Enhancement of hybridoma production

Abstract

Provided herein are methods for generating hybridomas and related methods for producing antigen-specific antibodies. In exemplary embodiments, the methods include (a) preparing an enriched population of IgG positive (IgG+) memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, where (i) no more than about 10% of the enriched population are IgM positive (IgM+) B cells and / or (ii) the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 0.5, and optionally greater than about 1 or greater than about 2; (b) bulk culturing the enriched population to obtain an expanded population; and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas. In exemplary aspects, the resulting hybridomas represent at least 10% or at least 15% of the IgG+ memory B cell repertoire produced by the immunized animal.

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] Therapeutic antibodies constitute a major class of drugs, many of which are derived from in vivo immunization platforms such as human antibody locus transgenic animals and rely on the capture of specific B cell clones that are activated in response to antigenic stimulation (Lu et al., Journal of Biomedical Science (2020) volume 27, Article number: 1). Antibodies generated in in vivo immune responses to immunization in rodents can be captured by immortalizing B cell populations from immune tissues, which are primarily located within the germinal centers (GCs) of the spleen and lymph nodes, but are also found in bone marrow, mucosa-associated lymphoid tissue (MALT), and circulating in the blood. The process of immortalization, known as hybridoma generation, creates hybrid cells that express membrane-bound clonal B cell receptors (BCRs) and even secrete the same antibody clonotype. Because the hybrids continually divide and secrete antibodies, clones with the required specificity can be identified and characterized without concern for loss of clones or lack of antibody material to test. Established hybrids are typically robust and continue to secrete antibody if kept under selective pressure. Hybrids respond well to freeze-thaw cycles and can survive decades of storage in liquid nitrogen. With the advent of Koehler and Milstein's hybridoma generation in 1975, immortalization of B cells by fusion with myeloma cells became a widely used method for antibody discovery from rodent species, most often mice (G. Koehler & C. Milstein. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (1975) 256, 495-497).

[0003] Both the in vivo generation of antigen-specific B cells for immortalization and the timing of immortalization are important aspects of hybrid generation. Typically, in rodents, responses occur over weeks or months with repeated immunization. In response to repeated antigen exposure, B cells expressing BCRs that recognize the antigen are stimulated to undergo isotype switching to express IgG and form GCs in secondary lymphoid organs (Akkaya et al., Nat Rev Immunol (2020) 20, 229-238). Within GCs, B cells mature and differentiate into memory cells or plasma cells through cycles of somatic hypermutation (SMH) of antigenic determinants and selection of higher affinity variants (Lau et al., Current Opinion in Immunology (2019); 63: 29-34). Memory cells express high levels of B220 / CD45R and IgG on the surface but do not secrete antibodies. When fully differentiated, memory cells are small and quiescent, but have great potential to proliferate in response to stimulation by cognate antigen or polyclonal activation. Clonal diversity within the memory cell pool is high. In contrast, plasma cells are large, highly activated, explosive B cells that secrete large amounts of antibodies. Both surface IgG and B220 / CD45R are downregulated. CD138 and TACI become highly expressed on the surface of plasma cells (Tellier et al., Eur J Immunol 47(8):1276-1279(2017)). Once plasma cells are terminally differentiated, they lose their ability to divide. Plasma cells have a short life span of days to weeks, unless sequestered in a highly specialized long-term survival niche. The diversity of the plasma cell compartment is low, and the in vivo selection process focuses on a relatively small number of high-affinity clones to mount an effective and rapid immune response to antigens. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Lu et al.,Journal of Biomedical Science(2020)volume 27,Article number:1 [Non-Patent Document 2] G.Koehler&C.Milstein.Continuous cultures of fused cells secreting antibody of predefined specificity.Nature(1975)256,495-497 [Non-Patent Document 3] Akkaya et al., Nat Rev Immunol(2020)20,229-238 [Non-Patent Document 4] Lau et al.,Current Opinion in Immunology(2019);63:29-34 [Non-Patent Document 5] Tellier et al.,Eur J Immunol 47(8):1276-1279(2017) Summary of the Invention [Problem to be solved by the invention]

[0005] Hybridoma generation can sample the immune repertoire, but the process is highly inefficient, with immortalization success rates ranging from 0.01% to 0.001%. This inefficiency of fusion can be countered by using larger immunized cohorts to increase the number of cells for fusion, or by working with larger animals such as rats. In view of the above, methods of enhanced hybridoma generation are needed. [Means for solving the problem]

[0006] The present disclosure provides an Enhanced Hybridoma Generation (EHG) method that offers advantages over those known in the art due to the depletion of IgM positive (IgM+) B cells, large scale, high efficiency, and specificity of the repertoire of hybridomas produced. Advantageously, the EHG method of the present disclosure provides enrichment of IgG positive (IgG+) memory B cells by efficient bead-based depletion of IgM+ B cells. IgM class switches to IgG+ B cells during culture, diluting the IgG+Ag+ memory B cell population with irrelevant or low affinity B cell clones. IgM+ B cells divide faster compared to IgG+ B cell clones. By depleting the majority of IgM+ B cells prior to culture, and then culturing the IgG+ B cell enriched population (allowing this population to make hundreds of clonal copies prior to fusion), the majority of clones of interest are immortalized. The result is a large hybridoma pool that facilitates the identification of rare Ag+ clones. The EHG method of the present disclosure provides large-scale bulk culture. Liters of bulk culture containing hundreds of thousands of highly selected IgG+ memory B cells allows for large-scale deep repertoire mining of many more animals. Thus, while prior art methods identify only a small number of antigen-binding clones, the EHG of the present disclosure identifies hundreds to thousands of unique binders daily. The EHG method of the present disclosure also includes immortalizing B cells before identifying antigen-binding clones. Hybridoma supernatants are screened. This sequence represents the reverse order of steps of prior art methods (see, e.g., Steenbakkers et al., J Immunol Methods 152(1):69-77 (1992)), in which B cells are screened for antigen binding before being immortalized. The sequence of immortalization and subsequent identification of antigen-binding clones of the present invention allows for highly efficient identification of antigen-binding clones. In the method of the present disclosure, an unlimited supply of cell supernatant (SN) is available for post-fusion screening and characterization. The sensitivity of the assay during SN screening is not limited. Hybridoma cultures can be expanded to produce high antibody concentrations.In the disclosed EHG method, the ability to identify antigen-binding clones is not limited by B cell antibody secretion rates, and rare binders can be identified by deep mining of hybrid pools. Deep mining by large-scale liquid handling plating and Ag+FACS sorting can be performed.

[0007] Thus, the present disclosure provides an enhanced hybridoma production method useful in antibody discovery. In an exemplary embodiment, the hybridoma production method includes (a) preparing an enriched population of IgG positive (IgG+) memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, the enriched population being substantially free of IgM+ B cells, (b) bulk culturing the enriched population on a large scale to obtain an expanded population, and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas. In an exemplary embodiment, (i) about 10% or less of the enriched population are IgM positive (IgM+) B cells, and / or (ii) the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 0.5, and optionally greater than about 1 or greater than about 2. Without being bound to a particular theory, hybridomas produced by the disclosed method allow for deep mining of the immune repertoire of immunized non-human animals without limitations imposed by B cell antibody secretion rates and / or low abundance of rare B cells. Advantageously, hybridomas produced by the disclosed method more fully represent the immune repertoire of immunized non-human animals. In exemplary embodiments, the proportion of immortalized IgG+ memory B cell repertoire captured by the disclosed method is maximized, and in various cases is at least about 15% (e.g., at least about 20% or at least about 25%) of the repertoire produced by the immunized animal. The disclosed method further allows for an unlimited supply of hybridoma supernatants that can be screened for antigen-specific antibodies. In exemplary embodiments, the disclosed method routinely yields hundreds, if not thousands, of antigen-specific hybridomas producing antigen-specific antibodies. The disclosed methods further result in higher fusion efficiency and more efficient capture of B cell clones in hybridoma pools.

