Non-terminal antibody generation method and single cell assay

Abstract

Provided herein is a method for monitoring the production of a selected antibody in a non-human animal, comprising: (a) immunizing the non-human animal with an immunogen; (b) obtaining a blood sample from the non-human animal comprising antibody-secreting cells (ASCs); and (c) individually assaying the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody. Also provided is a method for inducing antibody production in a non-human animal for the production of a selected antibody. In an exemplary embodiment, the method comprises performing cycles of (a)-(c) as described above, and repeating the cycle if the proportion of ASCs producing the selected antibody is below a threshold value. In various aspects, the cycle is repeated until the proportion of ASCs producing the selected antibody is equal to or greater than a threshold value. Further provided herein is a single cell assay.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 146,135, filed February 5, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Traditional animal-based antibody generation methods involve killing animals based on polyclonal serum titers against protein targets of varying complexity. While some antibody generation campaigns have simple design goals (e.g., binding to a target), most are more complex and require various characteristics in the desired antibodies (e.g., cross-reactivity, binding to a specific epitope, binding with specific affinity, etc.). Traditional antibody generation approaches rely on examining polyclonal secretory antibody (serum) responses to select animals for B cell harvesting and antibody generation. The "serum titer" approach is less than ideal because it measures the total reactivity of all antibodies secreted (i.e., it is a polyclonal mixture) and cannot be used to identify the B cell source of the detected antibodies (i.e., there are both physically and temporally detached from the source B cells). The lack of a direct correlation between phenotype (antibody titer measurement) and genotype (the critical source of B cells that code for the antibodies) makes it difficult to interpret the quality of the B cell response. Other than determining whether soluble antigen-specific antibodies are present in the serum, it is difficult to obtain any further useful information from this polyclonal analysis that can aid in animal selection.

[0003] Moreover, conventional methodologies are final with respect to the animal and therefore a "one-off" attempt to capture the relevant B-cell repertoire of an animal. The inability to capture this repertoire, which may be caused by technical issues, the selection of animals with suboptimal antibody production, and / or lack of sampling depth of the repertoire (i.e., the low efficiency of conventional viral immortalization and hybridoma processes that result in the fusion of very low B-cell repertoires (less than 0.1%)), results in the waste of valuable resources and forces the use of alternative immunized animals or completely new immunization campaigns. Moreover, conventional methods exclude the possibility of a continuous process of evolving antibody responses utilizing the immune system of the same animal.

[0004] Despite these limitations, conventional methods are widely used in part because they allow capture of an acceptable fraction of the immune repertoire and provide a renewable source of antibodies that can be easily scaled to fit downstream assays.

[0005] Increasingly, these traditional methods are too time-consuming to accommodate project timelines, capture the wrong B cell population, fail to adequately sample the B cell repertoire, or fail to allow real-time monitoring of evolving B cell responses. In addition, the challenging nature of many antibody target classes (e.g., complex membrane proteins, targets with minimal epitope space, proteins highly similar to orthologs, etc.) can make it difficult to elicit B cell responses in animals due to lack of robust immunogenicity. Combined with the extreme complexity of some antibody design goals, it can be difficult to generate immunized animals with the desired immune profile (i.e., B cell repertoire). Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above, there is a need for more efficient antibody generation methods, such as antibody generation methods that can better position traditional animal immunization and B cell methods for successful antibody generation, which would greatly enhance animal-based antibody generation. [Means for solving the problem]

[0007] For the first time, rationale, experimental methods, and data are provided that demonstrate a technique useful for antibody generation. In an exemplary embodiment, the method involves identifying antigen-specific antibodies directly from the peripheral blood of a living non-human animal. Advantageously, such methods provided herein allow for antibody generation without the need to kill the animal, as opposed to traditional methods that involve harvesting immune organs (e.g., spleen, lymph nodes, and bone marrow) after animal euthanasia. Because such methods are non-terminal (e.g., do not involve euthanasia of the antibody-producing animal), the method can be repeated multiple times on the same animal, for example, until the desired antibody is obtained. The ability to repeat the method on the same animal offers several advantages over traditional methods. For example, repeating the method on the same animal reduces the cost of the overall antibody generation process. Also, because the animal is alive, the disclosed methods allow for real-time, in-life sampling of the antibody repertoire, and strategic adjustments can be made to the immunization protocol (used for subsequent immunizations) based on the B cell response observed (in a previous immunization), for example, if the animal does not produce B cells expressing the desired target antibody. The method of the present invention thereby allows for rational repertoire formation and / or steering of immune responses with intent to meet antibody design goals. An exemplary process of the present disclosure is shown in Figures 1B-1E. Figure 1B shows an exemplary non-terminal method for monitoring immune responses, including screening single cells obtained from a blood sample of an immunized animal. Based on the results of the single cell screening, the animal may be subjected to repeated rounds of immunization (e.g., another immunization), followed by single cell screening of cells from a blood sample obtained from the immunized animal, or tissue may be harvested if the screening determines that the animal exhibits a desired phenotype. Figure 1C shows an exemplary non-terminal method for monitoring the production of selected antibodies, in which antibody-secreting cells (ASCs) purified from a blood sample obtained from an immunized animal are screened at the single cell level. The process is repeated until design goals are achieved and / or selected antibodies are produced.FIG. 1D shows an exemplary non-terminal method of inducing antibody production to produce a selected antibody, in which an animal is immunized using a primary strategy and ASCs obtained from PBMCs isolated from the immunized animal are screened for the desired phenotype. If the screen determines that the design goal has not been achieved, the animal is immunized with another strategy (e.g., different from the primary strategy) and ASCs obtained from PBMCs isolated from the immunized animal are screened for the desired phenotype. This process is repeated until the screen determines that the design goal has been achieved. If the design goal has been achieved, a final tissue can be harvested for antibody rescue using hybridomas, single cell platforms, or sequence-based generation. FIG. 1E shows an exemplary non-terminal method of screening animals and B cell profiling, in which a series of animals are immunized with an immunogen and ASCs obtained from blood samples obtained from each animal are screened and the B cell repertoire is profiled. In various aspects of the exemplary process, antibody secreting cells (ASCs), e.g., plasmablasts, are purified from the peripheral blood of immunized mice and then screened at single cell resolution for relevant activities or phenotypes. Compared to the traditional hybridoma production process (shown in FIG. 1A), which typically requires about 8 weeks and requires a high degree of technical skill, the disclosed process is less labor intensive and takes less time.

[0008] Thus, the present disclosure provides a method for monitoring the production of a selected antibody in a non-human animal. In an exemplary embodiment, the method includes (a) immunizing a non-human animal with an immunogen; (b) obtaining a blood sample from the non-human animal that contains antibody-secreting cells (ASC); and (c) assaying, e.g., individually, the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody. In various examples, the method further includes repeating (b) and (c) one or more times until a design goal is achieved, e.g., until the selected antibody is produced. Figure 1C illustrates this exemplary embodiment of the present disclosure. The present disclosure also provides a method for inducing antibody production in a non-human animal for the generation of a selected antibody. In an exemplary embodiment, the method comprises: (a) performing an initial immunization of a non-human animal with an immunogen; (b) obtaining a blood sample from the non-human animal comprising ASCs; (c) assaying, e.g., individually assaying, the ASCs present in the blood sample or a fraction thereof for the production of a select antibody; and (d) performing a cycle of steps if the proportion of ASCs producing the select antibody is below a threshold, the cycle comprising: (i) performing a subsequent immunization of the non-human animal with the immunogen if the proportion of ASCs producing the select antibody is below a threshold; (ii) obtaining a blood sample from the non-human animal comprising ASCs; and (iii) assaying, e.g., individually assaying, the ASCs present in the blood sample or a fraction thereof for the production of a select antibody.

[0009] In various embodiments, assaying includes single cell live cell assay. As used herein, the phrase "assaying ASCs individually" means assaying or analyzing ASCs at a single cell level or single cell resolution. In an exemplary embodiment, "assaying ASCs individually" provides results related to a single ASC. Optionally, multiple ASCs are assayed simultaneously. In various embodiments, multiple ASCs are assayed simultaneously individually. In an exemplary embodiment, a blood sample is obtained from a non-human animal in a non-terminal manner, e.g., the non-human animal is not killed during blood sample collection. In an exemplary embodiment, the method includes performing a non-terminal blood draw from the non-human animal. In various embodiments, the method includes applying the blood sample or a fraction thereof to a matrix and assigning a unique address of the matrix to each ASC. The result of the assay is optionally the identification of each ASC producing a selected antibody. In a particular embodiment, the result of the assay is the identification of a unique address of each ASC producing a selected antibody. In an illustrative example, the method includes at least one cycle of (i) performing a subsequent immunization of the non-human animal with an immunogen if the proportion of ASCs producing the selected antibody is below a threshold value, (ii) obtaining a blood sample from the non-human animal containing ASCs, and (iii) assaying, e.g., individually, the ASCs present in the blood sample or a fraction thereof for production of the selected antibody. Optionally, the cycle is repeated until the proportion of ASCs producing the selected antibody in the assay of (iii) is equal to or greater than the threshold value. In various examples, the cycle is repeated at least twice.

[0010] The immunogen of the subsequent immunization may be different from the immunogen of the first immunization in an exemplary embodiment. For example, in an exemplary embodiment, each subsequent immunization is different from the previous immunization in that (A) a different immunogen, adjuvant, and / or immunomodulator is administered to the non-human animal, (B) a different dose of immunogen is administered to the non-human animal, (C) the time between each administration of immunogen, adjuvant, immunomodulator is different, and / or (D) the route of each administration of immunogen, adjuvant, immunomodulator is different. Optionally, a different immunogen is used each time the non-human animal is immunized. Figure ID shows an exemplary method of inducing antibody production for the production of a selected antibody.

[0011] The present disclosure further provides a method of producing a selected antibody in a non-human animal, in an exemplary embodiment, the method comprises inducing antibody production in the non-human animal for the production of a selected antibody according to a method of the present disclosure for inducing antibody production, and then isolating the selected antibody and / or the ASC that produces the selected antibody. In an exemplary embodiment, the method comprises: (a) performing an initial immunization campaign on a non-human animal with an immunogen; (b) obtaining a blood sample from said non-human animal comprising antibody secreting cells (ASC); (c) assaying, e.g., individually assaying, the ASCs present in the blood sample or a fraction thereof for the production of a select antibody; and (d) performing a cycle of steps if the proportion of ASCs producing the select antibody is below a threshold, wherein the cycle comprises: (i) performing a subsequent immunization of the non-human animal with the immunogen if the proportion of ASCs producing the select antibody is below a threshold; (ii) obtaining a blood sample from said non-human animal comprising ASCs; and (iii) assaying, e.g., individually assaying, the ASCs present in the blood sample or a fraction thereof for the production of the select antibody; and (e) isolating the select antibody and / or the ASCs producing the select antibody. In various embodiments, the method includes (f) determining a nucleotide sequence encoding a heavy chain variable region of a selected antibody produced by an ASC (e.g., an isolated ASC producing a selected antibody) and a nucleotide sequence encoding a light chain variable region of the selected antibody produced by the ASC; (g) introducing into a host cell a first vector comprising a nucleotide sequence encoding the heavy chain variable region of the selected antibody and a second vector comprising a nucleotide sequence encoding the light chain variable region of the selected antibody; and (h) isolating the antibody produced by the host cell.

[0012] In an exemplary embodiment, the assay of the disclosed method includes (a) combining the ASC in the matrix with a reagent that binds to the selected antibody and generates a detectable signal, e.g., a fluorescent signal, upon binding to the selected antibody. In various embodiments, the assay of the disclosed method includes (a) combining the ASC in the matrix with at least one reagent that binds to the Fc domain of the selected antibody and at least one reagent to which the selected antibody binds (e.g., a reagent that binds to the antigen-binding domain of the selected antibody), at least one of which is coupled to a detectable label. In an exemplary example, the ASC is combined with a detection reagent that binds to the Fc domain of the selected antibody and includes a first detectable label, and a target to which the selected antibody binds (e.g., a reagent that binds to the antigen-binding domain of the selected antibody). Figures 2A-2C show exemplary assays in the context of the disclosed method. In various examples, the target is labeled with a second detectable label that is different from the first detectable label. In some examples, a capture reagent that binds to the Fc domain of the selected antibody and includes a solid support is further combined with the ASC, the detection reagent, and the labeled target. In various examples, the method further comprises (b) assaying for the first detectable label and the second detectable label; and (c) identifying locations within the matrix where the first detectable label and the second detectable label are detected, where each identified location represents the location of an individual ASC producing a select antibody. Figures 2A and 2B show an exemplary assay using such a labeled target and capture reagent. Figure 2A shows the matrix as a well. Figure 2B shows the matrix as a multi-pen tip or multi-well plate, where each ASC is placed within a single pen or well. In various examples, the target is expressed by cells, and the cells expressing the target are combined with the ASC and the detection reagent. In an exemplary embodiment, the method further comprises (b) assaying for the first detectable label, and (c) identifying locations within the matrix where the first detectable label is detected, where each identified location represents the location of an individual ASC producing a select antibody. Figure 2C shows an exemplary assay using cells expressing such a target.In an exemplary embodiment, the assay of the disclosed method includes: (a) combining the ASC in the matrix with (i) a capture reagent that binds to the selected antibody and includes a solid support; (ii) a detection reagent that binds to the selected antibody and includes a first detectable label; and (iii) a labeled target to which the selected antibody binds (wherein the labeled target includes a second detectable label that is different from the first detectable label); (b) assaying for the first detectable label and the second detectable label; and (c) identifying locations in the matrix where both the first detectable label and the second detectable label are detected (wherein each identified location represents the location of an individual ASC that produces the selected antibody). Optionally, the capture agent includes an antibody that binds to the antibody Fc domain bound to the solid support. The detection agent, in an exemplary embodiment, includes an antibody that binds to the antibody Fc domain bound to the first detectable label. In various embodiments, the antibody that binds to the antibody Fc domain of the capture agent is the same antibody as the detection agent. In an exemplary embodiment, the combining is performed in a well, and the capture agent forms a monolayer in the well. In various embodiments, the method includes identifying locations within the well where both the first detectable label and the second detectable label are detected, where each identified location represents the location of an individual ASC that produces a selected antibody.

[0013] The present disclosure further provides a single cell assay for identifying ASCs that produce select antibodies.The present disclosure provides a method of assaying for ASCs that produce select antibodies. In an exemplary embodiment, the assay or method comprises: (a) combining in a well: (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen (wherein the blood sample comprises ASCs); (ii) a detection reagent that binds to the selected antibody and comprises a first detectable label; and (iii) a target to which the selected antibody binds (wherein (A) the target is a labeled target that comprises a second detectable label different from the first detectable label, and a capture reagent that binds to the selected antibody and comprises a solid support are further combined in the well to form a monolayer in the well, or (B) the target is expressed on the surface of a cell, and the cells are combined in the well to form a monolayer in the well); (b) assaying for the first detectable label, and optionally, if the target is a labeled target, assaying for the second detectable label; and (c) identifying a location in the well where the first detectable label is detected, or where the first and second detectable labels are detected (wherein each identified location indicates the location of an individual ASC producing the selected antibody). In various embodiments, the assay or method comprises: (a) combining in a well (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen; (ii) a capture reagent comprising an antibody that binds to the Fc of an antibody bound to a solid support; (iii) a detection reagent comprising an antibody that binds to the Fc of the antibody bound to a first detectable label; and (iv) a labeled target comprising the immunogen or a portion thereof bound to a second detectable label different from the first detectable label (wherein the capture reagent forms a monolayer in the well); (b) assaying for the first detectable label; (c) assaying for the second detectable label; and (d) identifying locations in the well where both the first detectable label and the second detectable label are detected (wherein each identified location indicates the location of an individual ASC producing a selected antibody).In various embodiments, the assay or method includes (a) combining in a well (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen, (ii) a detection reagent that binds to the selected antibody, and (iii) cells that express a target bound by the selected antibody on their cell surface, the cells combining in the well to form a monolayer in the well; (b) assaying for a first detectable label; and (c) identifying locations in the well where both the first detectable label and the second detectable label are detected (where each identified location indicates the location of an individual ASC that produces the selected antibody).

[0014] In various embodiments of the disclosed method, the non-human animal is not subjected to removal of one or more secondary lymphoid organs or euthanasia. Also, in various examples, the ASCs from the blood sample are not used to generate hybridomas. In an exemplary embodiment, the non-human animal is one of a set of non-human animals, and the result of the assay is the identification of non-human animals in which the proportion of ASCs producing the selected antibody is below a threshold and / or which require further immunization. In another embodiment, the method includes sacrificing the non-human animal and harvesting tissue from the non-human animal if the proportion of ASCs producing the selected antibody is equal to or greater than a threshold. In various examples, the steps of the method are performed on a set of non-human animals, and the method includes profiling the B cell repertoire of the blood sample for each set of non-human animals and selecting a set of subsets having a target B cell profile. FIG. 1E illustrates such steps.

[0015] Rational immune repertoire generation and selection is a key component in animal-based antibody engineering. Despite advances from traditional B cell immortalization to direct B cell platforms such as (but not limited to) NanOBLAST (a nanofluidic Beacon® instrumented antibody engineering process) and microencapsulation, the diversity and quality of input B cells remain essential determinants in achieving antibody design goals. Traditional approaches to evaluate immunized animals rely on polyclonal secreted antibody (serum) testing to assess immune responses and select animals for B cell harvesting and antibody generation. The "serum titer" approach is less than ideal as it measures the total reactivity of all secreted antibodies and not the quality of the individual B cell sources of the detected antibodies. The lack of a direct correlation between antibody titer measurements and the critical B cell sources makes the quality of the B cell response difficult to interpret. Other than determining whether soluble antigen-specific antibodies are present in the serum, it is difficult to obtain additional useful information from this polyclonal analysis to aid in animal selection or immune steering strategies. Addressing these issues, an ASC assay is provided herein for investigating B cell responses of immunized animals using a sample derived from non-terminal peripheral blood. Thus, the present disclosure provides a method for screening non-human animals for antibody secreting cells (ASCs) that produce a select antibody. In an exemplary embodiment, the method comprises: (a) immunizing a series of non-human animals with an immunogen; (b) obtaining a blood sample containing ASCs from each non-human animal in the series; and (c) individually assaying the ASCs present in the blood sample or a fraction thereof for production of the select antibody, and for each non-human animal in the series, the proportion of ASCs that produce the select antibody is determined. In various embodiments, the screening method further comprises selecting the non-human animal for sacrifice and / or tissue harvesting if the proportion of ASCs that produce the select antibody is equal to or greater than a threshold. In various embodiments, the screening method further comprises selecting the non-human animal for subsequent immunization if the proportion of ASCs that produce the select antibody is less than a threshold.Thus, in various embodiments, the screening method distinguishes between animals to be killed and those to be subsequently immunized based on the proportion of ASCs producing the selected antibody.

