offered by way of illustration and not by way of limitation. [Example]

[0074] Example 1: Assay optimization The Carterra® LSA instrument (Salt Lake City, UT) was the instrument selected to perform binding and affinity measurements of immunogen-binding antibodies recovered from the supernatant of primary B cells. Therefore, the first step was to determine the optimal conditions for capturing low concentration antibodies from the supernatant of individually cultured B cells. The three conditions optimized in this example included capture antibody immobilization, minimum supernatant antibody concentration, and antibody printing time.

[0075] To prepare the antibody-immobilized surface, the Single Flow Channel (SFC) and 96-Print Head (96PH) of the Carterra® LSA instrument were primed with running buffer (HEPES-buffered Steinberg's Solution (HBS-T); 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20). The capture surface was prepared in the SFC by standard amine coupling. An HC30-M chip (Carterra® LSA catalog number 4279) was activated with a 10-minute injection of a freshly prepared 1:1:1 (v / v / v) mixture of 0.4 M EDC + 0.1 M NHS + 0.1 M MES pH 5.5. Monoclonal mouse anti-human Fc antibody (mAb1.35.1) was prepared at 100 μg / mL in 10 mM sodium acetate pH 4.5 (Carterra® Catalog No. 3628) and coupled to the HC30-M chip for 20 minutes. Excess reactive esters were blocked with a 7 minute injection of 1 M ethanolamine HCl pH 8.5 (Carterra® Catalog No. 3626).

[0076] To determine the minimum immunogen-conjugated antibody concentration and antibody printing time required, a Carterra® LSA 96PH was used to print known concentrations of immunogen-conjugated antibodies onto chips conjugated with mAb1.35.1 as described above for various times. Forty-eight antibody samples were diluted to 500, 100, 50, or 25 ng / mL in running buffer (HEPES-buffered Steinberg's Solution (HBS-T); 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20). The 500, 100, and 50 ng / mL samples were printed for 10 minutes, and the 25 ng / mL sample was printed for 30 minutes. The goal was to determine the minimum antibody concentration required to reach an antibody printing signal of at least 100 RU. It was found that a 25 ng / mL antibody sample printed for 30 minutes gave a signal of approximately 200-250 RU (see Figure 3), which compared favorably with the RU signal obtained from printing a 50 ng / mL antibody sample on the chip for 10 minutes.

[0077] To confirm that lower antibody concentrations at longer printing times were sufficient for affinity measurements, the following affinity assay was performed. After printing the antibody at 100, 50, or 25 ng / mL as described above, a non-regenerating kinetic assay was set up using SFC by sequentially injecting six concentrations of target immunogen (i.e., 100, 33.33, 11.11, 3.7, 1.23, and 0.41 nM) in a 1:3 dilution series starting from 100 nM. Dilutions were performed in running buffer, from low to high, without regeneration between injections. Association was over a 10-minute period, with a dissociation time of 20 minutes. Data were double-referenced by subtracting both the local reference and the zero nanomolar analyte concentration (buffer). The double-referenced data were globally fit to a 1:1 Langmuir binding model using the Carterra® Kinetic tool, and the k of each spot itself was calculated. a and k d Value in K DThis analysis shows that an antibody concentration of 25 ng / mL printed over 30 minutes gives similar affinity data as an antibody concentration of 100 ng / mL printed over 10 minutes (compare 100 ng / mL and 25 ng / mL sensorgrams in Figure 4). The data also show that an antibody concentration of 50 ng / mL printed over 10 minutes does not capture enough antibody for a good affinity measurement (see Figure 4, shaded graph).

[0078] Example 2 - Characterization of antibodies using antibody-containing supernatants from primary B cells A Carterra® LSA instrument was used to characterize the cross-reactivity, binding affinity, receptor-ligand (RL) blocking, and light chain composition of immunogen-binding antibodies recovered from primary XenoMouse® B cells in one continuous experimental run (shown in Figure 2). In this example, desirable antibody properties included high-affinity binding to an interleukin cytokine protein target (referred to herein as the "target protein") without binding to related interleukin cytokines (referred to herein as "off-target proteins"). In addition, it was desirable that candidate antibodies that bound to the target protein would block binding of the target protein to its receptor.