[0008] Thus, the present disclosure further provides a method of generating hybridomas. In an exemplary embodiment, the method includes: (a) preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 10% (optionally less than about 5%) of the cells of the enriched population are IgM+ B cells; (b) bulk culturing the enriched population to obtain an expanded population; and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas. In exemplary aspects, less than 2.5% or less than 1% of the cells of the enriched population are IgM+ B cells. In various aspects, the method includes removing more than about 90% (e.g., more than 95%, more than 98%, more than 99%) of the IgM+ cells and / or positively selecting for IgG+ cells to obtain the enriched population. In exemplary embodiments, (i) about 10% or less of the enriched population are IgM positive (IgM+) B cells, and / or (ii) the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than about 0.5, and optionally greater than about 1 or greater than about 2. In exemplary embodiments, the number of IgM+ B cells in the enriched population is low relative to the number of IgM+ B cells, and the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 1 or greater than 2. In various embodiments, the method includes bulk culturing the enriched population at a density of about 350 B220 positive B cells per mL to about 770 B220 positive B cells per mL. In an exemplary embodiment, bulk culturing of the enriched population is initiated at a seeding density of about 350 to about 700 B220 positive B cells per mL, optionally about 600 to about 650 B220 positive B cells per mL. In an exemplary case, the method includes fusing cells of the expanded population with myeloma cells at a ratio of B cells to myeloma cells in the range of 1:1 to 1:4 (e.g., 1:1.0, 1:1.5, 1:2.0, 1:2.5, 1:3.0, 1:3.5, or 1:4.0). In an exemplary embodiment, all cells of the expanded population (and thus all B cells of the expanded population) are combined with myeloma cells. For example, B cells of the enriched or expanded population are not selected for fusion with myeloma cells based on production of antibodies that bind to an antigen.Similarly, for example, the B cells of the enriched or expanded population are not assayed for the production of antigen-specific antibodies prior to fusion with myeloma cells. In various cases, the method involves screening the hybridomas for the production of antibodies, and, optionally, screening serum obtained from the immunized animal for the production of antigen-specific antibodies. In an exemplary case, the only screen for the production of antigen-specific antibodies that occurs after removal of lymphoid organs from the immunized animal is the screening of the hybridomas.

[0009] The present disclosure also provides a method of screening for hybridomas expressing antigen-expressing antibodies, comprising generating hybridomas according to the present disclosure, culturing the hybridomas in individual wells, and screening the supernatant of each well for antigen-specific antibodies. In various embodiments, about 1 to about 10 or about 1 to about 5 different clones of hybridomas are cultured in a single well.

[0010] Further provided is a method of producing an antigen-specific antibody, comprising generating hybridomas according to the present disclosure, culturing the hybridomas in individual wells, screening the supernatant of each well for antigen-specific antibody to identify hybridomas expressing the antigen-specific antibody, and expanding the cultures of the identified hybridomas to produce the antigen-specific antibody. [Brief description of the drawings]

[0011] [Figure 1A] An exemplary EHG method of the present disclosure is shown, and the following text describes this EHG method in detail. [Figure 1B] An exemplary enrichment process is shown that removes RBCs, non-B cells, and IgM+ cells and further positively selects for surface IgG+ cells to obtain an enriched population that can be used for bulk culture. [Figure 1C]FIG. 1 shows an exemplary enrichment process in which RBCs are removed from a pooled single cell suspension from the spleens of immunized animals, followed by combining the RBC-depleted SCS with SCS from the LNs of immunized animals. Non-B cells and IgM+ cells are removed from the combined SCS using a magnet. Surface IgG+ cells are positively selected using IgG microbeads. Release of surface IgG+ cells from the column yields an enriched population that can then be brought into bulk culture. [Diagram 2] 1 is a schematic of an exemplary method for enhancing hybridoma production. [Figure 3A-1] 1 is a series of FACS analysis plots of B cell markers to assess enrichment of IgG+ memory B cells prior to B cell culture and fusion. [Figure 3A-2] 1 is a series of FACS analysis plots of B cell markers to assess enrichment of IgG+ memory B cells prior to B cell culture and fusion. [Figure 3B-1] 1 is a series of FACS analysis plots of B cell surface markers to assess the enrichment process. The enriched population contains a high affinity IgG secreting plasma cell population and is applied to direct B cell discovery technology for antigen binding secretion assays. [Figure 3B-2] 1 is a series of FACS analysis plots of B cell surface markers to assess the enrichment process. The enriched population contains a high affinity IgG secreting plasma cell population and is applied to direct B cell discovery technology for antigen binding secretion assays. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure provides an Enhanced Hybridoma Generation (EHG) method that significantly enhances the efficiency of immune repertoire capture in transgenic animals. This is accomplished, at least in part, by expanding highly purified memory B cells in bulk culture for 4-6 days prior to fusion. In some embodiments, this is accomplished, at least in part, by expanding highly purified memory B cells in bulk culture for 6 days prior to fusion. In bulk culture prior to fusion, each memory B cell clone is estimated to divide 7-10 times (when expanding B cells in bulk culture for 6 days), generating 125-1000 clonal copies. Without being bound by any particular theory, the high B cell copy number, e.g., after 6 days of B cell bulk culture, the blasting phenotype of activated B cells, the size of the B cells during fusion (which approximates the size of the fusion partner, thereby promoting fusion), and the fact that the B cell membrane is fluid and therefore prone to fusion correction even after, e.g., 6 days of B cell culture, are all factors that overcome the inefficiency of the fusion event. The EHG process of the present disclosure is estimated to capture about 25% of the in vivo memory B cell immune repertoire, thereby resulting in a hybrid pool with deep diversity. In an exemplary embodiment, the EHG method includes harvesting immune tissues from immunized transgenic animals and processing cells from such tissues into a single cell suspension (Figure 1A). In various embodiments, non-B cells and IgM positive B cells are removed to specifically enrich for surface IgG positive memory B cells, and in various cases, about 99.9% of viable cells are removed from the immune tissue preparation, while 25-50% of the IgG+ memory B cell population is retained. The highly enriched memory B cell fraction is bulk cultured, in various embodiments, in the presence of an irradiated mouse T cell line (EL4B5), rabbit T cell supernatant (TSN), and microbeads with anti-IgG antibodies attached (e.g., anti-human IgG microbeads). During culture, the resting memory B cells are highly activated, and in some embodiments, divide 7-10 times. In various cases, after about 6 days of culture, the cells are harvested and fused with the P3 myeloma cell line to produce hybridomas.Fusion efficiency can be calculated by measuring clonal growth at low density plating from a portion of the hybridoma pool. Identification of antigen-specific clones begins here, using methods also employed in conventional hybridoma generation.

[0013] Without being bound by any particular theory, the EHG method described herein offers the unique ability to capture a much larger proportion of the immune repertoire generated in vivo in rodents compared to conventional hybridoma generation, and specifically targets the significantly deeper and more diverse IgG+ memory B cell compartment compared to the plasma cell compartment. The EHG method described herein advantageously overcomes the limitations of conventional methods (e.g., poor fusion efficiency and weak immune response, etc.), leading to the production of hybridoma pools with large and diverse antibody repertoires. The EHG method disclosed herein can be successfully applied to any transgenic mouse and rat model as well as wild-type strains. Notably, the EHG method disclosed herein is also effective in first generation human antibody transgenic animals such as XenoMouse®, where the immune response is less robust and antigen-reactive B cells are rare. The disclosed EHG method specifically immortalizes in vivo generated memory B cell populations, whereas traditional hybridoma generation as described in Kohler and Milstein, Nature (1975) 256, 495-497, is biased towards immortalizing plasma cell populations that represent different arms of the antigen-specific repertoire. It should be noted that even when applying the present EHG method to IgG+ memory cells, plasma cell populations can be successfully isolated from the same immune tissue before proceeding to EHG and captured by various single B cell techniques combined with direct molecular rescue. The disclosed EHG method allows for the investigation of both memory B cell and plasma cell compartments, adding depth to the capture of in vivo immune responses to antigens.