[0016] Consistent with the above, a method for selecting an immunized non-human animal for subsequent immunization is provided. In an exemplary embodiment, the method comprises monitoring the production of a selected antibody in the non-human animal according to any one of the methods disclosed herein, the method being carried out on a series of non-human animals, the number of ASCs producing the selected antibody being identified for each series of non-human animals, and if the proportion of ASCs producing the selected antibody for the animal is below a threshold, the animal is selected for subsequent immunization. Also provided herein is a method for selecting an immunized non-human animal for euthanasia and secondary lymphoid collection. In an exemplary embodiment, the method comprises monitoring the production of a selected antibody in the non-human animal according to any one of the methods disclosed herein, the method being carried out on a series of non-human animals, the number of ASCs producing the selected antibody being identified for each series of non-human animals, and if the proportion of ASCs producing the selected antibody for the animal is equal to or greater than a threshold, the animal is selected for euthanasia and secondary lymphoid collection. [Brief description of the drawings]

[0017] [Figure 1A] FIG. 1A illustrates a conventional method for producing transgenic mouse hybridomas. FIG. 1B illustrates a non-terminal method for monitoring immune responses. FIG. 1C illustrates a non-terminal method for monitoring production of select antibodies. FIG. 1D illustrates a non-terminal method for inducing antibody production for the production of select antibodies. FIG. 1E illustrates a non-terminal method for animal screening and B cell profiling. [Figure 1B]FIG. 1A illustrates a conventional method for producing transgenic mouse hybridomas. FIG. 1B illustrates a non-terminal method for monitoring immune responses. FIG. 1C illustrates a non-terminal method for monitoring production of select antibodies. FIG. 1D illustrates a non-terminal method for inducing antibody production for the production of select antibodies. FIG. 1E illustrates a non-terminal method for animal screening and B cell profiling. [Figure 1C] FIG. 1A illustrates a conventional method for producing transgenic mouse hybridomas. FIG. 1B illustrates a non-terminal method for monitoring immune responses. FIG. 1C illustrates a non-terminal method for monitoring production of select antibodies. FIG. 1D illustrates a non-terminal method for inducing antibody production for the production of select antibodies. FIG. 1E illustrates a non-terminal method for animal screening and B cell profiling. [Figure 1D] FIG. 1A illustrates a conventional method for producing transgenic mouse hybridomas. FIG. 1B illustrates a non-terminal method for monitoring immune responses. FIG. 1C illustrates a non-terminal method for monitoring production of select antibodies. FIG. 1D illustrates a non-terminal method for inducing antibody production for the production of select antibodies. FIG. 1E illustrates a non-terminal method for animal screening and B cell profiling. [Figure 1E] FIG. 1A illustrates a conventional method for producing transgenic mouse hybridomas. FIG. 1B illustrates a non-terminal method for monitoring immune responses. FIG. 1C illustrates a non-terminal method for monitoring production of select antibodies. FIG. 1D illustrates a non-terminal method for inducing antibody production for the production of select antibodies. FIG. 1E illustrates a non-terminal method for animal screening and B cell profiling. [Figure 2A]Figure 2A illustrates the application of an exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2B illustrates another exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2C illustrates yet another exemplary single-cell assay for identifying ASCs that produce select antibodies. [Figure 2B] Figure 2A illustrates the application of an exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2B illustrates another exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2C illustrates yet another exemplary single-cell assay for identifying ASCs that produce select antibodies. [Figure 2C] Figure 2A illustrates the application of an exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2B illustrates another exemplary single-cell assay for identifying ASCs that produce select antibodies. Figure 2C illustrates yet another exemplary single-cell assay for identifying ASCs that produce select antibodies. [Diagram 3] Fig. 3 is an explanatory diagram of an antibody that binds to an anti-idiotope antibody, showing the paratope, idiotype, and idiotope. [Figure 4] FIG. 4 is a graph of polyclonal titers of sera obtained from the indicated mice immunized with Antibody 1. [Figure 5A] Figure 5A illustrates the components of an exemplary single cell screen, and Figure 5B illustrates how the components of the single cell assay interact in the presence of an antibody that binds to an antigen. Figure 5C illustrates individual pens holding ASC-secreted antibodies that interact with polystyrene beads to create a fluorescent "bloom." IgG secretion and antigen-specific antibodies are detected by the assay. [Figure 5B]Figure 5A illustrates the components of an exemplary single cell screen, and Figure 5B illustrates how the components of the single cell assay interact in the presence of an antibody that binds to an antigen. Figure 5C illustrates individual pens holding ASC-secreted antibodies that interact with polystyrene beads to create a fluorescent "bloom." IgG secretion and antigen-specific antibodies are detected by the assay. [Figure 5C] Figure 5A illustrates the components of an exemplary single cell screen, and Figure 5B illustrates how the components of the single cell assay interact in the presence of an antibody that binds to an antigen. Figure 5C illustrates individual pens holding ASC-secreted antibodies that interact with polystyrene beads to create a fluorescent "bloom." IgG secretion and antigen-specific antibodies are detected by the assay. [Figure 6] FIG. 6 illustrates the dual bloom on individual pens holding single cells, export of the cells to wells, and PCR analysis for antibody cloning, expression, purification and analysis. [Figure 7A-C] Figure 7A illustrates the sandwich ELISA format used to select appropriate antibody pairs. Figure 7B is a graph of ELISA signal plotted as a function of antibody 1 concentration. Figure 7C is a graph of PD1 function plotted as a function of antibody concentration. [Figure 8A-D] Figure 8A is an image of a green fluorescent spot where ASC secreting antibody is located in a single well. Figure 8B is an image of a red fluorescent spot where antigen-specific antibody secreted by ASC is located in a single well. Figure 8C is an image of a colored spot where ASC secreting antibody is located in a single well, a spot where antigen-specific antibody secreted by ASC is located in a single well, and a spot where ASC secreting antigen-specific antibody is located in a single well. Figure 8D is an exemplary image of a labeled transfected cell with multiple fluorescent spots where an antigen expressed by 293T cells binds to an antibody produced by B cells and labeled with an Alexa 488-labeled goat anti-human Fc antibody. [Figure 9] FIG. 9 is a series of images of single cells of the indicated hybridoma clones (or unrelated clones), where RFU in the green channel (top) represents antibody secretion or RFU in the red channel (bottom) represents antigen (EGFR) binding. [Figure 10] FIG. 10 is a graph of the RFU green / RFU red ratio plotted as a function of the KD of the hybridoma. [Figure 11] Figure 11 is a schematic diagram of the immunization protocol used for all mice, with timing of bleeds and antigen transfers indicated. [Figure 12] Figure 12 is a graph of serum titers from the first bleed of mice in Groups 1 and 2. The graph plots human antigen titers versus cynomolgus antigen titers. [Figure 13] Figure 13 is a series of images of single cells with RFU on the red channel showing human antigen binding (left), green channel showing cynomolgus antigen binding (center), and combined channel showing human and cynomolgus antigen binding (right). Data for serum obtained from bleed 1. [Figure 14] Figure 14 is a graph of the percentage of antigen positive ASCs from mice in Group 1 (closed circles) and Group 2 (open circles) that react to human antigens only, cynomolgus antigens only, or both human and cynomolgus antigens. Data for cells from bleed 1 obtained by single cell Incucyte screening. [Figure 15] Figure 15 is a graph of the percentage of antigen positive ASCs in Group 1 (closed circles) and Group 2 (open circles) that react to human antigens only, cynomolgus antigens only, or both human and cynomolgus antigens. Data for cells from bleed 2 obtained by single cell Incucyte screening. [Figure 16]Figure 16 is a graph of the percentage of antigen positive ASCs in Group 1A (human boost) and Group 1B (cyno boost) that respond to human antigens only, cyno antigens only, or both human and cyno antigens. Data for cells from bleed 2 are shown in filled circles and data for cells from bleed 3 are shown in open circles. Data obtained by single cell Incucyte screening. [Figure 17] Figure 17 is a graph of the change in cross-reactive ASC frequency (relative to bleed 1) for Group 1A (human boost) and Group 1B (cyno boost). Data obtained by single cell Incucyte screening. [Figure 18] Figure 18 is a graph of serum titers of reactivity to cynomolgus antigens plotted as a function of serum titers of reactivity to human antigens. The percentage of cross-reactive ASC is indicated. Target animals selected for harvest are circled in red. [Figure 19] FIG. 19 is a schematic diagram of an immunization campaign involving immune steering towards the production of human / cynomolgus cross-reactive antibodies that bind to both the human and cynomolgus subdomain orthologs of a multidomain protein (antigen). [Figure 20] Figure 20 is a graph of serum titers of reactivity to cynomolgus antigens plotted as a function of serum titers of reactivity to human antigens. Serum from bleed 1. [Figure 21] Figure 21 is a series of images of single cells in RFU on green and red channels at t=0 hr (bottom) and t=23 hr (top) for human only binders, cyno only binders, and human / cyno cross-reactive binders. Data for serum obtained from bleed 1 using single cell Incucyte screening. [Figure 22] Figure 22 is a graph of the percentage of antigen positive ASCs reacting to human antigens only, cynomolgus antigens only, or both human and cynomolgus antigens. Data is shown for cells from bleed 1. Data obtained by single cell Incucyte screening. [Diagram 23]FIG. 23 is a graph of the change in cross-reactive ASC frequency (to an unrelated clone) in serum from bleed 1 and bleed 3. [Figure 24] Figure 24 is a graph of the percentage of ASCs secreting antibodies reactive to cynomolgus antigens only (filled circles) or both cynomolgus and human antigens (open squares) in bleed 1 and bleed 3. Data obtained by single cell Incucyte screening. [Diagram 25] Figure 25 is a graph of the percent of human-cynomolgus cross-reactive binders. Target animals selected for harvesting are indicated by boxes. Data obtained from single cell Incucyte screening. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Non-terminal monitoring and steering of B cell function and antibody production Antigen-specific B cells that have recently encountered antigen in the germinal centers (GCs) of secondary lymphoid organs (e.g., spleen and lymph nodes) are stimulated to divide and differentiate along multiple pathways. See, e.g., Klein and Dalla-Favera, Nature Reviews Immunol 8:22-33 (2008). The primary B cell lineage that secretes antibodies into serum in response to antigen challenge is the plasma cell. Plasma cell differentiation begins in secondary lymphoid organs, where cell-cell interactions within GCs drive B cells expressing antigen-specific antibodies on their surface to differentiate into immature plasma cells known as plasmablasts. Plasmablasts are rapidly dividing B cells that produce and secrete soluble antibodies. However, plasmablasts are transient in nature and require significant trophic support to continue to survive and proliferate. The primary survival niche for plasmablasts is the secondary lymphoid organs, but these cues are provisional and dependent on the presence of cognate antigens.

[0019] B cells use two major strategies to maintain long-term humoral memory for antigens: the formation of IgG+ memory B cells and the formation of long-lived mature plasma cells. Memory B cells express a cell surface-bound form of their cognate antibody, known as the B cell receptor (BCR), but do not secrete soluble antibody. These cells are found in various locations in the body and are abundant in secondary lymphoid organs. Upon re-encounter with antigen, memory B cells can be induced to proliferate (i.e., generate clones of themselves) and differentiate into antibody-secreting plasma cells. The other route to long-term memory is through the formation of long-lived mature plasma cells. Mature plasma cells require very specialized survival niches that provide nutritional support and can be found in inflamed tissues, specialized structures associated with the gut (gut-associated lymphoid tissue - GALT), and in the bone marrow. See, e.g., Fairfax et al., Semin Immunol 20(1):49-58 (2008). The local environment created by niche stromal cells provides the signals necessary to maintain the lifespan of terminally differentiated plasma cells.

[0020] For B cells to settle in stromal niches long-term, they must migrate to these destinations by blood. Indeed, after exposure to antigen in GCs, differentiation into plasmablasts, and subsequent proliferation in secondary lymphoid organs, a wave of migrating plasmablasts can be detected in the circulating blood. In mice, this surge of plasmablasts in the blood occurs 3-7 days after antigen exposure and decreases over time as they settle in appropriate niches and differentiate into long-lived plasma cells.

[0021] Provided herein are methods that involve capturing recent antigen-stimulated plasmablasts and plasma cells (antibody secreting cells, (ASC)) as they travel through the blood and identifying those cells that produce an antibody of interest, e.g., an antibody of choice. The disclosed methods utilize blood samples, which are advantageously less complex, as the cellular environment of blood is substantially less complex than that of secondary lymphoid organs, particularly from the standpoint of B cell lineage. The disclosed methods address the difficulty of accessing this population of ASCs, which has historically been challenging due to their relatively low overall abundance.

[0022] Thus, the present disclosure provides a method of monitoring the production of a selected antibody in a non-human animal. In an exemplary embodiment, the method comprises: (a) immunizing a non-human animal with an immunogen; (b) obtaining a blood sample from said non-human animal comprising antibody secreting cells (ASC); and (c) assaying (optionally individually) the ASC present in the blood sample or a fraction thereof for the production of the selected antibody. The present disclosure also provides a method of inducing antibody production in a non-human animal for the generation of a selected antibody. In an exemplary embodiment, the method comprises: (a) performing an initial immunization campaign on a non-human animal with an immunogen; (b) obtaining a blood sample from said non-human animal comprising antibody-secreting cells (ASC); (c) assaying (optionally assaying individually) the ASCs present in the blood sample or a fraction thereof for production of a select antibody; and (d) performing a cycle of steps if the proportion of ASCs producing the select antibody is below a threshold, wherein the cycle comprises: (i) performing a subsequent immunization of the non-human animal with the immunogen if the proportion of ASCs producing the select antibody is below a threshold; (ii) obtaining a blood sample from said non-human animal comprising ASCs; and (iii) individually assaying the ASCs present in the blood sample or a fraction thereof for production of a select antibody. In exemplary embodiments, the threshold is about 1% to about 10%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In exemplary embodiments, the threshold is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In other embodiments, the threshold is greater than 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0023] In an exemplary embodiment of such a method, the non-human animal is not euthanized or one or more secondary lymphoid organs are removed, or the secondary lymphoid organs are euthanized and harvested only after the animal is deemed to have a sufficient number of ASCs that produce the antibody of interest, e.g., the selected antibody. In an exemplary embodiment, the method is carried out using a series of non-human animals. In various examples, the result of the assay is the identification of a series of non-human animals in which the proportion of ASCs that produce the selected antibody is below a threshold and / or which require further immunization. Such non-human animals can then be subjected to a cycle of steps ((d), supra), e.g., to increase production of the selected antibody.

[0024] In another example, the result of the assay is the identification of a set of non-human animals in which the proportion of ASCs producing the selected antibody is equal to or greater than a threshold value. Such non-human animals can then be killed and tissues harvested from such non-human animals. In another embodiment, if the proportion of ASCs producing the selected antibody relative to the total number of ASCs assayed is equal to or greater than a threshold value, the method includes killing the non-human animal and harvesting tissues from the non-human animal.

[0025] Thus, the disclosed methods of monitoring and directing or steering the production of a selected antibody are highly efficient, as fewer animals are unnecessarily killed (e.g., animals with a percentage of ASCs producing the selected antibody below a threshold) and a higher percentage of immunized animals ultimately produce the selected antibody. Also, in various examples, such disclosed methods do not involve generating hybridomas, and therefore advantageously consume less time and materials.

[0026] immunization In various embodiments of the present disclosure, the method includes immunizing a non-human animal with an immunogen. As used herein, the term "immunization" refers to carrying out or performing an "immunization campaign" or "immunization protocol" or "campaign" to mount an immune response to said immunogen. In exemplary embodiments, the immune response includes a B cell immune response and / or a humoral immune response to said immunogen. In exemplary embodiments, the immune response mounted in the non-human animal includes production of antibody-secreting cells (ASCs), e.g., antibody-secreting plasma cells, plasmablasts, plasma cells (e.g., rapidly dividing B cells that produce and secrete high levels of soluble antibodies). In various examples, the immune response includes migratory ASCs (e.g., plasma cells, plasmablasts) that travel in the blood to secondary lymphoid organs. In various embodiments, the secondary lymphoid organs are lymph nodes (e.g., popliteal, inguinal, mesenteric, and brachial), spleen, Peyer's patches, or mucosal tissue. In illustrative examples, ASCs are produced about 1 to 7 days after antigen exposure. In some cases, ASCs, e.g., migrating plasmablasts, are found in the blood about 3 to about 7 days (e.g., about 3, about 4, about 5, about 6, about 7 days) after antigen exposure. In some examples, ASCs, e.g., migrating plasmablasts, are found in the blood about 8, about 9, or about 10 days after antigen exposure.

[0027] Suitable techniques for immunizing non-human animals are known in the art. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice, 3 rded., Academic Press Limited, San Diego, CA, 1996. Gene gun methods described, for example, in Barry et al., Biotechniques. 16(4):616-8, 620 (1994); Tang et al., Nature. 12; 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) may also be used to immunize non-human animals. Further, as exemplified herein, immunization can include administering to the non-human animal cells expressing the antigen, or administering antigen-loaded dendritic cells, tumor cell vaccines, or immune cell-based vaccines. See, e.g., Sabado et al., Cell Res 27(1):74-95(2017); Bot et al., “Cancer Vaccines” in Plotkin's Vaccines, 7 thed., Editors: Plotkin et al., Elsevier Inc., 2018, and Lee and Dy, "The Current Status of Immunotherapy in Thoraic Malignancies" in Immune Checkpoint Inhibitors in Cancer, Editors: Ito and Ernstoff, Elsevier Inc., 2019. In various examples, immunization can be performed by microneedle delivery (see, e.g., Song et al., Clin Vaccine Immunol 17(9):1381-1389(2010)), virus-like particles (VLPs) (see, e.g., Temchura et al., Viruses 6(8):3334-3347(2014)), or 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). Further strategies for immunization and immunogen preparation, including, for example, the addition of T cell epitopes to antigens, are described in Chen and Murawsky, Front Immunol 9:460 (2018).