[0079] Figure 5 shows the cumulative sensorgram of this experimental run, in which the cross-reactivity, binding affinity, RL blocking, and light chain composition of a panel of mAbs secreted from primary B cells were each assessed sequentially in a single run (from one B cell supernatant sample) over approximately 11 hours. Each component of the experimental run is described in more detail below, and the corresponding portions of the cumulative sensorgrams are shown in Figures 6-9.

[0080] To prepare the assay surface, the Single Flow Channel (SFC) and 96-Print Head (96PH) of the Carterra® LSA instrument were primed with running buffer (HEPES-buffered Steinberg's Solution (HBS-T); 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20). The capture surface itself was prepared in the SFC by standard amine coupling. An HC30-M chip (Carterra® LSA catalog number 4279) was activated with a 10-minute injection of a freshly prepared 1:1:1 (v / v / v) mixture of 0.4 M EDC + 0.1 M NHS + 0.1 M MES pH 5.5. Monoclonal mouse anti-HuFc antibody (mAb1.35.1) was prepared at 100 μg / mL in 10 mM sodium acetate pH 4.5 (Carterra #3628) and conjugated to the surface for 20 minutes. Excess reactive esters were blocked with a 7-minute injection of 1 M ethanolamine HCl pH 8.5 (Carterra #3626). The final conjugation yield was >1000 response units (RU).

[0081] Primary B cell-generated antibody samples containing >100 ng / mL of antibody were diluted approximately 1:4.5 (40 μL of supernatant + 140 μL of running buffer). The diluted samples were printed onto the HC30-M chip surface containing the mAb1.35.1 antibody using 96PH for 30 minutes.

[0082] After printing, the immobilized immunogen-binding antibodies were screened for cross-reactivity using a 100 nm injection of off-target protein using SFC. Figure 6 shows SPRi sensorgrams (graphs of response units (RU) versus time) of various antibody samples. Binding of a population of mAbs to off-target proteins is easily identified through an increase in SPRi signal (RU) upon introduction of the off-target protein.

[0083] Next, non-regenerating kinetic assays were set up using SFC by sequentially injecting six concentrations of target protein (i.e., 100, 33.33, 11.11, 3.7, 1.23, and 0.41 nM) in a 1:3 dilution series starting from 100 nM. Dilutions were made in running buffer and injected from low to high concentrations without regeneration between injections. Association was over a 10-minute period, with a dissociation time of 20 minutes. Figure 7 shows affinity sensorgrams for some of the antibodies tested. The top panel of the sensorgrams shows high-affinity binders (i.e., K<100 pM). D ) and the middle and bottom panels show high to medium affinity antibody candidates as evidenced by distinguishable off-rates.

[0084] Antibody binding to off-target proteins could potentially interfere with affinity measurements. However, because off-target binding was undesirable, any antibodies showing cross-reactive binding to off-target proteins were immediately eliminated from progression. Therefore, any affinity or RL blocking data (described below) were considered irrelevant for antibodies showing off-target protein binding.

[0085] Immediately after the kinetic cycle, a single 10-minute injection of target protein receptor at 100 nM in running buffer was performed to measure the RL blocking activity of the immunogen-bound antibody. The sensorgrams in Figure 8 show the responses of non-blocking, partially blocking, and fully blocking antibodies. The SPR sensorgrams show the binding response after first introducing the target protein to the HC30-M chip containing the immunogen-bound antibody (see the "Target Binding" section of the sensorgram). The target protein receptor was then introduced to the chip, and the binding response was recorded (see the "Receptor Binding" section of the sensorgram). Antibodies that block receptor binding to the target protein show negligible binding responses when the target protein receptor is introduced. Note that because the target protein receptor is significantly larger than the target protein, the binding signal generated from receptor-target binding is similarly large.