[0014] Considering the results of the conventional EHG method and the EHG method described herein, it must be understood that the B cell populations captured during fusion events are different. Conventional hybridoma generation is strongly biased towards plasma cells, while EHG is strongly biased towards memory cells. This is determined by the enrichment, size, and activation state of B cells. Fusion events are more likely to be successful between cells of the same size and with cells that have a more fluid plasma membrane, as seen in activated cells (Rems et al., Sci Rep (2013) 3, 3382). Since the fusion partner (e.g., myeloma cells) is close in size to plasma cells, in conventional hybrid generation, rare plasma cells are more likely to contribute to the hybrid pool than the smaller ones that are resting despite the higher number of memory cells. Thus, conventional hybridoma generation produces a relatively small hybrid pool with limited diversity (Dubois et al. Hum Antibodies (2016) Jun 8; 24 (1-2): 1-15).

[0015] In contrast, in EHG, the starting point was depletion of IgM+ B cells; a highly enriched IgG+ memory cell compartment. This depletion is important because mouse IgG+ B cells divide 7-9 times in 6 days of culture, whereas IgM+ B cells typically divide 9-10 times or more during culture and also isotype switch to become IgG+ B cells. When cultured together, IgM+ B cells dramatically reduce the proportion of high affinity IgG-derived antigen expressing B cells in the pool; this is the population that subsequently fuses. The result is a hybrid pool with great diversity and a repertoire distinct from that observed using conventional methods.

[0016] The immortalization of a comprehensive pool from a little-explored source, a pre-enriched IgG+ memory B cell compartment, containing multiple copies of antigen-specific B cells, to overcome the inefficiencies of native electrocellular fusion, is the first key feature that distinguishes EHG from conventional hybridoma generation. The memory B cell compartment is not selected in vivo for high affinity binding in a first-line response to pathogens, but may retain greater diversity and a smaller bias towards major antigenic determinants, providing an important repertoire not captured from in vivo generated plasma cells.

[0017] A second important feature is the ability to discover antibodies from very rare antigen-reactive B cells derived from animal models such as transgenic Xenomouse®. Despite the low immune cell numbers and less robust immune responses in transgenic platforms, the methods described herein to highly enrich for memory cells prior to fusion, followed by activation and expansion, allow for highly efficient capture of immune repertoires and the generation of large and diverse hybrid pools, which is not always possible using traditional hybrid generation in transgenic animals.

[0018] Consistent with the above, the present disclosure further provides methods of generating hybridomas (eg, hybridomas that produce antibodies with a desired antigen specificity). In an exemplary embodiment, the method includes: (a) preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein (i) less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%) of the cells of the enriched population are IgM positive (IgM+) B cells, and / or (ii) the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells of the enriched population is greater than about 0.5, and optionally greater than about 1 or greater than about 2; (b) bulk culturing the enriched population to obtain an expanded population; and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas. In various aspects, the method includes: (a) preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%) of the cells of the enriched population are IgM positive (IgM+) B cells; (b) bulk culturing the enriched population to obtain an expanded population; and (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas.

[0019] Preparation of enriched populations In various instances, the method includes preparing an enriched population of IgG+ memory B cells from a secondary lymphoid organ of one or more immunized non-human animals. In various aspects, the method includes preparing an enriched population of IgG+ memory B cells from a single cell suspension isolated from a secondary lymphoid organ of one or more immunized non-human animals. In various instances, the secondary lymphoid organ is the spleen, lymph nodes Peyer's patches, mucosal tissue (e.g., nasal associated lymphoid tissue, adenoids, and / or tonsils). Optionally, the secondary lymphoid organ is a lymph node (LN) (e.g., draining LN), and / or the spleen. In various aspects, the secondary lymphoid organ is or has been removed from the immunized non-human animal about 3 to about 5 (e.g., about 3, about 4, or about 5) days after immunization. Optionally, secondary lymphoid organs are harvested from at least 1 to about 15 (e.g., about 1 to about 10) immunized non-human animals about 3 to about 5 (e.g., about 3, about 4, or about 5) days after immunization. In an exemplary embodiment, the secondary lymphoid organs are harvested only from selected immunized non-human animals, and optionally, the selected immunized non-human animals are selected based on post-immunization serum antibody titer levels. For example, in an exemplary embodiment, the method includes immunizing an animal, and selecting only a portion of the immunized animals for secondary lymphoid organ harvesting. Optionally, the selected animals are those that exhibit a post-immunization serum antibody titer level equal to or greater than a threshold level. In various cases, the method includes immunizing an animal, and assaying serum from all immunized animals after immunization, and selecting only a portion of the immunized animals for secondary lymphoid organ harvesting, and the selection is based on post-immunization serum antibody titer levels. In various instances, the post-immunization serum antibody titer level is a post-immunization serum titer level of an antigen-specific antibody. In an exemplary embodiment, the method includes harvesting secondary lymphoid organs from the immunized non-human animal about 3 to about 5 (e.g., about 3, about 4, or about 5) days after immunization. In various instances, the method includes harvesting secondary lymphoid organs from at least 1, 2, 3, 4, or 5 immunized non-human animals about 3 to about 5 (e.g., about 3, about 4, or about 5) days after immunization. The immunized non-human animal can be any of those known in the art, including, but not limited to, any of the non-human animals described herein.In various aspects, the non-human animal is a mouse (e.g., XenoMouse®). Optionally, the non-human animal is a rat, e.g., a transgenic rat (e.g., UniRat®) or a wild type rat. In various aspects, the immunized non-human animal is immunized or has been immunized according to any protocol known in the art. In various aspects, the non-human animal is immunized or has been immunized according to any of the immunization protocols described herein. See, e.g., Immunization, below. In various aspects, the method further comprises: (a) immunizing one or more non-human animals with an immunogen; (b) optionally harvesting secondary lymphoid organs from the immunized non-human animals about 3 to about 5 days after immunization; (c) preparing a single cell suspension (SCS) from the secondary lymphoid organs harvested from each immunized non-human animal; and / or (d) combining the SCS from the secondary lymphoid organs of multiple immunized non-human animals to prepare a pooled SCS.

[0020] In an exemplary embodiment, the method includes preparing a single cell suspension (SCS) from a secondary lymphoid organ of an immunized non-human animal (optionally, a secondary lymphoid organ of a selected immunized non-human animal). In an exemplary case, the method includes preparing a single cell suspension comprising mixed immune cells obtained from the secondary lymphoid organ. The single cell suspension may be prepared by dissociating the organ on a slide according to methods known in the art, or may be prepared using a homogenizer (e.g., Dounce tissue grinder, disperser, microbead homogenizer, ultrasonic processor, blender) and / or a tissue dissociator (e.g., gentleMACS™ (Miltenyi Biotec, Bergisch Gladbach, Germany)) with or without the use of a tissue dissociation kit (e.g., MACS® Tissue Dissociation Kit (Miltenyi Biotec)). See, e.g., Reichard and Asosingh, Cytometry 95(2):219-226 (2019); Scheuermann et al., Current Directions in Biomedical Engineering 5(1):545-548 (2019). In an exemplary embodiment, the method includes preparing an SCS from the spleens of one or more immunized non-human animals and / or preparing an SCS from the draining lymph nodes from one or more immunized non-human animals. Optionally, two separate SCS are prepared: one from the pooled spleens of the immunized non-human animals and one from the pooled LMs of the immunized animals. In various cases, the SCS from the pooled spleens is subjected to an RBC depletion step, followed by combining the SCS from the pooled LNs to obtain a pooled single cell suspension or a bulk single cell suspension. Optionally, RBCs are removed using an RBC lysis buffer, such as BD Pharm Lyse™ (BD Biosciences, Franklin Lakes, NJ) or Red Blood Cell Lysing Buffer Hybri-Max™ (Millipore Sigma, St. Louis, MO).