[0028] In various embodiments, the method includes immunizing the non-human animal with an immunogen, the immunogen being administered to the non-human animal one or more times (e.g., two, three, four, five, or more times). In various embodiments, the immunogen is administered by injection, e.g., intraperitoneal, subcutaneous, intramuscular, intradermal, or intravenous injection. In various embodiments, the method includes immunizing the non-human animal by administering a series of injections of the immunogen. In exemplary embodiments, each administration, e.g., injection, is administered to the non-human animal at intervals of about 10 to about 18 days, optionally at intervals of about 12 to about 16 days, or at intervals of about 14 days. In exemplary embodiments, each administration, e.g., injection, is administered to the non-human animal more frequently than at intervals of about 10 to about 18 days. For example, in an exemplary embodiment, the administration interval of the immunogen to the non-human animal is about 1 day to about 9 days, optionally about 1 day to about 8 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 9 days, about 3 days to about 9 days, about 4 days to about 9 days, about 5 days to about 9 days, about 6 days to about 9 days, about 7 days to about 9 days, about 8 days to about 9 days, about 4 days to about 8 days, about 5 days to about 8 days, or about 6 days to about 8 days. The administration interval of the immunogen to the non-human animal may be even longer in various embodiments. For example, the administration interval of the immunogen to the non-human animal may be about 1 week to about 20 weeks or more, for example, about 1 month to about 20 months. Optionally, the interval between administration of the immunogen to the non-human animal is about 1 week to about 19 weeks, about 1 week to about 18 weeks, about 1 week to about 17 weeks, about 1 week to about 16 weeks, about 1 week to about 15 weeks, about 1 week to about 14 weeks, about 1 week to about 13 weeks, about 1 week to about 12 weeks, about 1 week to about 11 weeks, about 1 week to about 10 weeks, about 1 week to about 9 weeks, about 1 week to about 8 weeks, about 1 week to about 7 weeks, about 1 week to about 6 weeks, about 1 week to about 5 weeks, about 1 week to about 4 weeks, about 1 week to about 3 weeks, about 1 week to about 2 weeks. , about 2 weeks to about 20 weeks, about 3 weeks to about 20 weeks, about 4 weeks to about 20 weeks, about 5 weeks to about 20 weeks, about 6 weeks to about 20 weeks, about 7 weeks to about 20 weeks, about 8 weeks to about 20 weeks, about 9 weeks to about 20 weeks, about 10 weeks to about 20 weeks, about 11 weeks to about 20 weeks, about 12 weeks to about 20 weeks, about 13 weeks to about 20 weeks, about 14 weeks to about 20 weeks, about 15 weeks to about 20 weeks, about 16 weeks to about 20 weeks, about 17 weeks to about 20 weeks, about 18 weeks to about 20 weeks, or about 19 weeks to about 20 weeks.In various embodiments, the interval between administrations of the immunogen may be longer than 8 or 9 days. Optionally, the interval between administrations of the immunogen is about 1 month to about 8 months, about 1 month to about 7 months, about 1 month to about 6 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 2 months to about 9 months, about 3 months to about 9 months, about 4 months to about 9 months, about 5 months to about 9 months, about 6 months to about 9 months, about 7 months to about 9 months, about 8 months to about 9 months, about 4 months to about 8 months, about 5 months to about 8 months, or about 6 months to about 8 months.

[0029] In various examples, during immunization, each administration (e.g., injection) of the immunogen is performed with the same (A) immunogen, adjuvant, immunomodulator, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulator, or combination thereof, (C) administration route or method for delivering the immunogen, (D) administration site to the non-human animal, or (E) combination thereof. Alternatively, during immunization, each administration (e.g., injection) of the immunogen is performed with a different (A) immunogen, adjuvant, immunomodulator, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulator, or combination thereof, (C) administration route or method for delivering the immunogen, (D) administration site to the non-human animal, or (E) combination thereof. Optionally, the amount of the immunogen is reduced or increased by subsequent administrations, e.g., injections. In some aspects, every other administration, e.g., injection, contains a reduced or increased amount of the immunogen compared to the first and third injections. Exemplary immunizations are described in the Examples provided herein.

[0030] Non-human animals Advantageously, the methods of the present disclosure are not limited to any particular non-human animal. The non-human animal in exemplary aspects is any non-human mammal. In exemplary embodiments, the non-human animal is a mammal, including but not limited to mammals of the order Rodentia, such as mice, rats, guinea pigs, gerbils, and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals of the order Carnivora, including Felidae (cats) and Canidae (dogs), mammals of the order Artiodactyla, including Bovidae (cattle) and Suidae (pigs), or mammals of the order Perssodactyla, including Equidae (horses). In some aspects, the mammal is a mammal of the order Primates, Ceboids, or Simoids (monkeys), or a mammal of the order Anthropoids (great apes). In various embodiments, the non-human animal is a goat, a llama, an alpaca, a chicken, a duck, a fish (eg, salmon), a sheep, or a lamb.

[0031] In illustrative examples, the non-human animals used in the methods of the present disclosure are modified, e.g., genetically modified, to produce chimeric or fully human antibodies. Such non-human animals are referred to as transgenic animals. The production of human antibodies in transgenic animals is described in Bruggemann et al., Arch Immunol Ther Exp(Warsz) 63(2):101-108(2015). Any transgenic animal can be used in the present invention, including, but not limited to, transgenic chickens (e.g., OmniChicken®), transgenic rats (e.g., OmniRat®), transgenic llamas, and transgenic cows (e.g., Tc Bovine™). In certain embodiments, the non-human animal is a transgenic mouse, such as XenoMouse®, Alloy mouse, Trianni mouse, OmniMouse®, and HuMAb-Mouse®. The XenoMouse® is a species of transgenic mouse that produces fully human antibodies. A review of the XenoMouse® is provided by Foltz et al., Immunol Rev 270(1):51-64 (2016) and U.S. Pat. No. 5,939,598. In an exemplary embodiment, the non-human animal is a transgenic rat. In various embodiments, the transgenic rat is a Unirat® or OmniFlic®, which are described in Clarke et al., Front Immunol 9:3037 (2019); doi:10.3389 / fimmu.2018.03037 and Harris et al., Front Immunol 9:889 (2018): doi:10.3389 / fimmu.2018.00889, respectively.

[0032] In an exemplary embodiment, the method of the present disclosure is non-terminal with respect to a non-human animal. As used herein, the term "non-terminal" in the context of a non-human animal means that the life of the non-human animal is not terminated (e.g., not euthanized or otherwise killed or caused to die) while the method is being performed. In an exemplary embodiment, the non-human animal is not subjected to removal or euthanasia of one or more secondary lymphoid organs, although the present invention allows for procedures such as biopsying such organs (e.g., spleen).

[0033] immunogen Advantageously, the methods of the present disclosure are not limited to any particular immunogen. The immunogen in various embodiments can be any antigen, optionally a protein, or a fragment, fusion, or variant thereof. In various examples, the immunogen is a cytokine, lymphokine, hormone, growth factor, extracellular matrix protein, tumor-associated antigen, checkpoint inhibitor molecule, cell surface receptor, or ligand thereof. For purposes of illustrating exemplary immunogens only, the immunogen used to immunize a non-human animal can be a target or antigen bound by any one of the following antibodies: muromonab-CD3 (trade name Orthoclone) or muromonab-CD3 (trade name Orthoclone). Okt3®), abciximab (products marketed under the trade name Reopro®), rituximab (products marketed under the trade name MabThera®, Rituxan®), basiliximab (products marketed under the trade name Simulect®), daclizumab (products marketed under the trade name Zenapax®), palivizumab (products marketed under the trade name Synagis®), infliximab (products marketed under the trade name Remicade®), trastuzumab (products marketed under the trade name Herceptin®), alemtuzumab (products marketed under the trade name MabCampath®, Campath-1H®), adalimumab (products marketed under the trade name Humira®), tositumomab-I 131 (product marketed under the trade name Bexxar®), efalizumab (product marketed under the trade name Raptiva®), cetuximab (product marketed under the trade name Erbitux®), ibritumomab tiuxetan (product marketed under the trade name Zevalin®), omalizumab (product marketed under the trade name Xolair®), bevacizumab (product marketed under the trade name Avastin®), natalizumab (product marketed under the trade name Tysabri®), ranibizumab (product marketed under the trade name Lucentis®), panitumumab (product marketed under the trade name Vectibix®), eculizumab (product marketed under the trade name Soliris®), certolizumab pegol (product marketed under the trade name Cimzia®),golimumab (product marketed under the trade name Simponi®), canakinumab (product marketed under the trade name Ilaris®), catumaxomab (product marketed under the trade name Removab®), ustekinumab (product marketed under the trade name Stelara®), tocilizumab (product marketed under the trade name RoActemra®, Actemra®), ofatumumab (product marketed under the trade name Arzerra®), denosumab (product marketed under the trade name Prolia®), belimumab (product marketed under the trade name Benlysta®), raxibacumab, ipilimumab (product marketed under the trade name Yervoy®), and pertuzumab (product marketed under the trade name Perjeta®). In exemplary embodiments, the antibody is one of an anti-TNF alpha antibody, such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; an anti-IL1β antibody, such as canakinumab; an anti-IL12 / 23(p40) antibody, such as ustekinumab and briakinumab; and an anti-IL2R antibody, such as daclizumab.

[0034] Methods for preparing immunogens for use in the immunization step are known in the art. See, e.g., Fuller et al., Curr Protoc Mol Biol, Chapter 11, Unit 11.4, (2001); Monoclonal Antibodies: Methods and Protocols, 2 nded., Ossipow et al. (Eds.), Humana Press 2014. In various instances, the immunogen is mixed with an adjuvant or other solution prior to administration to the non-human animal. Many adjuvants are known in the art, and illustrative examples include oils, alum, aluminum salts, or lipopolysaccharides. In various aspects, the adjuvant is inorganic. In other aspects, the adjuvant is organic. In various embodiments, adjuvants include: alum, aluminum salts (e.g., aluminum phosphate, aluminum hydroxide), complete Freund's adjuvant, incomplete Freund's adjuvant, RIBI adjuvant system (RAS), lipid A, Sigma Adjuvant system®, TiterMax® Classic, TiterMax® Gold, Montanide vaccine adjuvants (e.g., Montanide 103, Montanide ISA720, Montanide incomplete Seppic adjuvant, Montanide Adjuvants such as ISA51), AF03 adjuvant, AS03 adjuvant, Specol, SPT, nanoemulsions, VSA3, oil or lipid based solutions (e.g., squalene, MF59®, QS21, saponin, monophosphoryl lipid A (MPL)), dicorynoic acid mycolic acid trehalose (TDM), sTDM adjuvants, virosomes, and PRR ligands. See, e.g., “Vaccine Adjuvants Review” at https: / / www.invivogen.com / review-vaccine-adjuvants and “Role of Adjuvants in Antibody Production”, The Protein Man's Blog: A Discussion of Protein Research, posted on June 2, 2016 at https: / / info.gbiosciences.com / blog / role-of-adjuvants-in-antibody-production.In various examples, adjuvants include surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.

[0035] Blood samples and their fractions Following immunization of a non-human animal, a blood sample containing antibody secreting cells (ASC) is obtained from the immunized non-human animal. ASCs are terminally differentiated cells of the humoral immune response, and ASCs differentiate from activated B cells in lymph nodes and circulate transiently in the blood. In exemplary embodiments, the blood sample is obtained from the non-human animal in a non-terminal manner, e.g., the non-human animal is not killed during blood sample collection. In exemplary examples, the method includes performing a non-terminal blood draw from the non-human animal. In exemplary embodiments, the blood sample is obtained from the non-human animal about 1 to about 2 days after the non-human animal is immunized. In various examples, the blood sample is obtained from the non-human animal about 3 to about 7 days (e.g., 3, 4, 5, 6, or 7 days) after immunization. If the immunogen is administered more than once during immunization, the blood sample is obtained from the animal in some embodiments about 3 to about 7 days after the last administration of the immunogen. In various embodiments, a blood sample is obtained from the non-human animal about 8 to about 12 days after immunization of the non-human animal, and in some embodiments, fewer ASCs are expected to be present in the blood sample.

[0036] In an exemplary embodiment, the blood sample comprises peripheral blood mononuclear cells (PBMCs). Optionally, the blood sample comprises B lymphocytes, also known as B cells. In various examples, the blood sample comprises ASCs of the plasma lineage, plasma cells and / or plasmablasts, e.g., migrating plasmablasts. In various embodiments, the ASCs are CD138+ B cells. Optionally, the ASCs comprise migrating plasmablasts.

[0037] The amount of blood sample that can be taken depends on the non-human animal. In various examples, the blood sample obtained from the non-human animal is less than 1 L, 500 mL, or 100 mL, optionally less than about 50 mL, less than about 25 mL, less than about 15 mL, or less than about 10 mL, or less than about 5 mL (e.g., about 4 mL, 3 mL, 2 mL, 1 mL, or less). In some examples, the blood sample obtained from the non-human animal is less than about 1 L, 500 mL, or 100 mL, optionally less than about 50 mL, less than about 25 mL, less than about 15 mL, or less than about 10 mL, or less than about 5 mL (e.g., about 4 mL, 3 mL, 2 mL, 1 mL, or less). In some examples, 500 μL or less of blood is obtained from the non-human animal. In embodiments where the non-human animal is a mouse, the blood sample obtained is less than 200 μL, less than 190 μL, less than 180 μL, less than 170 μL, less than 160 μL, less than 150 μL, less than 140 μL, less than 130 μL, less than 120 μL, less than 110 μL, less than 100 μL, less than 90 μL, less than 80 μL, less than 70 μL, less than 60 μL, less than 50 μL, less than 40 μL, less than 30 μL, less than 20 μL, less than 10 μL, less than 5 μL, less than 4 μL, less than 3 μL, less than 2 μL, or less than 1 μL. In other embodiments where the non-human animal is a mouse, the blood sample obtained is about 200 μL, 190 μL, 180 μL, 170 μL, 160 μL, 150 μL, 140 μL, 130 μL, 120 μL, 110 μL, 100 μL, 90 μL, 80 μL, 70 μL, 60 μL, 50 μL, 40 μL, 30 μL, 20 μL, 10 μL, 5 μL, 4 μL, 3 μL, 2 μL, or 1 μL. In illustrative examples, the blood sample obtained from the non-human animal is about 500 μL or less. Optionally, the volume of the blood sample is about 100 μL to about 250 μL. In various examples, the volume of the blood sample is 10% or less of the total volume of blood circulating in the animal. In various aspects, the volume of the blood sample is not more than 10% of the volume of blood circulating in the animal. In exemplary embodiments, about 10% or less of the total blood volume of the animal is collected. In various examples, the volume of the blood sample is about 9% or less, about 8% or less, about 7% or less, about 6% or less, or about 5% or less of the blood volume circulating in the animal. In various examples, the blood sample is 10% or less of the body weight of the animal.In various embodiments, the blood sample is no more than 9%, no more than 8%, or no more than 7% of the animal's body weight.

[0038] In exemplary embodiments, after a blood sample is obtained from a non-human animal, the blood sample is processed, e.g., concentrated or fractionated. In various examples, the method includes enriching the blood sample for ASCs, e.g., by depleting red blood cells, plasma, and / or platelets from the blood sample. In certain embodiments, the method includes a depletion step using an anti-IgM antibody to remove B cells that contain cell surface IgM. In exemplary examples, the method includes a selection step to perform cells expressing one or more cell surface markers that identify a specific B cell population of interest. In some embodiments, the cell surface markers are CD138, CD19, B220, IgG, TACI, SLAM7, BCMA, CD98, SCA-1, Ly6C1 / 2, etc. If PBMC-derived B cells are desired, the method includes selecting CD138-positive cells. In exemplary embodiments, the method includes removing one or more components of a blood sample obtained from a non-human animal prior to the assay. Optionally, red blood cells, plasma, and / or platelets are removed from the blood sample. In some embodiments, a fraction of the blood sample is prepared by selecting for CD138+ cells.

[0039] Single cell assay In various embodiments of the disclosed method, ASCs present in a blood sample or a fraction thereof are individually assayed for the production of a selected antibody. In various examples, the assay comprises a single-cell assay that analyzes one or more individual cells. In various examples, the assay comprises a live cell assay that analyzes one or more live cells. In exemplary embodiments, a plurality of cells, such as ASCs, present in a blood sample obtained from an immunized non-human animal are assayed simultaneously. In exemplary embodiments, more than about 10, more than about 100, more than about 500, more than about 1000, more than about 2000, more than about 3000, more than about 4000, more than about 5000, more than about 6000, more than about 7000, more than about 8000, more than about 9000, or more than about 10,000 ASCs are assayed simultaneously by single-cell live cell assay.

[0040] In various examples, the method includes applying a blood sample or a fraction thereof to a matrix and assigning a unique address of the matrix to each ASC. The matrix may be two-dimensional (where each unique address of the matrix is ​​defined by a position along the horizontal (X) and vertical (Y) axes) or the matrix is ​​a three-dimensional matrix, for example comprising a porous foam, gel or polymer (where each unique address of the matrix is ​​defined by a position along the width (X), height (Y) and depth (Z) axes). In various aspects, the result of the assay is the identification of each ASC-generated selected antibody, and in certain aspects, the result is the identification of a unique address of each ASC-generated selected antibody.

[0041] In an exemplary embodiment, the assay of the disclosed method includes (a) combining the ASC in the matrix with a reagent that binds to the selected antibody and produces a detectable signal, e.g., a fluorescent signal, upon binding to the selected antibody. In various embodiments, the assay of the disclosed method includes (a) combining the ASC in the matrix with at least one reagent that binds to the Fc domain of the selected antibody and at least one reagent to which the selected antibody binds (e.g., a reagent that binds to the antigen-binding domain of the selected antibody), at least one of which is bound to a detectable label. In an exemplary example, the ASC is combined with a detection reagent that binds to the Fc domain of the selected antibody and includes a first detectable label, and a target to which the selected antibody binds (e.g., a reagent that binds to the antigen-binding domain of the selected antibody). In various examples, the target is expressed by a cell, and the cell expressing the target is combined with the ASC and the detection reagent. In an exemplary embodiment, the method further includes (b) assaying for a first detectable label, and (c) identifying locations within the matrix where the first detectable label is detected, where each identified location represents the location of an individual ASC that produces a selected antibody.

[0042] In an illustrative example, the assay of the disclosed method includes (a) combining the ASC in the matrix with (i) a capture reagent that binds to the selected antibody and includes a solid support, (ii) a detection reagent that binds to the selected antibody and includes a first detectable label, and (iii) a labeled target to which the selected antibody binds (wherein the labeled target includes a second detectable label that is different from the first detectable label); (b) assaying for the first detectable label and the second detectable label; and (c) identifying locations in the matrix where both the first detectable label and the second detectable label are detected (wherein each identified location represents the location of an individual ASC that produces the selected antibody). Optionally, the capture agent includes an antibody that binds to an antibody Fc domain bound to a solid support. The solid support can be any solid support material, such as polymer beads, films, slides, well bottoms, etc., that immobilizes the anti-Fc domain antibody and the antibody to which the anti-Fc domain antibody binds. The detection agent, in an illustrative example, includes an antibody that binds to an antibody Fc domain bound to a first detectable label. In various embodiments, the antibody that binds to the antibody Fc domain of the capture agent is the same antibody of the detection agent, but the anti-Fc antibody of the capture reagent is not bound to a detectable label and the antibody of the detection reagent is not bound to a solid support.

[0043] In an illustrative example, the combining is performed in a well, and the capture agent forms a monolayer in the well. In various embodiments, the method includes identifying locations within the well where both the first detectable label and the second detectable label are detected, where each identified location represents the location of an individual ASC producing a select antibody.