[0086] Finally, sequential injection of anti-human kappa and anti-human lambda antibodies at 5 μg / mL over 20 minutes allowed identification of the light chain subtype of the immunogen-binding antibody, as shown in the sensorgram in FIG.

[0087] Based on the data generated in this experimental run of 1,222 antibody samples, it was determined that 1,021 (83.6%) of these antibodies showed no cross-reactive binding to non-target proteins, 182 (14.9%) of these antibodies showed the desired high-affinity target protein binding (i.e., affinity less than 100 pM), and 26 (2.1%) of these antibodies were target protein receptor-blocking antibodies. Thus, of the 1,222 antibodies screened, this screening method identified 26 potential lead candidates with the desired binding specificity, affinity, and blocking activity from primary B cell supernatants in just 11 hours.

[0088] Example 3 - Characterization of IL-11 antibodies using antibody-containing supernatants from primary B cells In this example, the Carterra® LSA instrument was used to characterize the binding affinity and receptor-ligand (RL) blocking activity of immunogen-binding antibodies recovered from primary XenoMouse® B cells. The target protein of the B cell-secreted antibodies in this example was interleukin-11 (IL-11).

[0089] To prepare the assay surface, the Single Flow Channel (SFC) and 96-Print Head (96PH) of the LSA instrument were primed with running buffer (HEPES-buffered Steinberg's Solution (HBS-T); 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20). The capture surface was prepared in the SFC by standard amine coupling. An HC30-M chip (Carterra® LSA catalog no. 4279) was activated with a 10-minute injection of a freshly prepared 1:1:1 (v / v / v) mixture of 0.4 M EDC + 0.1 M NHS + 0.1 M MES pH 5.5. The monoclonal mouse anti-HuFc antibody mAb1.35.1 was prepared at 100 μg / mL in 10 mM sodium acetate pH 4.5 (Carterra #3628) and allowed to conjugate for 20 minutes. Excess reactive esters were blocked with a 7 min injection of 1 M ethanolamine HCl pH 8.5 (Carterra #3626). The final conjugate yield was >1000 response units (RU).

[0090] B cell supernatant samples estimated to have antibody concentrations greater than 100 ng / mL were diluted approximately 1:6.6 (30 μL of supernatant + 170 μL of Carterra buffer (HEPES-buffered Steinberg's Solution (HBS-T); 50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Tween 20)) and printed using 96PH for 30 minutes. After printing, a non-regenerating kinetic assay was set up using SFC by sequentially injecting six concentrations of target (IL11, Sino Biological / 12225-HNCE) in a 1:3 dilution series starting from 100 nM (100, 33.33, 11.11, 3.7, 1.23, and 0.41 nM). Dilutions were performed in running buffer, from low to high concentrations, without regeneration between injections. The association time was 10 minutes, and the dissociation time was 20 minutes.

[0091] Immediately after the kinetic cycle, a single 10 min injection of IL11 receptor (R and D Systems / 8895-MR-MTO) at 100 nM in running buffer was performed to measure RL blocking activity.

[0092] Figure 10 shows cumulative sensorgrams from this experimental run, in which the binding affinity and RL blockade of a panel of IL-11 mAbs secreted from primary B cells were each assessed sequentially in a single run over only 4 hours. Figure 11 shows affinity sensorgrams for some of the antibodies tested. The leftmost panel of the sensorgram represents high-affinity binders (<100 pM), the middle panel represents intermediate-affinity binders (1 nM-100 pM), and the rightmost panel represents low-affinity binders (>1 nM).