[0021] In various aspects, the method includes one or more of: (i) immunizing a non-human animal with an immunogen; (ii) optionally harvesting a secondary lymphoid organ from the immunized non-human animal about 3 to about 5 days after the last boost with the immunogen; (iii) optionally preparing a single cell suspension from the secondary lymphoid organ by dissociating the organ on a slide or by using a homogenizer and / or tissue dissociator with or without a tissue dissociation kit; and (iv) combining multiple prepared single cell suspensions (e.g., from different immunized non-human animals) to obtain a pooled or bulk single cell suspension. In various aspects, an enriched population of IgG+ memory B cells is prepared from the single cell suspension (e.g., pooled or bulk single cell suspension) by removing red blood cells (RBCs), non-B cells, and / or IgM positive (IGM+) cells. In various aspects, RBCs are removed from the single cell suspension (e.g., pooled or bulk single cell suspension) by using an RBC lysis buffer (e.g., BD Pharm Lyse™ (BD Biosciences, Franklin Lakes, NJ) or Red Blood Cell Lysing Buffer Hybri-Max™ (Millipore Sigma, St. Louis, MO). In various cases, non-B cells are removed from the single cell suspension (e.g., pooled or bulk single cell suspension) by using one or more antibodies (e.g., antibody cocktails) that specifically bind to cell surface markers of non-B cells. In exemplary aspects, the non-B cells are one or more of T cells, monocytes, macrophages, natural killer (NK) cells, granulocytes, and RBCs. In exemplary cases, the one or more antibodies are linked to biotin. In various aspects, the method includes removing T cells, monocytes, macrophages, NK cells, RBCs, granulocytes, or a combination thereof from the single cell suspension.In an exemplary case, the method includes removing T cells, monocytes, macrophages, NK cells, RBCs, granulocytes, or combinations thereof from the single cell suspension using biotin-labeled antibodies and streptavidin-labeled beads (optionally streptavidin-labeled magnetic beads). In various aspects, the cell surface markers for non-B cells are NK1.1 (expressed by NK cells), CD90.2 (expressed by T cells), Ly-6G GR.1 (expressed by granulocytes and / or macrophages), CD3ε (expressed by T cells), CD4 (expressed by T cells), CD8a (expressed by T cells), CD11b (expressed by granulocytes, macrophages, dendritic cells, and / or NK cells), and TER119 (expressed by red blood cells).

[0022] In an exemplary embodiment, IgM+ cells are removed from a single cell suspension (e.g., a pooled single cell suspension or a bulk single cell suspension) by adding a biotinylated anti-IgM antibody (e.g., an anti-human IgM antibody). In various embodiments, a biotinylated antibody is added to the single cell suspension to allow the antibody to bind to cell surface markers on the non-B cells and / or IgM+ cells. Then, in various cases, magnetic beads linked to streptavidin (e.g., streptavidin magnetic beads) are added to allow the streptavidin to bind to the biotin of the biotinylated antibody. In an exemplary embodiment, a magnet is used to isolate and remove the magnetic beads that are linked to the non-B cells and / or IgM+ cells via the antibody. In various instances, the methods include removing a spleen from the immunized non-human animal and preparing an SCS from the spleen, removing LNs from the immunized non-human animal and preparing an SCS from the LNs, removing RBCs from the spleen-derived SCS, combining the LN-derived SCS with the RBC-depleted spleen-derived SCS to obtain a pooled SCS, removing IgM+ cells from the pooled SCS by adding a biotinylated anti-IgM antibody, and capturing the IgM+ cells with streptavidin magnetic beads.

[0023] Optionally, greater than 90% of IgM+ B cells are removed from the pooled SCS to obtain an enriched population. In various cases, greater than 95% (e.g., greater than 96%, greater than 97%, greater than 98%, or greater than 99%) of the IgM+ B cells of the pooled SCS are removed to obtain an enriched population that is substantially depleted of IgM+ cells. In various cases, the method includes removing IgM+ cells and / or selecting IgG+ cells. In various aspects, the method includes adding anti-IgG antibody-labeled magnetic beads (e.g., anti-human IgG antibody-labeled magnetic beads) to the enriched population that is substantially depleted of IgM+ cells to isolate memory B cells. In various aspects, the remaining portion (e.g., depleted of RBCs, non-B cells, and / or IgM+ cells) is incubated with microbeads linked to anti-IgG antibodies (e.g., anti-human IgG antibodies). In certain aspects, cells expressing IgG on the cell surface bind to the microbeads via the anti-IgG antibodies. In an exemplary embodiment, the microbead-antibody-cell mixture is added to a magnetic column that retains the microbeads bound to cells expressing surface IgG. The flow-through fraction, in various embodiments, contains cells negative for expression of surface IgG. In exemplary cases, the cells expressing surface IgG are released from the column to obtain an enriched population of IgG+ memory B cells. In various cases, the percentage of IgG+ cells increases as RBCs, non-B cells, and / or IgM cells are removed from the single cell suspension (e.g., pooled single cell suspension or bulk single cell suspension). In various embodiments, the enriched population of IgG+ memory B cells contains a higher percentage of IgG+ cells compared to that of the single cell suspension. For example, in various embodiments, the percentage of IgG+ cells (relative to total viable cell count) increases by at least 5-fold or 10-fold or more relative to the percentage of IgG+ cells (relative to total viable cell count) of the single cell suspension prior to enrichment. In various embodiments, less than 1% of the single cell suspension (before enrichment) are IgG+ cells, and the percentage of IgG+ cells (relative to the total viable cell number) increases to about 5%, about 10%, about 15%, or more after enrichment.In various cases, more than 5%, more than 10%, or more than 15% of the live cells of the enriched population are IgG+ cells, and / or more than 20%, more than 30%, more than 40%, or more than 50% of the cells of the enriched population are positive for B220 expression. B220 is a B cell marker. In various cases, less than 10% of the cells of the enriched population of IgG+ cells are IgM+ cells. In various aspects, less than 5% of the cells of the enriched population of IgG+ cells are IgM+ cells. Optionally, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the cells of the enriched population are IgM+ cells. In various cases, the ratio of IgG+ cells to IgM+ cells in the enriched population is increased by at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, or more, relative to the ratio of IgG+ cells to IgM+ cells in the single cell suspension prior to enrichment. Without being bound to a particular theory, a decrease in the percentage of IgM+ cells and an increase in the percentage of IgG+ cells in the enriched population of IgG+ cells advantageously allows the IgG+ cells to be expanded in bulk culture to obtain an expanded population that can then be fused with myeloma cells to obtain hybridomas.