[0044] In an exemplary example, the combination is performed in a micro- or nano-fluidic chamber, a micro- or nano-well device, a micro- or nano-capillary tube, or a nano-pen of a nano-fluidic chip. In an exemplary example, the combination is performed in a nano-pen of a nano-fluidic chip. In an exemplary example, the method includes identifying a position of each pen in the nano-fluidic chip where both the first detectable label and the second detectable label are detected (where each identified position indicates the position of an individual ASC producing a selected antibody). Optionally, a single ASC of the blood sample is moved into the pen of the nano-fluidic chip by optoelectro positioning (OEP). Such a technique is described in Winters et al., MAbs 11(6):1025-1035 (2016).

[0045] Antigen-specific B cells that have recently encountered antigen in germinal centers (GCs) of secondary lymphoid organs (e.g., spleen and lymph nodes) are stimulated to divide and differentiate along multiple pathways. The primary B cell lineage that secretes antibodies into serum in response to antigen challenge is the plasma cell. Plasma cell differentiation begins in secondary lymphoid organs, where cell-cell interactions within GCs drive B cells expressing antigen-specific antibodies on their surface to differentiate into immature plasma cells known as plasmablasts. Plasmablasts are rapidly dividing B cells that produce and secrete high levels of soluble antibodies. Following exposure to antigen in GCs, differentiation into plasmablasts, and subsequent proliferation, waves of migrating plasmablasts can be detected in the circulating blood. In mice, plasmablasts in the blood occur 3-7 days after antigen exposure and decline over time as they settle in appropriate niches and differentiate into long-lived plasma cells. The trafficking of recently stimulated antigen-specific plasmablasts in the blood can be used to assess animal immune responses and characteristics at the single cell level, rather than by investigating polyclonal serum titers. In an exemplary embodiment, a non-terminal bleed is taken from the mouse, washed to remove plasma and soluble antibodies, and peripheral blood mononuclear cells (PBMCs) are directly assayed for ASCs. Small antibody capture beads are added to capture and localize the antibodies secreted from the ASCs, thereby allowing characterization at the single cell level. Red blood cell (RBC) contamination interferes with fluorescent plaque formation, but can be mitigated by diluting the assay to a larger volume. However, this results in a larger plating volume and reduced assay throughput. Alternatively, RBCs can be directly removed or desired cells can be isolated from the blood sample prior to plating, thus reducing plating volume and increasing assay throughput. Suitable methods include, but are not limited to, RBC lysis, density gradient centrifugation (e.g., HetaSep, Ficoll®), and the use of negative selection (e.g., anti-mouse TER119 RBC depletion) or positive selection (e.g., mouse CD138+ isolation) cell isolation kits with or without the use of automated washing instruments (e.g., Curiox Laminar Wash™).Cells expressing the target of interest can also be used in place of beads. Such techniques can be used to investigate strategies for generating species-reactive antibodies in mice.

[0046] In an exemplary embodiment, human-cynomolgus cross-reactive antibodies can be produced by immunizing animals with alternating boosts of human and cynomolgus types of antigens. Reactivity to each antigen can be easily monitored using a simple binding assay with polyclonal sera from immunized animals. However, since animals are immunized with both antigens and each antigen has both common and unique epitopes, the polyclonal sera contain antibodies that react to both types of epitopes. Unfortunately, from this analysis alone, it is not possible to determine whether the seroreactivity observed to both antigens is truly due to cross-reactive antibodies or is derived from multiple antibodies with specificity for only one of the antigens. However, investigation of B cell responses using ASCs derived from PBMC populations overcomes this problem by localizing and screening antibody specificities from a single cell that can guide further immune repertoire formation or animal selection for antibody generation. Shaping the immune repertoire may include modifications to the immunization strategy, such as (but not limited to) switching to different forms of immunogens, adjuvants, immunomodulatory agents, antigen doses, timing of immunization, and routes of administration.

[0047] Thus, the present disclosure further provides a single cell assay for identifying ASCs that produce select antibodies.The present disclosure provides a method of assaying for ASCs that produce select antibodies. In an exemplary embodiment, the assay or method comprises: (a) combining in a well: (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen (wherein the blood sample comprises ASCs); (ii) a detection reagent that binds to the selected antibody and comprises a first detectable label; and (iii) a target to which the selected antibody binds (wherein (A) the target is a labeled target that comprises a second detectable label different from the first detectable label, and a capture reagent that binds to the selected antibody and comprises a solid support are further combined in the well to form a monolayer in the well, or (B) the target is expressed on the surface of a cell, and the cells are combined in the well to form a monolayer in the well); (b) assaying for the first detectable label, and optionally, if the target is a labeled target, assaying for the second detectable label; and (c) identifying a location in the well where the first detectable label is detected, or where the first and second detectable labels are detected (wherein each identified location indicates the location of an individual ASC producing the selected antibody).

[0048] In various embodiments, the assay or method comprises: (a) combining in a well (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen; (ii) a capture reagent comprising a solid that binds to the Fc of an antibody bound to a solid support; (iii) a detection reagent comprising an antibody that binds to the Fc of the antibody bound to a first detectable label; and (iv) a labeled target comprising the immunogen or a portion thereof bound to a second detectable label different from the first detectable label (wherein the capture reagent forms a monolayer in the well); (b) assaying for the first detectable label; (c) assaying for the second detectable label; and (d) identifying locations in the well where both the first detectable label and the second detectable label are detected (wherein each identified location indicates the location of an individual ASC producing a select antibody).

[0049] In various embodiments, the assay or method includes (a) combining in a well (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen, (ii) a detection reagent that binds to the selected antibody and includes a first detectable label, and (iii) cells that express a target to which the selected antibody binds on their cell surface, the cells combining in the well to form a monolayer in the well, (b) assaying for the first detectable label, and (c) identifying locations in the well where the first detectable label is detected (where each identified location indicates the location of an individual ASC that produces the selected antibody).

[0050] In exemplary embodiments, the first detectable label and / or the second detectable label of the labeled target comprises a chromophore or fluorophore. Optionally, the fluorophore is selected from the group consisting of xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas red), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), squaraine derivatives (e.g., Seta dyes and Square dyes), squaraine rotaxane derivatives (e.g., Tau dyes), naphthalene derivatives (e.g., dansyl derivatives and prodan derivatives), coumarin derivatives, oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), anthracene derivatives (e.g., anthraquinones, including DRAQ5, DRAQ7, and CyTRAK Orange), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin), or dipyrromethene derivatives (e.g., BODIPY, aza-BODIPY).In various examples, the first detectable label and / or the second detectable label of the labeled target may be a CF dye (Biotium), DRAQ or CyTRAK probe (BioStatus), BODIPY (Invitrogen), EverFluor (Setareh Biotech), Alexa Fluor (Invitrogen), Bella Fluor (Setareh Biotech), CyLight Fluor (Thermo Scientific, Pierce), Atto or Tracy (Sigma Aldrich), FluoProbe (Interchim), Abberior dyes (Abberior), DY or MegaStokes dyes (Dyomics), Sulfo Cy dyes (Cyandye), HiLyte Fluor (AnaSpec), Seta, SeTau, Square dyes (SETA BioMedicals), Quasar or Cal Fluor dyes (SETA BioMedicals), SureLight dyes (APC, RPE PerCP, Phycobiolisomes (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech, Greensea, Prozyme, Flogen), or Vio dyes (Miltenyi Biotec). In exemplary embodiments, the fluorophore includes 3-hydroxyisonicotinaldehyde, allophycocyanin (APC), aminocoumarin, APC-Cy7 conjugate, BODIPY-FL, Cascade Blue, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, fluorescein, FluorX, G-Dye100, G-Dye200, G-Dye300, G-Dye400, hydroxycoumarin, Lissamine rhodamine B, Lucifer Yellow, methoxycoumarin, NBD, Pacific Blue, Pacific Orange, PE-Cy5 conjugate, PE-Cy7 conjugate, PerCP, R-Phycoerythrin (PE), Red613, Texas Red, TRITC, TruRed, or X-Rhodamine.In various embodiments, assaying for the first detectable label and / or the second detectable label comprises detecting a signal from the first detectable label and / or the second detectable label. In an exemplary embodiment, the signal is a fluorescent signal. In an exemplary embodiment, assaying for the first detectable label and / or the second detectable label comprises quantifying the signal from the first detectable label and / or the second detectable label. In various examples, the method comprises quantifying the signal from the first detectable label and / or the second detectable label and normalizing the signal by expressing the signal from the first detectable label and the second detectable label as a ratio. In various embodiments, the ratio is (relative fluorescence units (RFU) of the signal from the first detectable label) / (RFU of the signal from the second detectable label), or the reciprocal thereof.

[0051] In exemplary embodiments, the ASCs are exposed to the detection reagent and / or target initially in the well or immediately prior to being added to the well. In various embodiments, the ASCs are incubated with the detection reagent and target for at least 30 minutes, at least 60 minutes, at least 90 minutes, or at least 120 minutes. Optionally, the selected antibody binds to a target that is the same as or similar to the immunogen used to immunize the non-human animal. In exemplary examples, the detection reagent comprises an antibody that binds to an antibody Fc domain bound to a first detectable label. Optionally, the antibody that binds to the antibody Fc domain of the capture agent is the same antibody as the detection agent. In various embodiments, a blood sample is obtained from the non-human animal about 3 to about 7 days after the immunization step. In various examples, the blood sample obtained from the non-human animal is about 500 μL or less, optionally about 100 μL to about 250 μL. In certain embodiments, the ASCs are CD138+ B cells. Optionally, the ASCs comprise migratory plasmablasts. In exemplary embodiments, the method further comprises removing one or more components of a blood sample obtained from the non-human animal prior to combination in the well. In some examples, red blood cells, plasma, and / or platelets are removed from the blood sample. In various embodiments, the fraction of the blood sample is a fraction of CD138. + The selected antibody is prepared by selecting cells. In various examples, the selected antibody binds to the target in the presence of one or more competing binders. Optionally, the competing binders are combined with the ASC, detection reagents, and cells expressing the target during the assay. In an exemplary embodiment, the selected antibody binds to the target with target affinity, and optionally, the KD of the selected antibody for the target is about 10 -11 M~about 10 -9 M. Optionally, in the first round, the assay is performed with a first amount of cells expressing the target, and in the second round, the assay is performed with a second amount of cells expressing the target (wherein the first amount is greater than the second amount). In some embodiments, in the third round, the assay is further performed with a third amount of cells expressing the target, the third amount being less than the second amount, and in each round, when the ASC binds to the labeled target, the ASC produces a selection antibody.

[0052] The assay of the disclosed method tests the production of selected antibodies by individual ASCs. The term "selected antibody" refers to an antibody that meets a design goal and / or exhibits a target phenotype. In various examples, the selected antibody binds to a target that may be the same as or similar to the immunogen used to immunize the non-human animal. The target may be any of the immunogens listed herein. In an illustrative example, the selected antibody is a target-specific antibody, e.g., an antigen-specific antibody. In various examples, the selected antibody has a binding affinity of at least about 10 to a target (or antigen). -9 In various embodiments, the selected antibody exhibits a binding affinity represented by a KD in the picomolar range (e.g., about 1×10 -12 M~9.9×10 -12 In various embodiments, the selected antibody binds to the target in the presence of one or more competing binders. In various examples, the competing binder is a component in human blood, e.g., human plasma or serum. In such examples, the competing binder, e.g., human serum, is combined with the ASC, capture reagent, detection reagent, and labeled target during the assay. See, e.g., Example 1. In various embodiments, the competing binder is a natural ligand that binds to a target in a human or non-human animal body, and the selected antibody that binds to the target blocks or inhibits the natural ligand from binding to the target. For example, the selected antibody is an anti-PD-1 antibody, and the competing binder is PD-L1 and / or PD-L2. In such examples, the competing binder, e.g., PD-L1 and / or PD-L2, is combined with the ASC, capture reagent, detection reagent, and labeled target during the assay. See, e.g., Example 7.

[0053] Screening of the disclosed method can be completed in larger wells (e.g., 4-well plates or OmniTray™) for subsequent molecular rescue of ASCs producing selected antibodies. Unlike ELISpot or FluoroSpot assays, the disclosed method is a homogenous live cell assay amendable to micromanipulation or micromanipulation with automated fluorescent single cell picking systems (e.g., CellCelector™) known in the art. A confluent monolayer of IgG capture beads immobilizes ASCs in place allowing for well-defined fluorescent plaques, pinpointing the location of individual ASCs producing selected antibodies.

[0054] The disclosed method can guide the selection and harvesting of animals that produce select antibodies for antibody generation. Conventional approaches to select animals are to investigate polyclonal serum titers, which measure the overall reactivity and quality of all secreted antibodies, rather than the quality of individual antibodies. Investigation of B cell responses using ASCs derived from PBMC populations can overcome this problem by identifying individual ASCs that produce select antibodies that would otherwise be difficult to interpret or hidden in polyclonal serum titers. Exemplary methods of animal selection for terminal tissue harvesting are described in Figures 1D and 1E and Examples 12 and 13.

[0055] In an exemplary embodiment, the selected antibody binds to the target with a target affinity, and optionally, the KD of the selected antibody for the target is greater than or equal to 10 -11 M~10 -9 In various examples, the KD of the selected antibody against the target is in the picomolar range or about 10 -12 In various examples, the KD of the selected antibody against the target is in the sub-picomolar range, e.g., <10 -12M. In various embodiments, the assay comprises combining a first amount of labeled target comprising an immunogen or a portion thereof bound to a second detectable label with an ASC, a capture reagent, and a detection reagent in a first round, and combining a second amount of labeled target comprising an immunogen or a portion thereof bound to a second detectable label with an ASC, a capture reagent, and a detection reagent in a second round (wherein the first amount is greater than the second amount). In some examples, the assay can comprise a third round using a third amount of labeled target (wherein the third amount is less than the second amount). The ASC identified by detecting the first detectable label and the second detectable label throughout each round can be the ASC that produces a selected antibody with high affinity to the target. See, for example, Example 8.

[0056] The selected antibody binds to the target with target affinity. In various embodiments, the assay comprises combining the ASC with a detection reagent that binds to the Fc domain of the selected antibody and includes a first detectable label, and a target to which the selected antibody binds as a second label. This is followed by quantification of the fluorescent signal and normalization of secretion by determining the ratio of IgG secretion first label RFU (relative fluorescent units) to target second label RFU. The ASC identified with the smallest ratio (IgG RFU / target RFU) may be the ASC that produces the selected antibody with high affinity to the target. The ASC IgG normalized RFU can be compared to ASCs of known target affinity from recombinant antibodies expressed in validated hybridomas or cell lines to provide a rough relative affinity ranking. See, for example, Example 11.

[0057] In various embodiments, the selected antibody binds to the target and its ortholog or paralog, and optionally the target is a human protein and the ortholog is a cynomolgus protein. In various examples, during the assay, a second labeled target is combined with the ASC, the capture reagent, the detection reagent, and the labeled target, where the second labeled target comprises an ortholog bound to a third detectable label different from the first detectable label and the second detectable label, and the method further comprises assaying for the third detectable label and identifying the location where the first detectable label, the second detectable label, and the third detectable label are detected, where each identified location indicates the location of an individual ASC that produces the selected antibody.

[0058] In various examples, the selected antibody binds to the target and does not bind to its ortholog or paralog. Optionally, during the assay, a second labeled target is combined with the ASC, capture reagent, detection reagent, and labeled target, where the second labeled target comprises an ortholog bound to a third detectable label different from the first detectable label and the second detectable label, and the method further comprises assaying for the third detectable label and identifying the position where only the first detectable label and the second detectable label are detected, but not the third detectable label, where each identified position represents the position of an individual ASC that produces the selected antibody. For example, see Example 6.

[0059] In various embodiments, the selection antibody binds to a portion of the target. Optionally, during the assay, a second labeled target is combined with the ASC, capture reagent, detection reagent, and labeled target, where the second labeled target comprises a portion of the target bound to a third detectable label different from the first detectable label and the second detectable label, and the method further comprises assaying for the third detectable label and identifying the location where the first detectable label, the second detectable label, and the third detectable label are detected, where each identified location represents the location of an individual ASC that produces the selection antibody. In various examples, the target is a protein that comprises multiple domains, and the selection antibody binds to only one domain of the target. In various embodiments, during the assay, the labeled target comprises the extracellular domain of the target bound to the second detectable label, and the second labeled target comprises one domain bound to the third detectable label. See, for example, Example 9. In various embodiments, the selected antibody binds to a conformational epitope formed during dimerization or multimerization of the target, and the target comprises a dimerization or multimerization domain. Optionally, during the assay, the labeled target comprises an extracellular domain of the immunogen bound to a second detectable label, and the second labeled target is combined with the ASC, the capture reagent, the detection reagent, and the labeled target, and the second labeled target comprises a dimerization or multimerization domain of the immunogen bound to a third detectable label different from the first detectable label and the second detectable label, and the method further comprises assaying for the third detectable label and identifying the position where the first detectable label, the second detectable label, and the third detectable label are detected (where each identified position indicates the position of an individual ASC that produces the selected antibody). For example, see Example 10.

[0060] Induction of antibody production by repeated immunization In various embodiments, the method includes repeatedly immunizing the non-human animal. As exemplified herein, in various embodiments, the method includes immunizing the non-human animal two or more times. In various embodiments, the method includes performing an initial immunization and one or more subsequent immunizations. In various examples, each subsequent immunization is repeated after obtaining a blood sample from the non-human animal and assaying for ASCs producing the selected antibody. In various embodiments, the non-human animal is immunized at least two or more times, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times. In various embodiments, the method includes performing an initial immunization and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 subsequent immunizations. Optionally, the immunization is repeated until ASCs producing the selected antibody are identified or the proportion of ASCs producing the selected antibody is equal to or greater than a threshold value. In various examples, each immunization is repeated with the same (A) immunogen, adjuvant, immunomodulatory agent, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulatory agent, or combination thereof, (C) timing of immunization, (D) route or method of administration for delivering the immunogen, (E) site of administration to the non-human animal, or (F) combination thereof, as compared to the previous immunization or primary immunization. Alternatively, each immunization is repeated with a different (A) immunogen, adjuvant, immunomodulatory agent, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulatory agent, or combination thereof, (C) timing of immunization, (D) route or method of administration for delivering the immunogen, (E) site of administration to the non-human animal, or (F) combination thereof, as compared to the previous immunization or primary immunization. In an exemplary embodiment, each time an animal is immunized as compared to a previous immunization or the initial immunization, the animal is immunized using a different (A) immunogen, adjuvant, immunomodulatory agent, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulatory agent, or combination thereof, (C) timing of immunization, (D) route or method of administration for delivering the immunogen, (E) site of administration into the non-human animal, or (F) combination thereof.In various embodiments, the immunizations are varied from one to the next, such that the immune response elicited in the non-human animal by the immunization is varied compared to the prior immune response elicited by the prior immunization. Without being bound to any particular theory, multiple different immunization campaigns are performed on the same animal to direct the immune response towards the production of selected antibodies.