[0093] The table in Figure 12 summarizes the affinity data and blocking activity of three candidate IL-11 antibodies (i.e., antibodies LIBC729450-1, LIBC729919-1, and LIBC729812-1). All three antibodies demonstrated high binding affinity, but only LIBC729450-1 functioned as a receptor-blocking antibody based on RU after introducing the target receptor protein (IL-11 receptor) to an HC30-M chip containing antibodies bound to the target protein (IL-11). Antibodies LIBC729919-1 and LIBC729812-1 functioned as non-blockers and partial blockers of receptor binding to IL-11 based on their RU values.

[0094] In this example, 96 antibody samples were screened. Of these, 23 antibodies had the desired binding affinity of less than 40 pM, and of these 23 antibodies, 11 blocked IL-11 receptor binding to IL-11. Thus, the method described herein identified 11 potential lead candidates with the desired binding specificity and blocking activity from primary B cell supernatants in just 4 hours.

Claims

1. 1. A method for characterizing the binding affinity of antibodies from a preparation of B cells, comprising: providing a preparation of non-immortalized B cells, wherein the B cells of said preparation secrete immunogen-binding antibodies; individually culturing the B cells of said preparation under conditions effective for said B cells to secrete immunogen-binding antibodies into the culture supernatant; recovering the culture supernatant containing the secreted antibodies from the individually cultured B cells; exposing each culture supernatant to increasing concentrations of the immunogen, a fragment of said immunogen, or a homologue of said immunogen; detecting, at each of the increasing concentrations, association and dissociation between the secreted antibody of the culture supernatant and the immunogen, fragment thereof, or homolog thereof; characterizing the binding affinity of the secreted antibody based on said detection; A method comprising:

2. providing a solid support comprising a plurality of reaction surfaces, wherein each reaction surface comprises a capture reagent immobilized thereon; contacting each of the recovered culture supernatants with the solid support under conditions effective for secreted antibodies from one of the recovered culture supernatants to bind to the immobilized capture reagent on one of the reaction surfaces to form an array of capture antibodies on the solid support, wherein the exposing, detecting, and characterizing are performed on the solid support. The method of claim 1 further comprising:

3. contacting the array of capture antibodies with candidate cross-reactive binding molecules prior to said exposing; determining the presence or absence of an interaction between said candidate cross-reactive binding molecule and said capture antibody; identifying the binding cross-reactivity of said capture antibody as a result of said determination; The method of claim 2 further comprising:

4. After said characterization, contacting said array of capture antibodies with one or more additional binding analytes; determining the presence or absence of an interaction between the one or more additional binding analytes and the capture antibody to characterize additional binding properties of the capture antibody; The method of claim 2 further comprising:

5. The method of claim 4 , wherein the one or more additional binding analytes comprise a binding partner of the immunogen, and the blocking activity of the capture antibody is characterized based on the determination.

6. 5. The method of claim 4, wherein the one or more additional binding analytes are agents that bind to antibody light chains, and the light chain composition of the captured antibody is characterized based on the determination.

7. The method of claim 2 , wherein the detecting and characterizing is performed using surface plasmon resonance imaging.

8. The contacting step comprises: Flowing each culture supernatant onto a reaction surface of a solid support; repeating the flushing step for at least 15 minutes; The method of claim 2 , comprising:

9. The method of claim 8, wherein the repeating step is carried out for about 20 minutes to about 40 minutes.

10. The method of claim 8 , wherein the flowing step comprises bidirectional flowing.

11. 3. The method of claim 2, wherein each of the culture supernatants contacted with the reaction surface of the solid support comprises a volume of about 150 μl to about 200 μl and contains about 20 ng / mL to about 100 ng / mL of secreted antibody.