[0024] In exemplary embodiments, the enriched population is prepared by removing IgM+ cells and / or selecting IgG+ cells. In various cases, the enriched population is prepared by negative selection of IgM+ cells and / or positive selection of IgG+ cells. Optionally, greater than 90% of IgM+ B cells are removed during negative and / or positive selection, and in some cases, greater than 95% of IgM+ B cells are removed during negative and / or positive selection. In various embodiments, greater than 98% of IgM+ B cells are removed during negative selection, and greater than 99% of IgM+ B cells are removed during negative and positive selection. Optionally, greater than 99% of IgM+ cells present in the single cell suspension are removed by negative and positive selection. In various cases, the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells is increased by at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold upon negative selection and positive selection.

[0025] Bulk Culture In an exemplary embodiment of the EHG method of the present disclosure, the enriched population is bulk cultured with anti-IgG antibody labeled beads (e.g., anti-human IgG antibody labeled beads) and feeder cells in cell culture medium containing rabbit T cell supernatant. "Bulk culture" means that the cell culture is not a clonal cell population. "Bulk culture" in various embodiments is a mixture of cells derived from secondary lymphoid organs extracted from multiple immunized non-human animals. In various cases, "bulk culture" as used herein refers to culturing a polyclonal mixture of surface IgG positive B cells under conditions that activate them and induce proliferation and differentiation. In some cases, the feeder cells express CD40L and / or are gamma irradiated. Optionally, the feeder cells are gamma irradiated CD40L positive EL4B5 feeder cells. In various embodiments, the myeloma cells are in logarithmic growth phase. In certain embodiments, the myeloma cells are P3 myeloma cells. In an exemplary embodiment, the enriched population of IgG+ cells is bulk cultured at a density of 350 B220 positive cells per mL to about 700 B220 positive cells per mL.Optionally, the enriched population of IgG+ cells is cultured at about 350 B220 positive cells per mL to about 650 B220 positive cells per mL, about 350 B220 positive cells per mL to about 600 B220 positive cells per mL, about 350 B220 positive cells per mL to about 550 B220 positive cells per mL, about 350 B220 positive cells per mL to about 500 B220 positive cells per mL, about 350 B220 positive cells per mL to about 450 B220 positive cells per mL, about 350 B220 positive cells per mL to about 400 B220 positive cells per mL. Culture the cells in bulk at densities of approximately 400 B220 positive cells per mL to approximately 700 B220 positive cells per mL, approximately 450 B220 positive cells per mL to approximately 700 B220 positive cells per mL, approximately 500 B220 positive cells per mL to approximately 700 B220 positive cells per mL, approximately 550 B220 positive cells per mL to approximately 700 B220 positive cells per mL, approximately 600 B220 positive cells per mL to approximately 700 B220 positive cells per mL, and approximately 650 B220 positive cells per mL to approximately 700 B220 positive cells per mL. Optionally, the enriched population of IgG+ cells is bulk cultured at a density of about 550 B220 positive cells per mL to about 650 B220 positive cells per mL, optionally about 625 B220 positive cells per mL. In various cases, the enriched population is bulk cultured at a seeding density of about 350 B220 positive B cells per mL to about 700 B220 positive cells per mL, optionally about 600 B220 positive cells per mL to about 650 B220 positive cells per mL.In various embodiments, the seeding density is from about 350 B220 positive cells per mL to about 650 B220 positive cells per mL, from about 350 B220 positive cells per mL to about 600 B220 positive cells per mL, from about 350 B220 positive cells per mL to about 550 B220 positive cells per mL, from about 350 B220 positive cells per mL to about 500 B220 positive cells per mL, from about 350 B220 positive cells per mL to about 450 B220 positive cells per mL, from about 350 B220 positive cells per mL to about 400 B220 positive cells per mL. positive cells, about 400 B220 positive cells per mL to about 700 B220 positive cells per mL, about 450 B220 positive cells per mL to about 700 B220 positive cells per mL, about 500 B220 positive cells per mL to about 700 B220 positive cells per mL, about 550 B220 positive cells per mL to about 700 B220 positive cells per mL, about 600 B220 positive cells per mL to about 700 B220 positive cells per mL, or about 650 B220 positive cells per mL to about 700 B220 positive cells per mL. Optionally, the seeding density is about 550 B220 positive cells per mL to about 650 B220 positive cells per mL, optionally about 625 B220 positive cells per mL. In various embodiments, the volume of the bulk culture is about 10 mL or more, about 20 mL or more, about 30 mL or more, about 40 mL or more, about 50 mL or more. In various embodiments, the volume of the bulk culture is more than 50 mL, more than 60 mL, more than 70 mL, more than 80 mL, or more than 90 mL. In various embodiments, the volume is more than 100 mL, more than 250 mL, more than 500 mL, more than 750 mL, more than 1.0 L, about 1.1 L, about 1.2 L, about 1.3 L, about 1.4 L, about 1.5 L, about 1.6 L, about 1.7 L, about 1.8 L, about 1.9 L, or about 2.0 L. The enriched population is bulk cultured in an exemplary case in a volume of about 50 mL to about 500 mL, optionally about 100 mL to about 300 mL. Optionally, the enriched population is bulk cultured for at least about 4 days, at least about 5 days, or at least about 6 days. In an exemplary embodiment, the enriched population of IgG+ cells is bulk cultured for at least about 4 days.In exemplary embodiments, the enriched population of IgG+ cells is bulk cultured for at least about 5 days. In exemplary embodiments, the enriched population of IgG+ cells is bulk cultured for at least about 6 days. In various cases, the cells of the enriched population undergo at least or about 5 cell divisions to about 12 cell divisions. In various cases, the cells of the enriched population undergo at least or about 6 cell divisions to about 12 cell divisions (e.g., at least or about 6 cell divisions to about 11 cell divisions, at least or about 6 cell divisions to about 10 cell divisions), optionally at least or about 7 cell divisions to about 10 cell divisions, e.g., about 7, about 8, about 9, or about 10 cell divisions, to obtain an expanded population.

[0026] cell fusion In exemplary embodiments, cells of the expanded population are fused with myeloma cells by electric cell fusion (ECF) to obtain hybridomas, and optionally, ECF is performed using an SDF fusion chamber. In various cases, all cells of the expanded population (including all B cells) are used for fusion with myeloma cells. In various embodiments, all cells of the expanded population (including all B cells) are combined and fused with myeloma cells. In exemplary cases, the method does not include selecting B cells for fusion with myeloma cells. In exemplary cases, the method does not include selecting B cells for fusion with myeloma cells based on production of antibodies that bind to antigens (e.g., antigen-specific antibodies). In exemplary embodiments, B cells of the enriched or expanded population are not screened for production of antibodies that bind to antigens (e.g., antigen-specific antibodies).

[0027] In various cases, the B cells are present in the ECF at a ratio of B cells to myeloma cells of about 1:1 to about 1:4 (e.g., 1:1.5, 1:2.0, 1:2.5, 1:3.0, 1:3.5, 1:4.0). In some embodiments, the ratio is about 1:2. Methods of electrical cell fusion for hybridoma production are described in the art. See, for example, Greenfield, Cold Spring Harbor Protocols; doi:10.1101 / pdb.prot103184 (2019). Optionally, the expanded population of cells is fused with myeloma cells in a volume of greater than 10 mL per fusion event. In various embodiments, the method further comprises separating or isolating the hybridomas from unfused cells. In various cases, the method comprises transferring the cells from the ECF chamber to a culture medium containing hypoxanthine azaserine (HA), which causes cell death of any unfused cells. In various embodiments, the method includes transferring the cells from the ECF chamber to a culture medium containing HA for more than about 3 days. The hybridomas in various cases are then stored under frozen conditions. Optionally, after cryopreservation, the hybridomas are plated in a multi-well plate or clonally antigen-sorted into a multi-well plate by FACS. In exemplary embodiments, each well contains up to five hybridoma clones per well. In exemplary cases, the supernatant from each well is used in one or more screening assays to detect and characterize the antibodies produced by the hybridomas in the well. In various embodiments, the supernatant is optionally assayed for antigen-specific antibodies by ELISA, FACS, or other techniques.