[0061] In an illustrative example, a method of inducing antibody production in a non-human animal for the production of a selected antibody includes performing a cycle of steps if the proportion of ASCs producing the selected antibody is below a threshold value, the cycle including: (i) performing a subsequent immunization of the non-human animal with an immunogen if the proportion of ASCs producing the selected antibody is below a threshold value; (ii) obtaining a blood sample from the non-human animal comprising ASCs; and (iii) individually assaying the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody.

[0062] In an exemplary example, the method includes cycles of (i) performing a subsequent immunization of the non-human animal with the immunogen if the percentage of ASCs producing the selected antibody is below a threshold value, (ii) obtaining a blood sample from the non-human animal containing ASCs, and (iii) individually assaying the ASCs present in the blood sample or a fraction thereof for production of the selected antibody. In various examples, the cycle is repeated at least once, twice, or three or more times. In various embodiments, the cycle is repeated until the percentage of ASCs producing the selected antibody in the assay of (iii) is equal to or greater than the threshold value. In exemplary embodiments, after repeated cycles, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% of the immunized non-human animals have a percentage of ASCs producing the selected antibody equal to or greater than the threshold value. In various embodiments, more than 75%, or more than 85%, or more than 90% of the immunized non-human animals have a percentage of ASCs producing the selected antibody equal to or greater than the threshold value. In exemplary embodiments, the immunogen of the subsequent immunization is different from the immunogen of the initial immunization. For example, in exemplary embodiments, each subsequent immunization differs from the prior immunization in that: (A) a different immunogen, adjuvant, and / or immunomodulatory agent is administered to the non-human animal; (B) a different dose of immunogen is administered to the non-human animal than in the initial immunization; (C) the time between each administration of the immunogen, adjuvant, and / or immunomodulatory agent used in the initial immunization is different; and / or (D) the route of administration of each administration of the immunogen, adjuvant, and / or immunomodulatory agent used in the initial immunization is different. Optionally, a different immunogen is used each time the non-human animal is immunized.

[0063] As discussed herein, immunization involves one or more administrations of an immunogen (optionally prepared with an adjuvant) to a non-human animal. The method of the present invention may include multiple immunization steps with different immunization conditions, which can be used to steer the immune response so that the immunized non-human animal ultimately produces antibodies with a desired phenotype. Depending on the phenotype, or combination of phenotypes, the immunization conditions can be varied during successive immunization steps so that the immune response is steered to produce antibodies with the desired phenotype. In an exemplary embodiment, the non-human animal can be immunized with alternating boosts of human and cynomolgus types of antigen to produce human-cynomolgus cross-reactive antibodies. For example, the immunization can include a total of four injections, with the first and third injections using recombinant human antigen and the second and fourth injections using recombinant cynomolgus antigen. An exemplary immunization is described in Example 4 herein. Example 5 describes a further method of making human-cynomolgus cross-reactive antibodies, in which different immunizations are used. In exemplary embodiments, to generate antibodies specific for a domain of a multidomain protein, immunization can occur with one or more of the following three types of immunogens: the complete extracellular domain of the multidomain protein, the domain, and / or the full-length protein. See, e.g., Example 9. In exemplary embodiments, to generate antibodies specific for an epitope formed upon dimerization or multimerization of a dimeric or multimeric protein, immunization can occur with one or more of the following three types of immunogens: the complete extracellular domain of the dimeric or multimeric protein, the multimerization domain, and / or the full-length protein. See, e.g., Example 10. Further exemplary immunizations are provided herein. See the Examples section.

[0064] Additional steps The methods disclosed herein may include further steps. In an exemplary example, the method includes assaying an antibody response to an immunogen after a blood sample is obtained. In an exemplary example, the method includes assaying an antibody titer of a blood sample after the blood sample is obtained. In an exemplary example, the method includes assaying the antigen specificity of antibodies present in the blood sample, optionally a binding assay using the immunogen.

[0065] In various embodiments, the method includes isolating the ASC that produces the selected antibody, or isolating the selected antibody. In various examples, the isolation of the antibody is accomplished by isolating a single ASC that produces the selected antibody. In various embodiments, isolating the ASC includes a dilution step, optionally a serial dilution step, where the cell concentration is reduced such that statistically, one cell is present in a given calculated volume, and the calculated volume is placed in a separate vessel or well of a multi-well plate. In various embodiments, isolating the ASC of the blood sample includes microfluidically transferring a single ASC into a well or into an air bubble, where the ASC is maintained in culture until the selected antibody is secreted into the culture medium and / or the ASC undergoes cell division. Optionally, maintained for at least or about 3 minutes to about 30 minutes, 6 hours, 24 hours, or more. In various embodiments, the isolation of the ASC is performed by microfluidics, magnetic, capillary action, gravity, FACS, or optoelectro positioning (OEP).

[0066] In various embodiments, the method of the disclosure includes determining the sequence of the heavy and light chain variable regions of an antibody having a target phenotype. Optionally, the sequencing is performed by RT-PCR. Optionally, the method further includes transfecting cells with nucleic acids encoding the heavy and light chain variable regions of an antibody having a target phenotype, culturing the transfected cells, and harvesting the antibody from the culture. In some embodiments, the steps of the method are performed on a series of non-human animals, and the method includes profiling the B cell repertoire of blood samples for each series of non-human animals, and selecting a series of subsets having a target B cell profile. Such a method is described in Example 1.

[0067] Also, in various aspects, the method includes one or more upstream or downstream steps involved in producing, purifying, and formulating the antibody. Optionally, the downstream step is any one of the downstream process steps described herein or known in the art. In an exemplary embodiment, the method includes a step for generating a host cell expressing an antibody having a target phenotype. The host cell is, in some aspects, a prokaryotic host cell, such as E. coli or Bacillus subtilis, or the host cell is, in some aspects, a eukaryotic host cell, such as a yeast cell, a filamentous fungal cell, a protozoan cell, an insect cell, or a mammalian cell (e.g., a CHO cell). Such host cells are described in the art. See, e.g., Frenzel, et al., Front Immunol 4:217 (2013). For example, in some examples, the method includes introducing into the host cell a vector that includes a nucleic acid that includes a nucleotide sequence encoding the antibody, or a light or heavy chain thereof. In an exemplary embodiment, the method includes maintaining the cells in cell culture. Optionally, such steps may include maintaining the cells in a culture medium that includes a particular temperature, pH, osmolality, dissolved oxygen, humidity, or one or more of glucose, fucose, lactate, ammonia, glutamine, and / or glutamate.

[0068] In exemplary embodiments, the methods disclosed herein include isolating and / or purifying ASCs that produce a selected antibody, or isolating and / or purifying a selected antibody from the culture. In exemplary aspects, the methods include one or more chromatography steps, including, but not limited to, affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In exemplary aspects, the methods include generating a crystalline biomolecule from a solution that includes a recombinant glycosylated protein.

[0069] The methods of the disclosure, in various embodiments, include one or more steps for preparing a composition, such as, in some embodiments, a pharmaceutical composition that includes a purified selected antibody.

[0070] In an exemplary embodiment, the method includes (a) immunizing an animal using a standard protocol, and (b) evaluating the serum for an antigen-specific antibody response. In an exemplary aspect, the method further includes selecting immunized animals for boosting with antigen, and collecting blood from these animals (e.g., about 4 days after boost). In various examples, the method includes removing red blood cells, plasma, and platelets from blood collected from the animal to enrich the blood for B cells. In an exemplary example, the method includes identifying antibody-secreting cells (ASCs) and isolating the ASCs as single cells to allow elucidation and / or characterization of antibodies produced by individual ASCs. Optionally, single cell isolation and screening is accomplished using methods known in the art, e.g., NanOBLAST (e.g., by a nanofluidic Beacon instrument), microencapsulation. In various aspects, antibodies produced and secreted by individual ASCs are evaluated for a target phenotype. Optionally, evaluation of the target phenotype is accomplished by using a variety of different screening strategies to identify one or more antibodies with the target phenotype, and the ASCs that produce and secrete the antibodies. In various examples, the method further includes isolating antibody VH and VL genes from the ASCs (that produce and secrete antibodies exhibiting the target phenotype), for example by single cell RT-PCR, and cloning the paired VH and VL gene sequences into cells for recombinant production.

[0071] Screening, Selection, and Profiling Methods The present disclosure provides a method of screening a non-human animal for antibody secreting cells (ASCs) that produce a selected antibody. In an exemplary embodiment, the method comprises monitoring the production of the selected antibody in the non-human animal according to the monitoring method of the present disclosure, the method being performed on a series of non-human animals, and for each series of non-human animals, the number of ASCs that produce the selected antibody is identified. In an exemplary embodiment, the method comprises (a) immunizing a series of non-human animals with an immunogen, (b) obtaining a blood sample containing ASCs from each non-human animal in the series, and (c) individually assaying the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody, and for each series of non-human animals, the proportion of ASCs that produce the selected antibody is determined. In various aspects, the screening method further comprises selecting the non-human animal for sacrifice and / or tissue harvesting (e.g., secondary lymphoid tissue harvesting) if the proportion of ASCs that produce the selected antibody is equal to or greater than a threshold value. The present disclosure further provides a method of selecting an immunized non-human animal for subsequent immunization. In various aspects, the screening method further comprises selecting the non-human animal for subsequent immunization if the proportion of ASCs producing the selected antibody is below a threshold. Thus, in various embodiments, the screening method distinguishes between animals to be killed and animals to be subsequently immunized based on the proportion of ASCs producing the selected antibody. In an exemplary embodiment, the method comprises monitoring the production of the selected antibody in the non-human animal according to the monitoring method of the present disclosure, which is performed on a series of non-human animals, and for each series of non-human animals, the number of ASCs producing the selected antibody is identified, and if the proportion of ASCs producing the selected antibody for the animal is below a threshold, the animal is selected for subsequent immunization. A method for selecting an immunized non-human animal producing a selected antibody for euthanasia and secondary lymphoid harvesting is also further provided herein.In an exemplary embodiment, the method includes monitoring production of a select antibody in a non-human animal according to the monitoring method of the present disclosure, which is performed on a series of non-human animals, and for each series of non-human animals, the number of ASCs producing the select antibody is identified, and if the proportion of ASCs producing the select antibody for an animal is equal to or greater than a threshold value, the animal is selected for euthanasia and secondary lymphoid harvest.

[0072] The disclosure further provides a method of profiling the B cell repertoire of a non-human animal. In an exemplary embodiment, the method comprises: (a) immunizing a non-human animal with an immunogen; (b) obtaining a blood sample from said non-human animal comprising antibody secreting cells (ASC); and (c) individually assaying the ASCs present in the blood sample or a fraction thereof for production of a select antibody. In various examples, the method is performed on a series of non-human animals, the method comprising profiling the B cell repertoire of the blood sample for each series of non-human animals and selecting a series of subsets having a target B cell profile. In an exemplary example, the subset is selected for re-immunization. In another example, the subset is selected for euthanasia and harvesting of secondary lymphoid organs.

[0073] Thus, the screening and selection methods described herein allow for the identification of non-human animals producing selected antibodies.An exemplary advantage of such methods is that non-human animals producing such antibodies can be identified prior to sacrifice and B cell harvesting.This helps to enrich the non-human animals producing selected antibodies, and thus the B cell pool, thus alleviating some of the inefficiencies of conventional downstream antibody creation methods.

[0074] Generation of antibodies The present disclosure further provides a method for producing a selected antibody in a non-human animal. In an exemplary embodiment, the method includes inducing antibody production in a non-human animal for the production of a selected antibody according to the disclosed method of inducing antibody production, and then isolating the selected antibody and / or the ASCs that produce the selected antibody. In an exemplary embodiment, the method includes (a) performing an initial immunization campaign on the non-human animal with an immunogen, (b) obtaining a blood sample containing antibody-secreting cells (ASCs) from the non-human animal, (c) individually assaying the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody, (d) performing a subsequent immunization of the non-human animal with the immunogen if the proportion of ASCs that produce the selected antibody is below a threshold value, and (e) isolating the selected antibody and / or the ASCs that produce the selected antibody. In various embodiments, the method includes repeating the cycle of (i) performing a subsequent immunization of the non-human animal with the immunogen if the percentage of ASCs producing the selected antibody is below a threshold value, (ii) obtaining a blood sample from the non-human animal containing ASCs, and (iii) individually assaying the ASCs present in the blood sample or a fraction thereof for the production of the selected antibody, until the percentage of ASCs producing the selected antibody is equal to or exceeds a threshold value. Methods for isolating ASCs producing the selected antibody or isolating the selected antibody are described herein. See, for example, the additional steps section.

[0075] In various embodiments, the method further includes (f) determining a nucleotide sequence encoding a heavy chain variable region of a selected antibody produced by the ASC (e.g., an isolated ASC producing a selected antibody) and a nucleotide sequence encoding a light chain variable region of a selected antibody produced by the ASC; (g) introducing a first vector comprising a nucleotide sequence encoding a heavy chain variable region of the selected antibody and a second vector comprising a nucleotide sequence encoding a light chain variable region of the selected antibody into a host cell; and (h) isolating the antibody produced by the host cell. Methods for determining the sequence of the heavy and light chain variable regions of an antibody are known in the art, including, for example, single-cell PCR. See, for example, Tiller et al., J Immunol Methods 350:189-193 (2009); and Winters et al., 2019 (supra). It is known to generate vectors comprising nucleotide sequences. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2012). In various embodiments, the method of generating the selected antibody comprises engineering the heavy and / or light chain sequences to obtain an engineered selected antibody. In various embodiments, the engineered selected antibody exhibits greater stability, e.g., during storage or manufacturing, formulation, packaging, shipping or administration, or under in vivo conditions, as compared to the non-engineered selected antibody. In various embodiments, the engineered selected antibody exhibits greater affinity for a target or its ortholog or paralog, as compared to the non-engineered selected antibody. Suitable techniques for isolating antibodies produced by host cells are described herein and known in the art. See, e.g., the Additional Steps section herein, and Low et al., J Chromatog B 848(1):48-63 (2007); Ngo et al., U.S. Pat. No. 4,933,435; and Ayyar et al., Methods 56(2):116-129 (2012).

[0076] In an exemplary embodiment of the method of generating a selected antibody of the present disclosure, the method includes (a) performing an initial immunization of a non-human animal with an immunogen; (b) obtaining a blood sample containing antibody secreting cells (ASCs) from the non-human animal; (c) individually assaying the ASCs present in the blood sample or a fraction thereof for production of the selected antibody; and (d) harvesting one or more secondary lymphoid organs from the non-human animal if the proportion of ASCs producing the selected antibody is equal to or greater than a threshold value. In various embodiments, immune cells are obtained from the harvested secondary lymphoid organs, and at least a portion of the immune cells, e.g., IgG-positive memory B cells, are used to generate hybridomas. Methods of generating hybridomas are known in the art and described herein. See, for example, the section herein entitled Enhancing Hybridoma Production, as well as Zhang, Methods Mol Ciol 01:117-135 (2012); Tomita and Tsumoto, Immunotherapy 3(3):371-380 (2011); Hnasko and Stanker, Methods Mol Biol 1318:15-28 (2015); and Zaroff and Tan, Biotechniques 67(3):90-92 (2019). In certain embodiments, the methods of the present disclosure further comprise producing a hybridoma.

[0077] antibody Although antibody structures vary from species to species, the term "antibody" as used herein typically refers to a protein having a conventional immunoglobulin type, including heavy and light chains, and including variable and constant regions. The antibodies obtained or isolated by the method can have a variety of uses. For example, the antibodies obtained by the method of the present invention can be used as therapeutic agents. The antibodies obtained by the method can also be used as non-therapeutic antibodies, such as reagents used in diagnostic assays, such as imaging assays, and other in vitro or in vivo immunoassays, such as Western blots, radioimmunoassays, ELISA, EliSpot assays, and the like. In various aspects, the antibodies can be monoclonal or polyclonal. In illustrative examples, the antibodies are mammalian antibodies, such as mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, pig antibodies, human antibodies, alpaca antibodies, camel antibodies, llama antibodies, and the like. In some aspects, the antibodies can be monoclonal or polyclonal antibodies, optionally produced by transgenic animals. In such an embodiment, the antibody produced is a chimeric antibody containing sequences of two or more species. In various examples, the antibody has a human IgG "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" chain (typically with a molecular weight of about 25 kDa) and one "heavy" chain (typically with a molecular weight of about 50-70 kDa). Human antibodies have a variable region and a constant region. In the human IgG type, the variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is primarily involved in antigen recognition, and is substantially different between other antibodies that bind different antigens. See, for example, Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4 thed. Elsevier Science Ltd. / Garland Publishing, (1999). Briefly, in the framework of a human antibody, the CDRs are embedded within the framework in the variable regions of the heavy and light chains, where they constitute the regions that play a major role in antigen binding and recognition. A human antibody variable region comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (termed framework regions 1-4, FR1, FR2, FR3 and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The embodiments of the present disclosure include all such classes or isotypes of human antibodies. The human light chain constant region can be, for example, a kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, in an exemplary embodiment, the antibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM, for example, any one of IgG1, IgG2, IgG3, or IgG4.

[0078] The antigen-binding protein may have a structure different from that of a human antibody. In an illustrative example, the antigen-binding protein includes only a heavy chain fragment, such as a heavy chain variable region, a heavy chain constant region CH2, and a heavy chain constant region CH3. In various examples, the antigen-binding protein includes a nanobody structure, such as those made by dromedaries, llamas, and sharks. See, for example, Leslie, Science, "Mini-antibodies discovered in sharks and camels could lead to drugs for cancer and other diseases", 2018, at https: / / www.sciencemag.org / news / 2018 / 05 / mini-antibodies-discovered-sharks-and-camels-could-lead-drugs-cancer-and-other-diseases.

[0079] The following examples are offered merely to illustrate the present invention and are not intended to limit its scope in any way. EXAMPLES

[0080] Example 1 This example describes an exemplary method for monitoring the production of selected antibodies in mice.

[0081] In this example, the antibody of choice was an anti-idiotypic antibody (anti-ID ab) that binds to the idiotope of a therapeutic human IgG antibody specific for PD-1 (hereafter referred to as "Antibody 1"). An idiotope is a unique structure formed by the variable region (paratope) of an antibody that is normally involved in binding to an antigen. Figure 3 shows the idiotope and paratope of Antibody 1 and the anti-ID antibody.

[0082] Immunization Protocol A soluble form of Antibody 1 was emulsified in adjuvant (Complete Freund's Adjuvant followed by Sigma Adjuvant System® (SAS, Catalog No. S6322; Sigma-Aldrich, St. Louis, MO)). The antibody-adjuvant mixture was then delivered to multiple strains of wild-type mice including Balb / c, CD1 and B6 / 129 mice. A complete immunization campaign consisted of three injections delivered at 2-week intervals over a period of 38 days. The first immunization consisted of 50 μg of Antibody 1 emulsified in 100 μl of Complete Freund's Adjuvant and injected subcutaneously into two spots on the dorsum of each mouse. After 14 days, 25 μg of Antibody 1 was suspended in 200 μl of Sigma Adjuvant System and 100 μl of this mixture was injected subcutaneously into two spots on the dorsum of each mouse and the remaining 100 μl was injected intraperitoneally. The third immunization was delivered 14 days later using the same route and adjuvant as the second, except that the total amount of Antibody 1 was reduced to 15 μg.