12. The solid support comprises 96 to 384 reaction surfaces, and the contacting step comprises: contacting each reaction surface with a different culture supernatant, thereby capturing antibodies from 96 to 384 different culture supernatants on the solid support; The method of claim 2 , comprising:

13. 1. A method for characterizing immunogen-binding antibodies from a preparation of B cells, comprising: providing a preparation of non-immortalized B cells, wherein the B cells of said preparation secrete immunogen-binding antibodies; individually culturing the B cells of the preparation under conditions effective for the B cells to secrete the immunogen-binding antibodies into the culture supernatant; recovering the culture supernatant containing the secreted antibodies from the individually cultured B cells; subjecting each recovered supernatant to two or more different binding assays to characterize the immunogen-binding antibodies from the B cell preparation; A method comprising:

14. 14. The method of claim 13, wherein the two or more binding assays are selected from a binding affinity assay, a binding avidity assay, a binding cross-reactivity assay, an assay for determining immunogen binding conditions, a blocking activity assay, an assay for determining antibody chain composition, an epitope binding assay, and an antibody-antibody cross-competition assay.

15. 14. The method of claim 13, wherein one of the two or more binding assays is a binding affinity assay.

16. The subjecting step comprises: providing a solid support comprising a plurality of reaction surfaces, wherein each reaction surface comprises a capture reagent immobilized thereon; contacting each of the recovered culture supernatants with the solid support under conditions effective for antibodies secreted from the recovered culture supernatants to bind to the immobilized capture reagent on the reaction surface, thereby forming an array of capture antibodies on the solid support; exposing the array of capture antibodies to a first binding analyte; detecting the presence or absence of an interaction between the first binding analyte and the capture antibody to determine a first binding property of the antibody; sequentially repeating said exposing and said detecting with at least a second binding analyte to determine at least a second binding characteristic of said antibody; 14. The method of claim 13, comprising:

17. 17. The method of claim 16, wherein the first and second binding analytes are biomolecules independently selected from proteins, nucleic acid molecules, carbohydrates, and lipids.

18. 18. The method of claim 17, wherein the first and second binding analytes are independently selected from the group consisting of biomolecules suitable for characterizing the binding cross-reactivity of the capture antibody, biomolecules suitable for characterizing the binding affinity of the capture antibody, biomolecules suitable for characterizing the binding avidity of the capture antibody, one or more biomolecules suitable for characterizing the blocking activity of the capture antibody, biomolecules suitable for characterizing the epitope binding of the capture antibody, one or more biomolecules suitable for characterizing the antibody cross-competition of the capture antibody, and one or more biomolecules suitable for characterizing the chain composition of the capture antibody.

19. 17. The method of claim 16, further comprising sequentially repeating the exposing and detecting with a third binding analyte, and optionally a fourth binding analyte, to determine additional binding properties of the antibody.

20. The method of any one of claims 16 to 19, wherein the first binding analyte comprises one or more biomolecules suitable for characterizing the binding cross-reactivity of the capture antibody.

21. 21. The method of claim 20, wherein the first binding analyte comprises a biomolecule that is homologous to an immunogen.

22. The method of any one of claims 16 to 19, wherein the second binding analyte is a biomolecule suitable for characterizing the binding affinity of the capture antibody.

23. 23. The method of claim 22, wherein the second binding analyte is an immunogen or a fragment of the immunogen.

24. 23. The method of claim 22, wherein the second binding analyte is a homolog of an immunogen or a fragment thereof.

25. 20. The method of claim 19, wherein the third binding analyte is a biomolecule suitable for characterizing the blocking activity of the capture antibody.

26. 26. The method of claim 25, wherein the third binding analyte is a binding partner of an immunogen.

27. 26. The method of claim 25, wherein the fourth binding analyte comprises one or more biomolecules suitable for characterizing the antibody chain composition of the capture antibody.

28. 28. The method of claim 27, wherein the one or more biomolecules are suitable for characterizing antibody light chain composition and are selected from anti-lambda chain antibodies, anti-kappa chain antibodies, and combinations thereof.

29. selecting one or more candidate antibodies from the array of capture antibodies for sequencing based on said detection; sequencing the selected one or more candidate antibodies; The method of any one of claims 16 to 28, further comprising:

30. The method of any one of claims 16 to 28, wherein the detection is performed using surface plasmon resonance imaging (SPRi).