[0028] In various embodiments, the method further comprises screening the hybridomas for production of antibodies that bind to the antigen, and / or culturing the hybridomas in multiple plate wells and screening the supernatants of each well for antigen-specific antibodies. Optionally, the screening comprises an immunoassay to detect binding of the antibody to the antigen, which in various embodiments is a fluorescence activated cell sorting (FACS) analysis. In various embodiments, the screening for cells that produce antigen-specific antibodies is performed only after obtaining the hybridomas, and not before obtaining the hybridomas. Optionally, the assaying for antigen-specific antibodies is performed only after removing the secondary lymphoid organs and only after obtaining the hybridomas. In an exemplary case, the screening for antigen-specific antibodies is performed only after obtaining the hybridomas, and only before removing the secondary lymphoid organs. Optionally, the screening for antigen-specific antibodies is performed only before removing the secondary lymphoid organs. Optionally, the screening for antigen-specific antibodies is performed before removing the secondary lymphoid organs includes titer analysis of serum obtained from living immunized animals.

[0029] In an exemplary embodiment, a method for generating hybridomas that produce antigen-specific antibodies includes: a. immunizing one or more non-human animals with an immunogen; b. isolating a secondary lymphoid organ from the immunized non-human animal; c. Preparing a single cell suspension (SCS) from secondary lymphoid organs isolated from each immunized non-human animal; d. Preparing a pooled SCS by combining all the SCS prepared in (c); e. removing greater than 95% of IgM+ cells from the pooled SCS and / or positively selecting surface IgG+ cells from the pooled SCS to obtain an enriched population of IgG positive (IgG+) memory B cells; i. about 10% or less of this enriched population are IgM positive (IgM+) B cells; and / or ii. the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 0.5, and optionally greater than 1 or greater than 2; To get; f. Bulk culturing the enriched population to obtain an expanded population; g. fusing cells of this expanded population with myeloma cells to obtain hybridomas; and h. Identifying hybridomas that produce antigen-specific antibodies by culturing single hybridomas in individual wells and screening the supernatants from each well for antigen-specific antibodies. Includes.

[0030] In exemplary embodiments of the disclosed methods of generating hybridomas that produce antigen-specific antibodies, the resulting hybridomas represent at least 15% of the IgG+ memory B cell repertoire produced by the immunized animal. In various cases, the resulting hybridomas represent at least 20% of the IgG+ memory B cell repertoire produced by the immunized animal. Optionally, the resulting hybridomas represent at least 25% of the IgG+ memory B cell repertoire produced by the immunized animal.

[0031] In exemplary instances of the EHG method of the present disclosure, more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the total IgG+ memory B cells from the in vivo repertoire are recovered and bulk cultured after enrichment. In other exemplary aspects of the EHG method of the present disclosure, more than 10% (e.g., more than 15%, more than 20%, more than 25%) of the total IgG+ memory B cells from the in vivo repertoire are recovered and bulk cultured after enrichment. Exemplary examples of the EHG method of the present disclosure capture more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% of the memory B cell repertoire of pooled B cells from immunized animals. Other exemplary examples of the EHG method of the present disclosure capture more than 10% (e.g., more than 15%) of the memory B cell repertoire of pooled B cells from immunized animals. In various embodiments, the EHG method of the present disclosure generates more than 100,000 unique hybridomas. In certain cases, the EHG method of the present disclosure produces more than 150,000 or more than 200,000 unique hybridomas. In various cases, the fusion efficiency achieved by the EHG method of the present disclosure is at least 0.10%. In other cases, the fusion efficiency achieved by the EHG method of the present disclosure is greater than 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, or 0.09%. In other cases, the fusion efficiency achieved by the EHG method of the present disclosure is greater than 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, or 0.20%. In certain embodiments, the fusion efficiency achieved by the EHG method of the present disclosure is greater than 0.140%.

[0032] Further provided herein is a method of screening for hybridomas expressing antigen-specific antibodies. In an exemplary embodiment, the method includes: (a) generating hybridomas according to any one of the disclosed methods of generating hybridomas; (b) culturing the hybridomas in wells, optionally with each well containing up to 5 hybridomas; and (c) screening or assaying the supernatant of each well for antigen-specific antibodies. In an exemplary embodiment, the method includes: (a) preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 5% of the cells of the enriched population are IgM+ B cells; (b) bulk culturing the enriched population to obtain an expanded population; (c) fusing the cells of the expanded population with myeloma cells to obtain hybridomas; (d) culturing the hybridomas in wells; and (e) screening the supernatant of each well for antigen-specific antibodies. Screening, in various embodiments, involves ELISA, or binding to streptavidin beads coated with the target antigen, FACS detection of antibody binding to cells transfected with the target, or another high throughput microscopy technique.

[0033] The present disclosure also provides a method for producing antigen-specific antibodies. In an exemplary embodiment, the method includes: (a) preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 10% (e.g., less than about 5%) of the cells of the enriched population are IgM+ B cells; (b) bulk culturing the enriched population to obtain an expanded population; (c) fusing cells of the expanded population with myeloma cells to obtain hybridomas; (d) culturing the hybridomas in wells; (e) screening the supernatant of each well for antigen-specific antibodies to identify hybridomas expressing the antigen-specific antibodies; and (f) expanding the hybridomas identified in (e) to produce the antigen-specific antibodies.

[0034] immunization In various aspects of the disclosure, the disclosure includes immunizing a non-human animal with an immunogen. As used herein, the term "immunizing" refers to carrying out or executing an "immunization campaign" or an "immunization protocol" or a "campaign" to mount an immune response against said immunogen. In exemplary aspects, the immune response includes a B cell immune response and a humoral immune response against said immunogen. Suitable techniques for immunizing non-human animals are known in the art. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, 3 rd ed., Academic Press Limited, San Diego, CA, 1996. Gene gun methods may also be used to immunize non-human animals, as 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). Additionally, as exemplified herein, immunizing can include administering cells expressing the antigen to the non-human animal, 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 instances, immunizing can be performed by microneedle delivery (see, e.g., Song et al., Clin Vaccine Immunol 17(9):1381-1389(2010)); by virus-like particles (VLPs) (see, e.g., Temchura et al., Viruses 6(8):3334-3347(2014)); or by any means known in the art. See, e.g., Shakya et al., Vaccine 33(33):4060-4064 (2015) and Cai et al., Vaccine 31(9):1353-1356 (2013). Further strategies for immunization and immunogen preparation, including, for example, adding T cell epitopes to antigens, are described in Chen and Murawsky, Front Immunol 9:460 (2018).