[0083] Bridging ELISA analysis of serum titers from each mouse was performed essentially as described in Winters et al., mAbs 11(6):1025-1035 (2019) to confirm antigen reactivity and inform animal selection for the final boost. As shown in Figure 4, serum titer levels were highest in CD1 mice, but all immunized mice, including Balb / c and B6 / 129 mice, showed serum titer levels higher than controls (no serum). Four days prior to non-terminal peripheral blood mononuclear cell (PBMC) collection, 50 μg of antibody 1 suspended in 150 μl phosphate-buffered saline (PBS) was injected into each animal (N=12) by intraperitoneal route to stimulate antigen-specific antibody-secreting cells (ASCs).

[0084] Blood collection and concentration Blood was collected from each animal and processed for single cell isolation and screening. Table 1 lists the mouse strains and the amount of blood collected from each mouse.

[0085] [Table 1]

[0086] The collected blood was then processed to enrich the B cell pool. First, red blood cells (RBCs), platelets, and serum plasma were removed from the collected blood using a RedSift cell processor instrument (Aviva Systems Biology, Corp., San Diego, CA). Then, EasySep™ Mouse B Cell Isolation Kit (STEMCELL Technologies, Inc., Vancouver, British Columbia) was run according to the manufacturer's protocol to further enrich for B cells. An additional step was performed using an in-house generated rat anti-mouse IgM mAb (clone 8M3.1) to effectively remove naive B cells expressing cell surface IgM. This additional step allowed further enrichment of antigen-specific class-switched IgG-secreting cells within the ASC population.

[0087] The enriched B cell pool was incubated with a fluorescently labeled anti-CD138 antibody to label cells of the plasma B cell lineage. Although high levels of CD138 expression have proven to be the most reliable indicator of IgG secretion from PBMC-derived B cells, it is contemplated herein that other cell surface markers (e.g., B220, CD19, IgG.TACI, SLAM7, BCMA, CD98, SCA-1, Ly6C1 / 2, etc.) or combinations of markers may be used to identify specific B cell populations of interest. Tellier et al., Eur J Immunol 47(8):1276-1279(2017).

[0088] Single-cell screening assays The fluorescently labeled cells were then loaded onto an OptoSelect™ chip (Berkeley Lights, Inc., Emeryville, CA) using the Beacon® optofluidic platform (Berkeley Lights, Inc., Emeryville, CA). The platform steers individual B cells into individual pens of the OptoSelect™ chip by optoelectronic positioning (OEP). The chip has 3513 individual pens, each with a volume of approximately 740 picoliters and a unique pen identification number. ASCs loaded onto the chip were identified by expression of CD138 using the on-board optics of the Beacon® optofluidic platform. Using this technique, individual B cells were sequestered into separate pens of the chip such that antibodies secreted by individual B cells were isolated. Antibodies produced and secreted by one B cell were not mixed with antibodies produced and secreted by another B cell. This "one ASC per pen" relationship allows phenotypic characterization of antibodies produced by single B cells, and because ASCs have specific genotypes, phenotype-genotype correlations can be made. Due to the extremely small volume of the pen and the rapid secretion rate of plasmablasts and plasma cells (WenerFaver, et al., Eur J Immunol 23(8):2038-2040(1993)), the concentration of ASC-derived antibodies in each pen rose rapidly. Levels of antibodies sufficient for screening the desired characteristics (e.g., phenotype) of selected antibodies were reached within 5-15 minutes.

[0089] ASCs expressing relevant antigen-specific antibodies (selection antibodies) can be identified using a series of iterative homogenous screens. Depending on the desired antibody properties of the selection antibodies, these screens may be simple binding assays (e.g., antigen binding, species cross-reactivity, etc.) or may be designed to identify antibodies that achieve additional design goals (e.g., ligand blocking, competition, function, etc.). Here, a homogenous bead-based competitive assay was performed to identify ASCs secreting anti-ID ab against antibody 1 (selection antibody). The assay is shown in Figure 5A-5C. In this assay, a capture reagent containing an anti-mouse IgG antibody (anti-mu IgG) bound to 3.2 μm polystyrene beads (Spherotech Inc, Lake Forest, IL) was mixed with a detection reagent containing anti-mouse IgG labeled with fluorophore A, a labeled target containing antibody 1 labeled with fluorophore B, and an excess of human serum (10% normal human serum). See Figure 5A. Serum was included to provide competitive binding conditions. Without wishing to be bound by any particular theory, under such competitive binding conditions, antibodies specific for epitopes outside the desired therapeutic IgG paratope do not bind to the labeled target (Fluorescent B-Labeled Antibody 1).

[0090] This assay mixture was then flowed through the chip microfluidic channels such that a bead was placed at the mouth of each pen containing an individually isolated ASC (Figure 5B). The pens containing cells that secreted the antibodies were then detected using the fluorescence imaging capabilities of the Beacon® optofluidic platform (Berkeley Lights, Inc., Emeryville, CA) and a filter capable of detecting Fluorescence A. As the antibody levels from the ASCs increased, they diffused out of the pen's mouth, where they were captured (and concentrated) by the capture reagent. As the amount of antibody bound to the beads increased, the anti-mouse IgG antibodies bound to Fluorescence A were then concentrated, resulting in a characteristic fluorescent "bloom" pattern that was concentrated at the mouth of the pen of interest (the pen containing the ASC secreting the IgG antibody; Figure 5C). The 82 pens harboring the ASCs secreting the IgG antibody were identified by the Fluorescence A bloom and their pen identification numbers were recorded. To identify the ASCs secreting antibodies specific to Antibody 1 (the selected antibody), a second fluorescence filter cube was used to detect the Fluorescence B signal. Twenty-three ASCs expressing select antibodies specific to Antibody 1 that bound to the labeled target in the presence of human serum were shown by fluorescence B bloom (Figure 5C).

[0091] Sequencing, cloning, and recombinant expression To validate the selected antibodies, 23 ASCs were individually transferred from the OptoSelect™ chip pen using OEP and exported into separate wells of a standard 96-well plate containing cell lysis buffer using the integrated microfluidics of the Beacon® optofluidic platform (Figure 6). For the antibodies produced by the ASCs in each well, the sequences of the corresponding antibody heavy chain (HC) and light chain (LC) variable regions were determined by single-cell RT-PCR, following the protocol substantially described in Winters et al., 2019 (supra). The sequences were then cloned into mammalian expression vectors carrying the antibody constant regions. One vector carried the HC variable region and the antibody constant region, and the second vector carried the LC variable region and the antibody constant region. The recombinant antibody HC / LC pairs were then transfected into 293T cells and expressed in the culture supernatant as soluble antibodies.

[0092] The antibodies in the culture supernatants were then tested for binding to Antibody 1 by sandwich ELISA in the presence of human serum, as substantially described in Winters et al., 2019 (supra) and shown in Figure 7A. Using this method, nine antibodies were identified that had the desired characteristics and could act as anti-ID antibodies to Antibody 1. Of the nine anti-ID candidates, a single antibody (Ab287) had the best profile, exhibiting a potential lower limit of quantification (LLOQ) of 0.5 ng / ml in clinical patient samples. The performance of Ab287 in the sandwich ELISA in the presence of serum from different sources is shown in Figure 7B. As expected for an anti-ID binding to the paratope of Antibody 1, the anti-ID antibody Ab287 blocked Antibody 1 from binding to its target (PD-1) with an EC50 of 233.9 nM (Figure 7C). Ab287 was expected to measure free bioactive Antibody 1 in clinical samples and was therefore selected for further development.

[0093] Example 2 This example describes another exemplary method for monitoring the production of selected antibodies in mice.

[0094] Immunization Protocol In this example, the antibody of choice was an anti-human EGFR antibody. CD1 mice were immunized with the soluble extracellular domain of human EGFR (huEGFR) a total of four times at two-week intervals. The first immunization consisted of 50 μg of human huEGFR emulsified in 100 μl of complete Freund's adjuvant and was injected subcutaneously into two spots on the back of each mouse. 14 days later, 25 μg of huEGFR was suspended in 200 μl of Sigma Adjuvant System, and 100 μl of this mixture was injected subcutaneously into two spots on the back of each mouse, and the remaining 100 μl was injected intraperitoneally. The third immunization was delivered 14 days later, using the same route and adjuvant as the second, except that the total amount of huEGFR was reduced to 15 μg. The fourth boost contained 50 μg of huEGFR in the absence of adjuvant and was delivered by both subcutaneous and intraperitoneal routes.

[0095] Blood collection and concentration Blood was collected 1–8 days after the final boost. ASCs were enriched using a magnetic CD138 positive selection kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer's protocol.

[0096] Single-cell screening assays The enriched B cells were mixed with a capture reagent comprising goat anti-human Fc bound to beads, a detection reagent comprising a goat anti-human Fc antibody labeled with Alexa 488, which produces a green fluorescent signal, and a labeled target comprising EGFR labeled with Alexa 594, which produces a red fluorescent signal. This mixture was then transferred to a single well of a 384-well plate and the components of the mixture were allowed to settle in the well for approximately 10 minutes.

[0097] To identify specific ASCs, imaging of the cells was performed using an Incucyte live cell analysis system. Figure 8A provides an exemplary image of green fluorescent signal indicative of antibody secretion, and Figure 8B provides an exemplary image of red fluorescent signal demonstrating antigen specificity of the antibody. Figure 8C provides an exemplary analysis composite image of the same well shown in Figures 8A-8B, where the green fluorescent signal indicative of antibody secretion is shown in magenta, the red fluorescent signal is shown in cyan, and the overlap of the green and red signals is shown in royal blue. From this imaging assay, 10 cells were found to exhibit antibody secretion, with only one cell shown as secreting antibody that was antigen (EGFR) specific.

[0098] Example 3 In this example, we describe another single cell imaging assay to identify select ASCs, where the target is expressed by cells in their native conformation.

[0099] To generate anti-CB-1 antibodies, mice were immunized with CB-1. Blood samples were taken from the immunized mice and then enriched for IgG-secreting B cells, substantially as described in Example 2. 293T cells transfected with a vector encoding full-length CB1 using 293fectin were washed with culture medium and then passed through a 40 μm strainer. A mixture of enriched B cells, CB-1-expressing 293T cells, and Alexa 488-labeled goat anti-human Fc antibody was then added to the wells and allowed to settle as a monolayer. Fluorescent signals on the transfected cell surface were detected using an Incucyte imaging system to identify specific ASCs. Figure 8D is an exemplary image of labeled transfected cells with multiple fluorescent spots, in which the antigen expressed by 293T cells binds to the antibody produced by B cells and labeled with Alexa 488-labeled goat anti-human Fc antibody. These results demonstrated that the enriched B cell pool contained cells secreting antibodies specific to CB-1.

[0100] Example 4 This example describes an exemplary method of inducing antibody production in a non-human animal to generate a selected antibody, in this example a human-cynomolgus cross-reactive IgG antibody specific for TNF-alpha.

[0101] To generate human-cynomolgus cross-reactive antibodies, animals are immunized with alternating boosts of human and cynomolgus versions of antigens. This immunization method is based on the assumption that epitopes shared between human and cynomolgus antigens are consistently presented to the immune system during each boost, allowing for sustained stimulation of relevant B cells that code for cross-reactive antibodies. Reactivity to each antigen can be easily monitored using simple binding assays and polyclonal sera from immunized animals. However, since animals are immunized with both antigens and each antigen has both common and unique epitopes, polyclonal sera contain antibodies that react with human antigens, antibodies that react with cynomolgus antigens, and / or antibodies that react with human and cynomolgus antigens. The presence of cross-reactive antibodies cannot be determined by assaying polyclonal sera. This problem is overcome by examining isolated single ASCs derived from a PBMC population. Single cell assays screen for ASCs that secrete truly cross-reactive antibodies.

[0102] Immunization Protocol A complete immunization campaign consists of four injections, delivered at two-week intervals over a period of 50 days. The first and third injections use complete Freund's adjuvant followed by recombinant human TNF-alpha (catalog no. 300-01A; PeproTech®; Rocky Hill, NJ) emulsified with Sigma Adjuvant System® (catalog no. S6322; Sigma-Aldrich, St. Louis, MO). The second and fourth injections use complete Freund's adjuvant followed by recombinant cynomolgus TNF-alpha (catalog no. RP1021Y-005, Kingfisher Biotech, Inc., St. Paul, MN) emulsified with Sigma Adjuvant System® (catalog no. S6322; Sigma-Aldrich, St. Louis, MO). For the first injection, approximately 50 μg of human TNF is suspended in adjuvant and injected subcutaneously into two spots on the dorsal side of each mouse. 14 days later, a second injection with 50 μg of cynomolgus TNF suspended in adjuvant is injected subcutaneously into two spots on the dorsal side of each mouse. 14 days after the second injection, a third injection containing 25 μg of human TNF is suspended in 200 μl of Sigma Adjuvant System, 100 μl of this mixture is injected subcutaneously into two spots on the dorsal side of each mouse, and the remaining 100 μl is injected intraperitoneally. 14 days later, a fourth injection containing 25 μg of cynomolgus TNF is suspended in 200 μl of Sigma Adjuvant System, 100 μl of this mixture is injected subcutaneously into two spots on the dorsal side of each mouse, and the remaining 100 μl is injected intraperitoneally. Bridging ELISA analysis of serum titers from each mouse is performed to confirm antigen reactivity and inform animal selection for non-terminal antibody generation. Four days prior to non-terminal peripheral blood mononuclear cell (PBMC) collection, each animal (N=12) is injected intraperitoneally with a solution containing 25 μg human TNF and 25 μg cynomolgus TNF suspended in 150 μl PBS to stimulate antigen-specific antibody-secreting cells (ASCs).

[0103] Blood collection and enrichment and single cell screening assays Blood is drawn from each mouse and enriched for B cells substantially as described in Example 1. Labeled cells are loaded onto an OptoSelect™ chip (Berkeley Lights, Inc., Emeryville, Calif.) using the Beacon® optofluidic platform, and individual B cells are sequestered into separate pens of the chip such that antibodies secreted by individual B cells are isolated.

[0104] To identify ASCs secreting the antibodies of choice (anti-TNF antibodies reactive to human TNF and cynomolgus TNF), a homogeneous bead-based competitive assay as described in Example 1 is performed. First, a capture reagent containing beads bound to anti-mouse IgG is mixed with a detection reagent containing Fluor A-labeled anti-mouse IgG, a labeled target containing human TNF labeled with Fluor B, and an excess of human serum. This assay mixture is then flowed through the chip microfluidic channels such that a bead is placed in the mouth of each pen containing individually isolated ASCs. Fluor A blooms indicate pens carrying ASCs secreting IgG antibodies, while Fluor B blooms indicate pens carrying ASCs secreting antibodies that bind to human TNF. The pen ID numbers of the pens indicated by each bloom type are identified and recorded.

[0105] In the second part of the bead-based assay, add detection reagent containing cynomolgus TNF labeled with fluorescent C. Fluorescent C bloom indicates pens harboring ASCs secreting antibodies that bind to cynomolgus TNF. Record the pen ID number of the pen indicated by fluorescent C bloom.

[0106] The pens that are described as positive for all three blooms (fluorescence A bloom, fluorescence B bloom, and fluorescence C bloom) are selected as candidate ASCs that secrete the selected antibody. The candidate ASCs are individually transferred from the pen of the OptoSelect™ tip using OEP and exported to individual wells of a standard 96-well plate containing cell lysis buffer using the integrated microfluidics of the Beacon® optofluidic platform, substantially as described in Example 1. The HC and LC variable regions of the antibodies produced by each candidate ASC are determined by single-cell RT-PCR. The sequences are cloned into vectors, which are then transfected into 293T cells. The antibodies in the culture supernatant are collected and then tested for cross-reactivity to human TNF and cynomolgus TNF in functional assays.

[0107] If no pen is positive for all three blooms, identify a pen that is double positive for Fluorescent A Bloom and Fluorescent B Bloom. Alternatively, identify a pen that is double positive for Fluorescent A Bloom and Fluorescent C Bloom. Mice whose blood contains ASCs of the double positive pen are selected for a second immunization campaign. For mice that have obtained Fluorescent A and Fluorescent B double positive ASCs, the second immunization campaign includes the same immunization campaign as the first campaign (above), but the first and third injections are performed with half the amount of human TNF.

[0108] In mice from which double-positive ASCs (fluorescence A and fluorescence C) were obtained, the second immunization campaign included the same immunization campaign as the first campaign (above), but the second and fourth injections were performed with half the amount of cynomolgus TNF.

[0109] All steps following immunization (from blood sampling to bead-based assay) are then carried out as described in this example. Pens that are described as positive in all three blooms (fluorescence A bloom, fluorescence B bloom, and fluorescence C bloom) are selected as candidate ASCs that secrete antibodies with the target phenotype. The variable regions are sequenced and cloned into vectors, the vectors are transfected into cells for recombinant antibody production, and the recombinantly produced antibodies are tested for the target phenotype.

[0110] If triple positive pens are still not identified, a third immunization campaign is designed and performed on the same mice that received the second immunization campaign. In the third immunization campaign, the second and fourth injections are performed with increasing amounts of cynomolgus TNF for fluorescence A / fluorescence B double positive mice, and the first and third injections are performed with half or quarter amounts of human TNF for fluorescence A / fluorescence C double positive mice, and the first and third injections are performed with increasing amounts of human TNF, and the second and fourth injections are performed with half or quarter amounts of cynomolgus TNF for fluorescence A / fluorescence C double positive mice. Following the third campaign, all steps following immunization (from blood collection to bead-based assay) are then performed as described in this example. Pens that are described as positive in all three blooms (fluorescence A bloom, fluorescence B bloom, and fluorescence C bloom) are selected as candidate ASCs that secrete antibodies with the target phenotype. If triple positive pens are still not identified after the third campaign, a fourth immunization campaign is designed and carried out. This process is repeated until antibodies with the target phenotype are identified.

[0111] This method advantageously provides the capability of long-term live B cell profiling, enabling repertoire steering. The developing B cell response of immunized animals is monitored in real time, and this information is used to iteratively modify immunization strategies. Because this method is non-terminal, the animal is not killed, and the power of the immune system can be harnessed to continue to evolve B cell responses toward a desired outcome. Modifications to immunization strategies include (but are not limited to) different forms of immunogen, adjuvants, immunomodulators, doses of antigen, timing of immunization, and routes of administration. In this scenario, initial immunization attempts using human antigens failed to elicit B cells that produce antibodies that cross-react with the cynomolgus antigen, as determined by non-terminal ASC screening of PBMCs. This method provides repertoire quality information, and can therefore be used to modify immunization strategies. In this example, the immunogen could be switched from the human antigen to the cynomolgus orthologue, and the immunization campaign continued until B cells expressing cross-reactive antibodies were identified. Animals in which the desired B cell repertoire has been induced can be used for antibody generation using conventional strategies or non-terminal ASC methods as described herein.