31. 17. The method of claim 16, wherein each collected supernatant contains about 20 ng / mL to about 100 ng / mL of secreted antibody in a volume of about 150 μl to about 200 μl.

32. 17. The method of claim 2 or 16, wherein the immobilized capture reagent comprises an anti-Fc specific antibody.

33. 17. The method of claim 2 or 16, wherein the immobilized capture reagent comprises an anti-light chain specific antibody.

34. The contacting step comprises: flowing the recovered supernatant onto a reaction surface of a solid support; repeating said flushing for at least 15 minutes; 17. The method of claim 16, comprising:

35. 35. The method of claim 34, wherein the repeating step is carried out for about 20 minutes to about 40 minutes.

36. 35. The method of claim 34, wherein the flowing step comprises bidirectional flowing.

37. 14. The method of claim 13, wherein the subjecting step is carried out in less than 12 hours.

38. The solid support comprises 96 to 384 reaction surfaces, and the contacting step comprises: contacting each reaction surface with a different culture supernatant, thereby capturing antibodies from 96 to 384 different culture supernatants on the array; 17. The method of claim 16, comprising:

39. 14. The method of claim 1 or 13, wherein the B cell preparation is a primary B cell preparation.

40. 14. The method of claim 1 or 13, wherein the preparation of B cells is from a subject immunized with the immunogen.

41. 41. The method of claim 40, wherein the immunized subject is selected from a human, a non-human primate, a horse, a pig, a cow, a goat, a sheep, a llama, a camel, a rabbit, a dog, a rat, and a mouse.

42. 14. The method of claim 1 or 13, wherein the preparation of B cells is from a subject with an autoimmune disorder.

43. 14. The method of claim 1 or 13, wherein the B cells of the preparation produce human antibodies.

44. 14. The method of claim 1 or 13, wherein the immunogen-binding antibody is a human antibody.

45. 1. A method for characterizing a binding protein, comprising: providing one or more samples, wherein each sample comprises from about 1 ng to about 25 ng of binding protein; contacting the one or more samples with a solid support comprising a plurality of reaction surfaces, each reaction surface comprising a capture reagent immobilized thereon, said contacting being carried out under conditions effective to cause binding proteins from the sample to bind to the immobilized capture reagents on the reaction surfaces, thereby forming an array of captured binding proteins on the solid support; subjecting the array of captured binding proteins to two or more different binding assays to characterize the binding proteins in each of the one or more samples; A method comprising:

46. The subjecting step comprises: exposing the array of captured binding proteins to a first binding analyte; detecting the presence or absence of an interaction between the first binding analyte and the captured binding protein to determine a first binding property of the binding protein; sequentially repeating said exposing and said detecting with at least a second binding analyte to determine at least a second binding property of said binding protein; 46. ​​The method of claim 45, comprising:

47. The contacting step comprises: flowing the sample over a reaction surface of a solid support surface; repeating said flushing for at least 15 minutes; 46. ​​The method of claim 45, comprising:

48. 48. The method of claim 47, wherein the repeating step is carried out for about 20 minutes to about 40 minutes.

49. 49. The method of claim 47 or 48, wherein the flowing step comprises flowing bidirectionally.

50. 50. The method of any one of claims 45 to 49, wherein each sample contains from about 3 ng to about 20 ng of binding protein.

51. 50. The method of any one of claims 45 to 49, wherein each sample contains about 20 ng / mL to about 100 ng / mL of binding protein in a volume of about 150 μl to about 200 μl.

52. 46. ​​The method of claim 45, wherein the two or more binding assays are selected from a binding affinity assay, a binding avidity assay, a binding cross-reactivity assay, an assay for determining immunogen binding conditions, a blocking activity assay, an assay for determining antibody chain composition, an epitope binding assay, and an antibody-antibody cross-competition assay.

53. 46. ​​The method of claim 45, wherein one of the two or more binding assays is a binding affinity assay.

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