[0035] In various aspects, the method includes immunizing a non-human animal with an immunogen, which is administered to the non-human animal one or more times (e.g., 2, 3, 4, 5 or more times). In various aspects, the immunogen is administered by injection, e.g., intraperitoneally, subcutaneously, intramuscularly, intradermally, or intravenously. In various aspects, the method includes immunizing the non-human animal by administering a series of injections of the immunogen. In exemplary aspects, each administration (e.g., injection) is administered to the non-human animal about 10 to about 18 days apart, optionally about 12 to about 16 days apart, or about 14 days apart. In exemplary aspects, each administration (e.g., injection) is administered to the non-human animal more frequently than about 10 to about 18 days apart. For example, in exemplary embodiments, the timing between administrations of the immunogen to the non-human animal is about 1 to about 9 days apart, optionally about 1 to about 8 days, about 1 to about 7 days, about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 9 days, about 3 to about 9 days, about 4 to about 9 days, about 5 to about 9 days, about 6 to about 9 days, about 7 to about 9 days, about 8 to about 9 days, about 4 to about 8 days, about 4 to about 8 days, or about 6 to about 8 days. The timing between administrations of the immunogen to the non-human animal is longer in various embodiments. For example, the timing between administrations of the immunogen to the non-human animal can be about 1 to about 20 weeks or longer, for example, about 1 to about 20 months. Optionally, the timing between administrations of the immunogen to the non-human animal is from about 1 week to about 19 weeks, from about 1 week to about 18 weeks, from about 1 week to about 17 weeks, from about 1 week to about 16 weeks, from about 1 week to about 15 weeks, from about 1 week to about 14 weeks, from about 1 week to about 13 weeks, from about 1 week to about 12 weeks, from about 1 week to about 11 weeks, from about 1 week to about 10 weeks, from about 1 week to about 9 weeks, from about 1 week to about 8 weeks, from about 1 week to about 7 weeks, from about 1 week to about 6 weeks, from about 1 week to about 5 weeks, from about 1 week to about 4 weeks, from about 1 week to about 3 weeks, from 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.Optionally, from about 1 week to about 8 days, from about 1 day to about 7 days, from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, from about 2 days to about 9 days, from about 3 days to about 9 days, from about 4 days to about 9 days, from about 5 days to about 9 days, from about 6 days to about 9 days, from about 7 days to about 9 days, from about 8 days to about 9 days, from about 4 days to about 8 days, from about 5 days to about 8 days, or from about 6 days to about 8 days. In various cases, during immunization, each administration (e.g., injection) of immunogen is carried out with the same (A) immunogen, adjuvant, immunomodulatory agent, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulatory agent, or combination thereof, (C) route of administration or method of delivery of immunogen, (D) site of administration in the non-human animal, or (E) combination thereof. Alternatively, one or more administrations (e.g., injections) of the immunogen during immunization are performed with different (A) immunogen, adjuvant, immunomodulatory agent, or combination thereof, (B) amount or dose of immunogen, adjuvant, immunomodulatory agent, or combination thereof, (C) route of administration or method of delivery of the immunogen, (D) site of administration in the non-human animal, or (E) combinations thereof. Optionally, the amount of immunogen decreases or increases with subsequent administrations (e.g., injections). In some aspects, every other administration (e.g., injection) decreases or increases the amount of immunogen compared to the first and third injections. Exemplary immunizations are described in the Examples provided herein.

[0036] Non-human animals Advantageously, the method of the present disclosure is not limited to any particular non-human animal. The non-human animal is, in exemplary embodiments, any non-human animal. In exemplary embodiments, the non-human animal is a mammal, such as, but not limited to, a mammal of the order Rodentia, such as mouse, rat, guinea pig, gerbil, and hamster, a mammal of the order Logomorpha, such as rabbit, a mammal of the order Carnivora, including Felidae (cats) and Canidae (dogs), a mammal of the order Artiodactyla, including Bovidae (cattle) and Suidae (pigs), or a mammal of the order Perssodactyla, including Equidae (horses). In some embodiments, the non-human mammal is a mammal of the order Primates, Ceboids, or Simoids (monkeys), or Anthropidae (great apes). In various aspects, the non-human animal is a goat, a llama, an alpaca, a chicken, a duck, a fish (eg, salmon), a sheep, or a lamb.

[0037] In exemplary cases, the non-human animals used in the methods of the present disclosure are modified (e.g., genetically altered) 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 may 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 strain of transgenic mouse that produces fully human antibodies. The XenoMouse® is described generally in 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. The transgenic rat is, in various embodiments, a Unirat® or OmniFlic® (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).

[0038] immunogen Advantageously, the methods of the present disclosure are not limited to any particular immunogen. The immunogen, in various aspects, can be any antigen (optionally a protein) or fragment, fusion, or variant thereof. In various cases, the immunogen is a cytokine, lymphokine, hormone, growth factor, extracellular matrix protein, tumor-associated antigen, checkpoint inhibitor molecule, cell surface receptor, or ligand thereof. Solely for purposes of illustrating exemplary immunogens, the immunogen used in immunizing a non-human animal can be a target or antigen bound by any one of the following antibodies: 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.

[0039] Methods for preparing immunogens for use in the immunization process 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 cases, 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 in exemplary cases include oils, alum, aluminum salts, or lipopolysaccharides. In various aspects, the adjuvant is inorganic. In alternative aspects, the adjuvant is organic. In various embodiments, adjuvants include: alum, aluminum salts (e.g., aluminum phosphate, aluminum hydroxide), Freund's complete adjuvant, Freund's incomplete adjuvant, RIBI adjuvant system (RAS), lipid A, Sigma Adjuvant System®, TiterMax® Classic, TiterMax® Gold, Montanide vaccine adjuvants (e.g., Montanide 103, Montanide ISA 720, Montanide incomplete Seppic adjuvant, Montanide ISA51), AF03 adjuvant, AS03 adjuvant, Specol, SPT, nanoemulsions, VSA3, oil or lipid based solutions (e.g., squalene, MF59®, QS21, saponin, monophosphoryl lipid A (MPL)), trehalose dicorynomycolate (TDM), sTDM adjuvants, virosomes, and PRP 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 instances, adjuvants include surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol, BCG (bacilli Calmette-Guerin), and Corynebacterium parvum.

[0040] antibody Although the structure of antibodies varies between species, as used herein, the term "antibody" generally refers to a protein having a conventional immunoglobulin type, typically including heavy and light chains and including variable and constant regions. The antibodies obtained or isolated by the present method may have various uses. For example, the antibodies obtained by the present method may be used as therapeutic agents. The antibodies obtained by the present method may also be used as non-therapeutic antibodies, for example, as reagents used in diagnostic assays, such as diagnostic 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 may be monoclonal or polyclonal. In exemplary cases, 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 antibody may be a monoclonal or polyclonal antibody, optionally produced by a transgenic animal. In such embodiments, the antibody produced is a chimeric antibody containing sequences of more than one species. In various cases, 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 format, the variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and differs substantially among 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. Human antibody variable regions contain 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. Human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining the antibody's isotype 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. 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 exemplary embodiments, the antibody is of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4.

[0041] The antigen-binding protein may have a structure different from that of a human antibody. In an exemplary case, the antigen-binding protein includes only a heavy chain fragment (e.g., a heavy chain variable region, a heavy chain constant region CH2, a heavy chain constant region CH3). In various cases, the antigen-binding protein includes the structure of a small antibody or nanobody, 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.

[0042] Exemplary embodiments In the following, exemplary embodiments of the present disclosure are described.

[0043] 1. A method for producing a hybridoma, comprising: a. preparing an enriched population of IgG positive (IgG+) memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 5% of the cells in said enriched population are IgM positive (IgM+) B cells; b. bulk culturing the enriched population to obtain an expanded IgG+ memory B cell population; and c. fusing cells of the expanded IgG+ memory B cell population with myeloma cells to obtain hybridomas. The method includes:

[0044] 2. The method of embodiment 1, wherein the secondary lymphoid organs are (i) secondary lymphoid organs isolated from the immunized non-human animal about 3 to about 5 days after immunization, and / or (ii) secondary lymphoid organs isolated from at least 1, 2, 3, 4, or 5 immunized non-human animals.

[0045] 3. The method of embodiment 1 or 2, comprising preparing an enriched population of IgG+ memory B cells from a single cell suspension prepared from said secondary lymphoid organs.

[0046] 4. The method of any one of embodiments 1 to 3, wherein the secondary lymphoid organs are the spleen and / or lymph nodes.