[0112] Example 5 This example describes another method of inducing antibody production in a non-human animal to generate a selected antibody, in this example a human-cynomolgus cross-reactive IgG antibody specific for antigen X.

[0113] In this example, we describe the identification of antibodies that cross-react with both the human and cynomolgus (cyno) orthologues of antigen X. Orthologues have low sequence homology, and therefore the generation of cross-reactive antibodies is rare. Immunization with one antigen may produce some cross-reactive antibodies, but they will be below the detection level of standard serum titers. Alternatively, co-immunization with both human and cynomolgus antigens will generate antibodies that bind primarily to the human or cynomolgus orthologue, but few that cross-react. Standard serum titers do not distinguish between mice that have generated cross-reactive antibodies and mice that have generated antibodies that bind independently to the human or cynomolgus antigen. Therefore, single cell screening is required to identify true cross-reactive antibodies in responding mice. This is combined with selective amplification of the B cells of interest for efficient recovery.

[0114] Mice are immunized with human antigen X for a total of four injections at two-week intervals. For the first boost, 50 μg of human antigen X is emulsified in 100 μl of complete Freund's adjuvant and the mixture is administered subcutaneously. 14 days later, 25 μg of human antigen X is suspended in 200 μl of Sigma Adjuvant System and 100 μl is injected subcutaneously and 100 μl is injected intraperitoneally. For the third injection, 15 μg of human antigen X is emulsified in Sigma Adjuvant system and injected both subcutaneously and intraperitoneally as described for the second boost. A final boost of 50 μg of human antigen X is injected intraperitoneally without adjuvant.

[0115] Blood is collected 4 days after the final boost, with a final volume of 10% of rodent body weight. CD138+ B cells are magnetically isolated and added to a mixture of capture reagents comprising anti-human IgG antibodies bound to beads, detection reagents comprising anti-human IgG antibodies labeled with Alexa488, and separately labeled fluorescent human antigen X and cynomolgus antigen X. This mixture is plated as a monolayer on a microtiter plate and then incubated to allow antibody and antigen capture. Antigen-specific cross-reactive ASCs are identified as double-stained fluorescent plaques using cell imaging, substantially as described in Example 2.

[0116] Animals that have produced B cells secreting cross-reactive antibodies are then immunized once a week for an additional four weeks with alternating subcutaneous administrations of cynomolgus and human antigens in combination with the Sigma Adjuvant system®. Blood is taken three days after the final boost and B cells are isolated for screening against human and cynomolgus antigens.

[0117] Animals that show increased numbers of cross-reactive antibodies are euthanized for tissue harvest and antibody generation. Animals with low ratios of cross-reactive to monoreactive antibodies are immunized with additional doses of human and cynomolgus orthologues of antigen X for an additional 3 weeks, followed by single cell screening for cross-reactive antibodies. This process continues until a threshold % of ASCs producing the selected antibody (human-cynomolgus cross-reactive antibody) is met.

[0118] The described method can be applied to any antibody discovery campaign that requires cross-reactivity against multiple orthologs or paralogs of a protein. The need to generate antibodies that cross-react with different species is common for efficacy and safety studies. Single B cell screening can be applied to programs that require cross-reactivity against rats, rabbits, guinea pigs, dogs, cats or pigs, as common examples.

[0119] Example 6 This example describes a method for inducing antibody production in a non-human animal to generate select antibodies, in this example, antibodies that bind only to one paralog of a protein, antigen X, but not to a closely related family member, antigen Y.

[0120] Due to the similarity between antigen X and antigen Y, animals immunized with antigen X show polyclonal serum cross-reactivity to both proteins. Therefore, direct single-cell screening is required to identify mice that are likely to generate biased antibody responses against the family member of interest. Selected animals are further immunized using a different immunization protocol to steer the immune response to maximize the generation of B cells that produce antibodies that react only to antigen X.

[0121] Rodents are immunized with antigen X subcutaneously twice weekly for four weeks. The priming immunogen complex contains 10 μg of antigen combined with Freund's complete adjuvant, while the boosting complex contains 5 μg of antigen combined with Sigma Adjuvant System®. Four days after the final boost, blood is collected and CD138+ B cells are isolated from the serum.

[0122] The cells are assayed by single cell assay as described in Example 1, screened for binding to antigen X using fluorescent A tagged antigen X and / or screened for binding to antigen Y using fluorescent B labeled antigen Y. Antigen A bloom indicates pens containing ASC secreting antibodies that secrete antibodies specific to antigen X, and fluorescent B bloom indicates pens containing ASC secreting antibodies that bind to antigen Y. Pens that are only fluorescent A bloom positive (but not fluorescent B bloom) are exported from the pen by OEP to wells for single cell PCR, substantially as described in Example 1. Antibodies produced by the ASC are assayed for binding to antigen X and non-binding to antigen Y.

[0123] If the pen is not single positive for Fluorescent A Bloom, the conserved domain of antigen X is bioinformatically identified. Animals are subcutaneously immunized once a week with the conserved antigen X domain in combination with Sigma Adjuvant System®, with three further boosts. Three days after the last boost, rodents are bled and B cells are enriched, essentially as described in Example 1. Cells are screened at the single cell level to identify B cells that secrete antibodies that bind only to antigen X, but not to antigen Y. In the single cell assay, the conserved antigen X domain, labeled with fluorescent C, is used as the labeling target. Fluorescent C Bloom indicates pens that contain ASCs that secrete the selected antibody (antibody specific for antigen X and that does not cross-react with antigen Y).

[0124] Rodents with improved numbers of B cells expressing the antibody of choice are euthanized for tissue collection, and mice with unimproved numbers are immunized a third time.

[0125] Example 7 This example describes a method for inducing antibody production in a non-human animal to generate select antibodies, in this example, that compete with native human PD-L1 and native human PD-L2 for binding to human PD-1.

[0126] Rodents are immunized twice weekly with decreasing doses of PD-1 antigen as described in Example 6.

[0127] Individual B cells are transferred to pens of the chip to achieve a ratio of one cell to one pen. A bead-based assay is performed with a capture reagent comprising beads bound to anti-mouse IgG, a detection reagent comprising fluoro-A labeled anti-mouse IgG antibodies, a labeled target comprising human PD-1 labeled with fluoro-B, and an excess of human serum. This assay mixture is then flowed through the chip microfluidic channels such that a bead is placed in the mouth of each pen containing individually isolated ASCs. Fluorescent A bloom indicates a pen holding an ASC that secretes IgG antibodies, and fluorescent B bloom indicates a pen holding an ASC that secretes antibodies that bind to PD-1. Record the pen ID numbers of pens that show fluorescent A bloom, fluorescent B bloom, or double positive fluorescent A and fluorescent B bloom.

[0128] The bead-based assay is performed a second time, only this time increasing amounts of PD-L1 are added to the assay. Fluorescence A / fluorescence B double bloom identifies pens containing ASCs producing PD-1 specific antibodies, and maintenance of signal intensity in the presence of PD-L1 indicates that the recombinantly produced antibodies outcompete PD-L1 for binding to PD-1. Record the pen number of pens that show double bloom with maintained signal intensity in the presence of PD-L1.

[0129] The bead-based assay is performed a third time, only this time increasing amounts of PD-L2 are added to the assay. Fluorescence A / fluorescence B double bloom identifies pens containing ASCs producing PD-1 specific antibodies, and maintenance of signal intensity in the presence of PD-L2 indicates that the recombinantly produced antibodies have outcompeted PD-L2 for binding to PD-1. Record the pen number of the pen that shows double bloom with maintained signal intensity in the presence of PD-L2. Desirably, there are pens identified as containing ASCs producing PD-1 specific antibodies that can outcompete both PD-L1 and PD-L2 for binding to PD-1.

[0130] Pens that are double positive for Fluorescence A and Fluorescence B bloom are noted, and the HC and LC variable regions of the antibodies from these pens are sequenced. The sequences are cloned into a vector, and the vector is transfected into 293T cells for the production of recombinant antibodies. The antibodies are harvested from the supernatant of the 293T cell culture and then tested for binding to recombinant PD-1 with increasing amounts of PD-L1, where PD-1 is labeled with a fluorophore that emits a signal at a given wavelength, and the recombinantly produced antibodies are bound to beads as in an immunoprecipitation assay. The labeled PD-1 is mixed with the antibody bound to the beads. The beads are washed for non-specific binding. Immune complexes containing labeled PD-1 and recombinantly produced antibodies are detected by detecting the signal at a given wavelength. This procedure is then performed with increasing amounts of PD-L1 and / or PD-L2. Maintenance of signal strength in the presence of PD-L1 and / or PD-L2 indicates that the recombinantly produced antibody outcompetes PD-L1 and PD-L2 for binding to PD-1.

[0131] Animals producing B cells that bind PD1 but do not fully compete with PD-L1 or PD-L2 are immunized with 2.5 μg of PD1 in combination with Sigma Adjuvant System® and given three further boosts. Mice are bled three days after the last boost and the isolated B cells are screened as above.

[0132] Example 8 This example describes a method for inducing antibody production in a non-human animal to generate a selected antibody, in this example, an antibody that has a particular binding affinity for antigen X.

[0133] The goal of this experiment is to identify antibodies that bind antigen X with sub-picomolar affinity. Affinity can only be measured at the clonal source of the antibody, and therefore serum cannot be used to identify mice that generated high affinity antibodies. Traditionally, mice are euthanized to obtain B cells for hybridoma fusion and characterization, eliminating any further immune steering. Combining real-time non-terminal B cell harvesting and interrogation with adaptive immunization strategies offers significant advantages over traditional methods for generating high affinity antibodies, as it exploits the competitive in vivo environment to evolve and induce higher affinity B cell clones.

[0134] Rodents are subcutaneously immunized with decreasing doses of antigen X for a total of four boosts every two weeks. The priming immunogen contains 40 μg of antigen X combined with Freund's complete adjuvant. The three subsequent boosts contain 20 μg, 10 μg or 5 μg of antigen X combined with Sigma Adjuvant Systems®. Four days after the final boost, blood is collected from the rodents and B cells from the collected blood sample are enriched substantially as described in Example 1. The cells are penned and screened for binding to fluorescently labeled antigen X using a microfluidic device substantially as described in Example 1. ASCs producing anti-antigen X antibodies are identified and then transferred from the pen to wells for molecular recovery and the affinity of the recombinant clones is determined substantially as described in Example 1.

[0135] Affinities are determined by KinExA (Sapidyne) or Carterra high throughput screening devices (Carterra).

[0136] Animals that have generated B cells expressing high affinity antibodies are then boosted weekly with 2.5 μg of antigen in combination with the Sigma Adjuvant System®, followed by three further boosts. Mice are bled three days after the final boost, and isolated B cells are screened for binding to antigen X, essentially as described in Example 1.

[0137] B cells are then exported for another round of sequencing, cloning, expression, and affinity characterization. Rodents with B cells that match the affinity bar are euthanized for tissue collection. Rodents that generate B cells that do not meet the affinity requirements are boosted once a week with 2.5 μg of antigen combined with Sigma Adjuvant System®, with three additional boosts. Animals are screened and boosted until the design goal is achieved. In one screening round, enriched B cells are subjected to the single-cell assay described in Example 2, except that the labeled target contains antigen X labeled with Alexa 594 instead of EGFR. Spots that show signal overlap identify ASCs that secrete antibodies specific to antigen X. The single-cell imaging assay is repeated with an amount of antigen X labeled with Alexa 488 that is approximately 1 / 10 less than the amount of antigen X labeled with Alexa 594. Spots that retain overlapping signals (from Alexa 594 and Alexa 488) indicate a higher affinity for antigen X, and therefore the desired high affinity for the target.

[0138] Example 9 This example describes a method for inducing antibody production in a non-human animal to generate a selected antibody, which in this example is a domain-specific antibody.

[0139] This example describes the identification of antibodies against domain 1 of the multi-domain human protein, protein Z. Immunization with the complete extracellular domain of protein Z produces an unbalanced immune response, with some domains being highly over-expressed and domain 1 antibodies undetectable at polyclonal serum titer levels. Immunization with domain 1 alone does not generate antibodies capable of recognizing the native extracellular domain. Single cell screening is required to identify mice that produced rare domain 1 antibodies after immunization with the native extracellular domain of the protein. These B cells are amplified with subsequent boosting of domain 1 peptide and full-length protein to expand domain 1-reactive B cells, but do not generate a de novo response to the peptide alone.

[0140] Mice are immunized with Protein Z for a total of four boosts spaced two weeks apart. The priming boost contains 50 μg of antigen emulsified in Complete Freund's Adjuvant (CFA) and is administered subcutaneously. Subsequent boosts contain 25 μg and 15 μg of antigen emulsified in SAS, respectively, half administered intraperitoneally and half subcutaneously. Mice are then boosted with 50 μg of antigen in PBS and blood is collected four days later. CD138+ B cells are isolated and added to a mixture of IgG capture beads, separately labeled fluorescent domain 1 peptide, and complete extracellular domain. This mixture is plated as a monolayer on a microtiter plate and then incubated to allow antibody and antigen capture, substantially as described in Example 2. ASCs producing domain 1-specific antibodies are identified as double-stained fluorescent plaques using cell imaging (Example 2).

[0141] Mice that generate domain 1-specific B cells are boosted with 5 μg of domain 1 peptide twice a week for 2 weeks. Mice are then boosted with 50 μg of complete soluble extracellular domain in PBS and blood is taken 4 days later. Mice are screened for binding to both domain 1 and full-length protein. Animals of interest are euthanized for tissue processing and screening. This process is repeated for other mice until the design goal is achieved.

[0142] The described method can be applied to antibody discovery campaigns where the immune response is primarily against regions of a protein of little interest. A common example is a protein with an immunodominant region that is over-represented in the antibody repertoire. The immune response needs to be steered away from that domain and towards the region of interest.

[0143] Example 10 This example describes a method for inducing antibody production in a non-human animal to generate a select antibody, in this example, an antibody that binds to the multimerization domain of a multimeric antigen.

[0144] This experiment describes the identification of antibodies that bind to a heterotrimeric transmembrane protein. Immunization with the native protein fails to elicit an immune response. Mice immunized with only the soluble domain develop an immune response, but the antibodies do not recognize the native conformation of the protein. Mice are immunized with a series of immunogens engineered to be increasingly closer to the native conformation: Immunogen 1 contains a protein composed of the extracellular domain of antigen X, Immunogen 2 contains a protein that binds to a multimerization domain to form a heterotrimeric complex, and Immunogen 3 contains DNA encoding the complete complex.

[0145] Rodents are subcutaneously immunized with 5 μg of Immunogen 1 in complex with Sigma Adjuvant System®. Animals are boosted twice weekly for a total of six times. Four days after the last boost, blood is collected from the mice and CD138+ B cells are magnetically isolated. Cells are added to a mixture of IgG capture beads and separately labeled fluorescent Immunogen 1 and Immunogen 2. This mixture is plated as a monolayer on a microtiter plate and then incubated to allow antibody and antigen capture, essentially as described in Example 2. Antigen-specific ASCs are identified as double-stained fluorescent plaques using cell imaging (Example 2). Mice that produce rare antibodies that naturally cross-react with Immunogen 2 are identified for further steering. These mice are further boosted with 5 μg of Immunogen 2 in complex with Sigma Adjuvant System® twice weekly for a total of six times to amplify the response. Four days after the last boost, rodents are bled and B cells are isolated for single cell screening against Immunogen 2 and Immunogen 3. Animals harboring B cells encoding antibodies that recognize Immunogen 3 are genetically immunized with a plasmid encoding the full complex. These rodents are boosted twice weekly with gene gun bullets for a total of six times. Blood is taken from the mice for single cell screening, and mice of interest are euthanized and tissues harvested. Mice producing weaker responses are boosted and screened until the design goal is achieved.

[0146] Example 11 This example describes an exemplary application of a single cell assay to rank ASCs by the affinity of antibodies secreted by the ASCs.

[0147] Hybridoma clones producing EGFR-specific antibodies were isolated and the EGFR-binding properties of each clone were determined using the Octet® Biolayer Interferometry Platform (Satorius). The range of EGFR-binding affinities (KD4.7×10 -10 ~1.1×10 -8Five hybridoma clones producing antibodies against the IgG antibody were selected for evaluation in the single cell assay. The selected hybridoma clones and the binding characteristics of the antibodies produced by each are listed in Table 2.

[0148] [Table 2]

[0149] A single cell screening assay was performed using the hybridoma clones in Table 2 as described in Example 2. Briefly, the hybridoma 12B4.1 clone was mixed with a capture reagent comprising goat anti-human Fc bound to beads, a detection reagent comprising goat anti-human Fc antibody labeled with Alexa 488, which generates a green fluorescent signal, and a labeled target comprising EGFR labeled with Alexa 594, which generates a red fluorescent signal. The mixture comprising the capture reagent, detection reagent, labeled target, and 12B4.1 clone was then transferred to a single well of a 384-well plate. These steps were performed with each of the hybridoma clones in Table 2 such that each well of the plate contained a mixture containing a single hybridoma clone (e.g., one well with hybridoma 1C2.1 clone, one well with hybridoma 7C11.1 clone, one well with hybridoma 2G8.1 clone, one well with hybridoma 12B4.1 clone, and one well with hybridoma 7.35.4 clone). After the mixture components were placed in the wells, the cells were imaged using an Incucyte live cell analysis system to determine green and red fluorescent relative fluorescence unit (RFU) values ​​for six individual cells (ASC) in each well. Example images are shown in Figure 9. RFU values ​​were recorded and the ratio of green RFU (indicative of IgG secretion) to red RFU (indicative of EGFR binding) was determined for data normalization (Table 3). Normalization of RFU is important because IgG secretion levels can be affected by cell health, cell cycle, and other characteristics of ASCs. The RFU ratios of the six clones of a single hybridoma were averaged and reported as the mean ratio (Table 3).

[0150] [Table 3]

[0151] [Table 4]

[0152] The ratio means for each hybridoma were then plotted as a function of its KD value from Table 2 (Figure 10). As shown in Figure 10, the ratio means and KD values ​​(determined with the Octet® Biolayer Interferometry Platform (Satorius)) were compared to determine statistical significance (R 2 = 0.932). Taken together, these results indicate that the normalized RFU of single clones is useful for ranking antibody affinities. Furthermore, these results suggest that the single cell assay of the present disclosure can be used to rank individual ASCs by the affinity of the antibodies they secrete.

[0153] Example 12 This example describes how to induce an immune response in mice to generate antibodies that cross-react with both the human and cynomolgus monkey (cyno) orthologues of an antigen. This example explores the impact of different immunization strategies on the formation of cross-reactive antibodies and the ability of single-cell screening strategies to detect those changes. This example also demonstrates the application of single-cell assays in immune steering.

[0154] immunization CD1 mice were immunized every 2 weeks with human orthologues of antigens. For the first boost, mice were immunized subcutaneously with 25 μg of human antigen in Complete Freund's Adjuvant (CFA). The second boost contained 25 μg of human antigen combined with 50% Sigma Adjuvant System (SAS), half administered subcutaneously and half intraperitoneally. The third dose contained 15 μg of human antigen combined with 50% SAS, half administered subcutaneously and half intraperitoneally. Mice were rested for 14 weeks and then boosted intraperitoneally with 25 μg of human antigen (in phosphate buffered saline (PBS)) without adjuvant. Blood was collected 4 days after the boost for serology and single cell screening (Figure 11, bleed 1).

[0155] Mice were then divided into two groups: Group 2 was immune steered toward the production of human / cynomolgus cross-reactive antibodies by boosting subcutaneously with cynomolgus antigen once a week, while Group 1 served as the control group and was boosted with human antigen. Both groups of mice were bled 4 days after the 8th boost (FIG. 11, bleed 2) and blood was prepared for analysis.

[0156] After the eighth boost, mice from group 1 were split into two subgroups (groups 1A and 1B) and given a single boost of either cynomolgus antigen (group 1B) or human antigen (group 1A) without adjuvant. Mice from both subgroups were bled four days after the boost (Figure 11, bleed 3) and blood was prepared for analysis.

[0157] Blood preparation and cell enrichment Blood was drawn at the times indicated in Figure 11 (Draw 1, Draw 2, and Draw 3). In each case, the blood was centrifuged to separate serum from the blood cells. Serum was used for serum titer analysis as described below, and blood cells were enriched for ASCs by enriching for CD138+ B cells using a modified version of the standard protocol for the CD138 Enrichment Kit (STEMCELL Technologies, Inc., Vancouver, British Columbia).

[0158] Single-cell screening assays Single-cell screening assays were performed with the enriched CD138+ B cell population as described in Example 2. Briefly, the enriched CD138+ B cell population was mixed with a capture reagent comprising goat anti-mouse IgG Fc coupled to 3.4 μm polystyrene beads (Spherotech Inc, Lake Forest, IL), cynomolgus antigen labeled with Alexa 488, which produces a green fluorescent signal, and human antigen labeled with Alexa 594, which produces a red fluorescent signal, to a final concentration using B cell medium as diluent. The use of different fluorescent signals (green for cynomolgus antigen and red for human antigen) allowed the single cell assay to distinguish between single cells that bind only the human ortholog, single cells that bind only the cynomolgus ortholog, and single cells that bind both orthologs. The mixture was transferred to a single well of a 384-well plate, with a final enriched B cell concentration of approximately 2-3 μL of cell mixture per well. The components of the mixture were allowed to sit in the wells for approximately 10 minutes, after which the cells were imaged using an Incucyte live cell analysis system. The RFU values ​​of green and red fluorescence were determined.

[0159] Serum titer analysis Sera from bleeds 1 and 3 were diluted to final concentrations of 1:100, 1:1000 and 1:10,000 and then added to beads with captured biotinylated antigen plated in a V-bottom 96-well plate. The mixture was incubated for 1 hour at room temperature. The beads were then washed and resuspended in 30 μg of goat anti-mouse IgG Fc (Jackson Immunoresearch) to a final concentration of 5 μg / mL. After 15 minutes of incubation, the beads were washed and resuspended in FACS buffer. The plates were then prepared for flow cytometry.

[0160] Results and Discussion Mice in groups 1 and 2 were initially boosted in the same manner, and serum titer analysis showed that all mice generated strong immune responses to both the human and cynomolgus orthologues, but the titer data could not distinguish cynomolgus-only binders from human-cynomolgus cross-reactive binders (Figure 12, bleed 1). Single cell screening was used to identify the proportion of antibodies in each individual mouse that bound only human, only cynomolgus, or both cynomolgus orthologue and human orthologue (Figures 13 and 14, bleed 1). The dominant immune response was against the human orthologue, but cross-reactive antibodies could be readily identified in some mice (Figure 14).

[0161] The immune conditions were then shifted to determine whether single cell screening could detect changes in the antibody repertoire following immune steering. Group 1 mice continued to receive boosts with the human ortholog, while group 2 mice were boosted with the cynomolgus ortholog (Figure 11, bleed 2). The single cell screening assay was able to detect large changes in the proportion of antibodies bound to the cynomolgus ortholog vs. the human ortholog in group 2 mice (Figure 15), with the immune response now dominated by cynomolgus-only antibodies. Group 1 control mice had similar responses at bleeds 1 and 2 (Figures 14 vs. 15). There was no significant change in the production of cross-reactive antibodies in either group. These data indicate that the single cell screening assay has the resolution to detect changes in the immune response and that small changes in immunization strategy can shape the antibody repertoire.

[0162] Group 1 mice were then boosted one last time to determine whether the immune response could be directed toward increasing cross-reactive antibodies with minimal cynomolgus-only responses. Previous data showed that there was a strong de novo response to cynomolgus antigens and multiple boosts would not produce a necessary response. Therefore, to minimize the de novo response, group 1 mice were boosted once with 25 μg of protein without adjuvant. Group 1A received a boost with the human ortholog and group 1B received a boost with the cynomolgus ortholog. There was no change in the percentage of cross-reactive antibodies in mice that received the human boost (group 1A) compared to bleed 1 (Figures 16-17). The lack of variation in mice boosted only with human antigens highlights the reproducibility of single-cell screening. In contrast, the group boosted with cynomolgus (group 1B) saw a large increase in the percentage of cross-reactive antibodies (Figure 16) and a range of 2- to 16-fold increases in cross-reactive antibodies (Figure 17). The proportion of antibodies that bound only to cynomolgus monkeys was minimal, indicating that the single boost minimizes de novo generation of cynomolgus-only antibodies. All mice in the cynomolgus boost group (Group 1B) now achieved the design goal, but standard polyclonal serology did not identify the top animals (Figure 18). This is likely because the serum contained a polyclonal mixture of all antibodies generated by the immunization campaign, and newly formed ASCs contributed only a very small proportion to the repertoire.

[0163] Taken together, these results suggest that differential immune responses can be achieved by immune steering, for example by boosting with different orthologous antigens. These results further support that, unlike polyclonal serum titer analysis, which cannot distinguish between antibodies that bind only to the human ortholog, only to the cynomolgus ortholog, and antibodies that bind to both orthologs (cross-reacting with both the human and cynomolgus orthologs), the single cell screening assay of the present disclosure has the necessary resolution and sensitivity to detect changes in immune responses and immune repertoires, allowing for tracking or monitoring of antibody production, even when only small changes in immunization strategy occur.

[0164] Example 13 This example describes a method to steer the immune response to increase the proportion of antibodies that cross-react with both the human and cynomolgus orthologs of a multidomain protein.

[0165] Previous attempts have been made to generate human / cynomolgus cross-reactive antibodies against an antigen that is a multi-domain protein. The homology between the cynomolgus orthologue and the human orthologue is less than 80%. In previous attempts, human antigen boosting was alternated with cynomolgus antigen boosting, and although this approach resulted in a strong de novo response to each orthologue protein, very few cross-reactive antibodies were produced. In this example, mice were first boosted with the complete extracellular domain of the human protein (antigen 1), followed by boosting with a subdomain of the protein (antigen 2). The cynomolgus subdomain and the human subdomain have more than 80% homology.

[0166] immunization CD-1 mice were immunized with antigen 1 every 2 weeks for a total of 4 boosts (Figure 19). The first boost was 50 μg of antigen 1 emulsified in CFA and injected subcutaneously. The second boost was 25 μg of antigen 1 combined with 50% SAS, half delivered subcutaneously and the other half intraperitoneally. The third boost was 15 μg of antigen 1 combined with 50% SAS, half injected subcutaneously and half injected intraperitoneally. The fourth boost was 25 μg of antigen 1 without adjuvant. Four days later blood was taken for single cell screening (bleed 1). Two additional boosts were administered after bleed 1, each with 25 μg of antigen 2, and blood was taken 4 days later (bleed 2 and bleed 3).

[0167] Blood preparation and cell enrichment As shown in Figure 19, blood was collected three times throughout the immunization campaign (bleed 1, bleed 2, bleed 3). In each case, blood was centrifuged to separate serum from blood cells. Serum was used for serum titer analysis as described below, and blood cells were enriched for ASCs by enriching for CD138+ B cells using a modified version of the standard protocol for the CD138 enrichment kit (STEMCELL Technologies, Inc., Vancouver, British Columbia).

[0168] Single-cell screening assays Single-cell screening assays were performed with enriched CD138+ B cell populations as described in Example 2. Briefly, enriched CD138+ B cell populations were mixed with capture reagents including goat anti-mouse IgG Fc bound to 3.4 μm polystyrene beads (Spherotech Inc, Lake Forest, IL), cynomolgus antigens labeled with Alexa 488 to produce a green fluorescent signal, human antigens labeled with Alexa 594 to produce a red fluorescent signal, and at least 100-fold molar excess of HexaHis protein (GenScript RP11737) to compete with the His-tag specific titer, and final concentrations using B cell medium as diluent. The use of different fluorescent signals (green for cynomolgus antigens and red for human antigens) allowed the single-cell assay to distinguish between single cells that bind only the human orthologue, single cells that bind only the cynomolgus orthologue, and single cells that bind both orthologues. The mixture was transferred to a single well of a 384-well plate, with a final concentrated B cell concentration of approximately 2-3 μL of cell mixture per well. After the mixture components were allowed to sit in the well for approximately 10 min, the cells were imaged using an Incucyte live cell analysis system. RFU values ​​for green and red fluorescence were determined.

[0169] Serum titer analysis Sera were diluted to final concentrations of 1:100, 1:1000 and 1:10,000 and then added to beads with captured biotinylated antigen plated in V-bottom 96-well plates. The mixture was incubated for 1 hour at room temperature. The beads were then washed and resuspended in 30 μg of goat anti-mouse IgG Fc (Jackson Immunoresearch) to a final concentration of 5 μg / mL. After 15 minutes of incubation, the beads were washed and resuspended in FACS buffer. The plates were then prepared for flow cytometry.

[0170] Results and Discussion Serum titers from bleed 1 were able to detect a robust immune response to the human antigens, with low but detectable binding to the cynomolgus antigens (Figure 20). However, the polyclonal titer data was unable to distinguish between antibodies that bound only to the cynomolgus antigens and those that could cross-react to both humans and cynomolgus. In contrast, single cell screening was able to detect binding to only humans, only cynomolgus, and both human and cynomolgus cross-reactive antibodies (Figures 21-22). Both serum titers and single cell screening showed that the predominant immune response was restricted to antibodies that bound only to the human orthologues. Although some cross-reactive antibodies were generated, the majority of the cynomolgus response was unable to bind to the human orthologues and required further immunization.

[0171] Mice were then boosted with a subdomain of the human antigen (antigen 2) that has a higher degree of human / cynomolgus homology compared to the full-length protein. Mice were initially injected intraperitoneally with 25 μg of antigen 2 without adjuvant. Mice were then bled (bleed 2) and the sample was screened for binding to the cynomolgus and human orthologs. This boost did not generate a sufficiently strong immune response to reliably identify cross-reactive antibodies. Thus, mice were boosted once more with 25 μg of antigen 2 combined with 50% SAS. Mice were bled 4 days after the boost (bleed 3) and CD138+ASC were isolated and screened as above. This experiment highlights the value of using non-terminal sampling to allow for adjustments and boost expansion when necessary.

[0172] Boosting with antigen 2 strongly increased serum titers to the cynomolgus orthologue of the antigen (Figure 23). Consistent with this observation, the single cell assay also detected an increase in both cynomolgus-only and cynomolgus-human cross-reactive antibodies in bleed 3 compared to bleed 1 (Figure 24). Animals of interest could then be identified by plotting the ratio of cynomolgus-only binding versus binding to both human and cynomolgus orthologues. This plot revealed that some mice respond only to the cynomolgus orthologue, while others have strong cross-reactivity. This cannot be discerned using standard polyclonal serology.

[0173] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference herein and was set forth in its entirety herein.

[0174] The use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (and particularly with respect to the claims which follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated.

[0175] The recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each of the separate values ​​falling within that range, and each of the endpoints, all of which are incorporated herein as if each separate value was individually set forth herein.

[0176] All of the methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0177] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the above description. The inventors expect that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced in other forms than those specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements is encompassed in the present disclosure in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A method for monitoring the production of a selected antibody in a non-human animal, comprising: a. immunizing a non-human animal with an immunogen; b. obtaining a blood sample containing antibody secreting cells (ASCs) from said non-human animal; c. individually assaying the ASCs present in said blood sample or a fraction thereof for the production of a selected antibody; The method includes:

2. 1. A method for inducing antibody production in a non-human animal for the production of a selected antibody, comprising: a. performing an initial immunization of a non-human animal with an immunogen; b. obtaining a blood sample containing antibody secreting cells (ASCs) from said non-human animal; c. individually assaying the ASCs present in said blood sample or a fraction thereof for the production of a selected antibody; d. performing a cycle of steps if the percentage of ASCs producing the selected antibody is below a threshold value; wherein the cycle comprises: i. if the proportion of ASCs producing the selected antibody is below a threshold, then performing a subsequent immunization of the non-human animal with an immunogen; ii. obtaining a blood sample containing ASCs from the non-human animal; iii. individually assaying the ASCs present in said blood sample or a fraction thereof for the production of selected antibodies; The method includes:

3. 3. The method of claim 1 or 2, wherein the cycle is repeated until the number of ASCs producing the selected antibody, as assayed in (iii), is equal to or greater than a threshold value.

4. The method of any one of claims 1 to 3, wherein the immunogen of the subsequent immunization is different from the immunogen of the initial immunization.

5. 5. The method of any one of claims 2-4, wherein each subsequent immunization differs from the previous immunization in that: (A) a different immunogen, adjuvant, and / or immunomodulatory agent is administered to the non-human animal; (B) a different dose of immunogen is administered to the non-human animal; (C) the time between administration of immunogen, adjuvant, immunomodulatory agent is different; and / or (D) the route of administration of immunogen, adjuvant, immunomodulatory agent is different.

6. 1. A method for producing a selected antibody in a non-human animal, comprising: a. performing an initial immunization of a non-human animal with an immunogen; b. obtaining a blood sample containing antibody secreting cells (ASCs) from said non-human animal; c. individually assaying the ASCs present in said blood sample or a fraction thereof for the production of a selected antibody; d. if the proportion of ASCs producing the selected antibody is below a threshold, performing a cycle of steps, wherein said cycle comprises: i. if the proportion of ASCs producing the selected antibody is below a threshold, then performing a subsequent immunization of the non-human animal with an immunogen; ii. obtaining a blood sample containing ASCs from the non-human animal; iii. individually assaying the ASCs present in said blood sample or a fraction thereof for the production of selected antibodies; Including, e. isolating the selected antibody and / or ASCs producing the selected antibody; The method includes:

7. The selected antibody is (i) binds to a target that is the same as or similar to the immunogen used to immunize the non-human animal; (ii) binds to a target and its orthologs or paralogs; (iii) binds to a target and not its ortholog or paralog; (iv) binding to a portion of said label; and / or (v) binds to a conformational epitope formed upon dimerization or multimerization of the target, the target comprising a dimerization or multimerization domain; The method according to any one of claims 1 to 6.

8. The method of any one of claims 1 to 7, wherein the blood sample is obtained from the non-human animal about 3 to about 7 days after the immunization step.

9. The method of any one of claims 1 to 8, wherein the ASCs are CD138+ B cells.

10. The method of any one of claims 1 to 9, wherein the ASCs comprise migrating plasmablasts.

11. The method of any one of claims 1 to 10, further comprising the step of removing one or more components of the blood sample obtained from the non-human animal prior to assaying.

12. The method according to any one of claims 1 to 11, wherein the non-human animal is not subjected to removal of one or more secondary lymphoid organs or to euthanasia.

13. The method of any one of claims 1 to 12, wherein ASCs from the blood sample are not used to generate hybridomas.

14. 14. The method of any one of claims 2 to 13, wherein the non-human animal is one of a set of non-human animals and the result of the assay is the identification of non-human animals having a number of ASCs producing a selected antibody below a threshold and / or requiring further immunization.

15. 15. The method of any one of claims 2 to 14, wherein the method steps are performed on a series of non-human animals, comprising profiling the B cell repertoire of the blood sample for each series of non-human animals, and selecting a series of subsets having a target B cell profile.

16. 1. A method for screening a series of non-human animals for antibody secreting cells (ASCs) which produce a selected antibody, comprising: monitoring the production of selected antibodies in a series of non-human animals according to the method of any one of claims 1 to 15, A method for identifying, for each non-human animal in said series, the number of ASCs that produce said selected antibody.

17. 1. A method for selecting an immunized non-human animal for subsequent immunization, comprising: monitoring the production of selected antibodies in non-human animals according to the method of any one of claims 1 to 16, wherein for each non-human animal the number of ASCs producing said selected antibodies is identified. selecting an animal for subsequent immunization if the proportion of ASCs producing a selected antibody is below a threshold value; The method includes:

18. 1. A method for selecting an immunized non-human animal for euthanasia and secondary lymph collection, comprising: monitoring the production of selected antibodies in non-human animals according to the method of any one of claims 1 to 17, wherein for each non-human animal the number of ASCs producing said selected antibodies is identified. selecting an animal for euthanasia and secondary lymph collection if the proportion of ASCs producing a selected antibody is equal to or greater than a threshold value; The method includes:

19. 1. A method for assaying ASCs producing a selected antibody, comprising: a. combining in a well (i) a blood sample, or a fraction thereof, obtained from a non-human animal immunized with an immunogen, wherein the blood sample contains antibody-secreting cells (ASCs), (ii) a detection reagent that binds to the selected antibody and that contains a first detectable label, and (iii) a target to which the selected antibody binds; Where: (A) the target is a labeled target comprising a second detectable label different from the first detectable label, and a capture reagent that binds to the selected antibody and comprises a solid support is further combined in the well to form a monolayer in the well; or (B) the target is expressed on the surface of the cells, and the cells are combined in a well to form a monolayer in the well. b. assaying for said first detectable label, and optionally, if said target is a labeled target, assaying for said second detectable label; c. identifying locations within the well where the first detectable label is detected, or locations within the well where the first and second detectable labels are detected, where each identified location indicates the location of an individual ASC producing a selected antibody; The method includes:

20. 20. The method of any one of claims 1 to 19, wherein the blood sample obtained from the non-human animal is about 500 μL or less, optionally about 100 μL to about 250 μL.

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