[0047] 5. The method of any one of embodiments 1 to 4, wherein the non-human animal is a mouse or a rat.

[0048] 6. The method of any one of embodiments 1-5, comprising removing non-B cells, red blood cells (RBCs), IgM positive (IgM+) cells, or a combination thereof, from a single cell suspension prepared from said secondary lymphoid organ, optionally comprising removing said non-B cells, RBCs, IgM+ cells, or a combination thereof, using an antibody specific for one or more cell surface markers expressed by said non-B cells, RBCs, IgM+ cells, or a combination thereof.

[0049] 7. The method of embodiment 6, wherein the cell surface markers are human IgM, CD90.2, Ly-6G GR.1, NK-1.1, CD3 epsilon, CD4, CD8a, CD11b, and / or TER119.

[0050] 8. The method of embodiment 6 or 7, wherein the antibody is linked to biotin, and the method comprises removing the non-B cells, RBCs, and / or IgM+ cells using streptavidin-labeled beads, optionally streptavidin-labeled magnetic beads.

[0051] 9. The method of any one of embodiments 1 to 8, optionally comprising selecting surface IgG+ cells by using anti-IgG antibody labeled beads, optionally anti-human IgG antibody labeled magnetic beads.

[0052] 10. The method of any one of embodiments 1-9, wherein at least 10% of the cells in the enriched population are IgG+ B cells and / or more than 20% of the cells in the enriched population are positive for B220 expression.

[0053] 11. The method according to any one of embodiments 1 to 10, comprising bulk culturing the enriched population of IgG+ cells with anti-human IgG antibody-labeled beads and feeder cells in cell culture medium comprising rabbit T cell supernatant.

[0054] 12. The method of embodiment 11, wherein the feeder cells express CD40L and / or are gamma irradiated.

[0055] 13. The method of embodiment 12, wherein the feeder cells are gamma-irradiated CD40L-positive EL4B5 feeder cells.

[0056] 14. The method of any one of embodiments 1 to 13, wherein the myeloma cells are in logarithmic growth phase.

[0057] 15. The method of any one of embodiments 1 to 14, wherein the myeloma cells are P3 myeloma cells.

[0058] 16. The method of any one of embodiments 1 to 15, wherein the enriched population is bulk cultured at a density of about 350 B220 positive B cells per mL to about 700 B220 positive B cells per mL, optionally about 600 B220 positive cells per mL to about 650 B220 positive cells per mL.

[0059] 17. The method of any one of embodiments 1 to 16, wherein the enriched population is bulk cultured in a volume of about 50 mL or more.

[0060] 18. The method of any one of embodiments 1 to 17, wherein the enriched population is bulk cultured for at least about 4 days, at least about 5 days, or at least about 6 days, optionally for about 6 days.

[0061] 19. The method of any one of embodiments 1 to 18, wherein the cells of the enriched population are subjected to at least about seven cell divisions to obtain the expanded IgG+ memory B cell population.

[0062] 20. The method according to any one of embodiments 1 to 19, wherein cells of the expanded IgG+ memory B cell population are fused with myeloma cells by electrocellular fusion (ECF) to obtain hybridomas.

[0063] 21. The method of any one of embodiments 1-19, further comprising transferring the hybridoma and any unfused cells to a selection medium, optionally wherein the selection medium contains HA.

[0064] 22. The method of any one of embodiments 1 to 21, comprising storing the hybridoma under freezing conditions.

[0065] 23. The method of any one of embodiments 1 to 22, further comprising culturing the hybridomas in multiple plate wells and screening the supernatant of each well for antigen-specific antibodies.

[0066] 24. A method for screening for a hybridoma expressing an antigen-specific antibody, comprising: a. Producing a hybridoma according to the method of any one of embodiments 1-23; b. culturing a single hybridoma in an individual well; and c. Screening the supernatants of each well for antigen-specific antibodies. The method includes:

[0067] 25. A method for screening for a hybridoma expressing an antigen-specific antibody, comprising: a. preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 5% of the cells in the enriched population are IgM+ B cells; b. bulk culturing the enriched population to obtain an expanded IgG+ memory B cell population; c. fusing cells of the expanded IgG+ memory B cell population with myeloma cells to obtain hybridomas; d. culturing a single hybridoma in an individual well; and e. Screening the supernatants of each well for antigen-specific antibodies. The method includes:

[0068] 26. A method for producing an antigen-specific antibody, comprising: a. preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 5% of the cells in the enriched population are IgM+ B cells; b. bulk culturing the enriched population to obtain an expanded IgG+ memory B cell population; c. fusing cells of the expanded IgG+ memory B cell population with myeloma cells to obtain hybridomas; d. culturing a single hybridoma in an individual well; e. screening the supernatants of each well for antigen-specific antibodies to identify hybridomas expressing antigen-specific antibodies; and f. Growing the culture of the hybridoma identified in (e) to produce an antigen-specific antibody. The method includes:

[0069] 27. The method of any one of embodiments 1 to 26, wherein less than about 3% of the cells in the enriched population are IgM+ B cells.

[0070] 28. The method of embodiment 27, wherein less than about 2% of the cells in the enriched population are IgM+ B cells.

[0071] 29. The method of embodiment 28, wherein less than about 1% of the cells in the enriched population are IgM+ B cells.

[0072] The following examples are presented merely to illustrate the present invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0073] Example 1 This example describes an exemplary method for generating hybridomas of the present disclosure.

[0074] immunization A cohort of seven transgenic XenoMouse® G2-KL (XMG2-KL) animals (capable of producing human antibodies with either kappa or lambda light chains) was immunized with native antigen Z stably expressed on CHO cells. Animals were initially immunized subcutaneously in the left thigh (SQ / LT) with 4×10 6 immunized with 2×10 cells and adjuvant Alum / CpG ODN, then administered 2×10 6 The animals were boosted twice weekly with 100 cells, with the final boost being given 4 days before harvesting the animals and removal of the spleen and draining lymph nodes.

[0075] Isolation & Enrichment To isolate memory B cells, the spleens and lymph nodes (LNs) of seven immunized animals were harvested and processed into single cell suspensions (SCS) for each organ type: one SCS from the spleens of all animals, and one SCS from the LNs of all animals. Red blood cells (RBCs) were removed from the SCS from the spleens using BD Pharm Lyse™ (BD Biosciences, Franklin Lakes, NJ). All SCS were then combined into one pooled SCS for further processing. B cells were enriched using a four-step enrichment procedure, with the first two steps removing IgM-positive (IgM+) cells, among other cells, and the last two steps positively selecting from surface IgG-positive (IgG+) B cells.

[0076] In the first step, cells were incubated with a biotinylated antibody cocktail for 20 min at 4° C. The antibody cocktail contained biotinylated antibodies that specifically bind to cell surface markers on non-B cells (e.g., T cells, monocytes, macrophages, natural killer (NK) cells, granulocytes, and RBCs) and human IgM-positive B cells. The antibody cocktail contained antibodies specific for NK1.1 (expressed by NK cells), IgM (expressed by IgM positive B cells), CD90.2 (expressed by T cells), Ly-6G GR.1 (expressed by granulocytes and / or macrophages), CD3ε (expressed by T cells), CD4 (expressed by T cells), CD8a (expressed by T cells), CD11b (expressed by granulocytes, macrophages, dendritic cells, and / or NK cells), and TER119 (expressed by red blood cells). After incubation with the cocktail, the cells were washed to remove unbound antibodies. In the second step, the washed cells were incubated with streptavidin magnetic beads (e.g., Streptavidin Dynabeads (ThermoFisher Scientific, Pleasanton, CA)) for 10 min at 4° C. to bind the streptavidin magnetic beads to the biotinylated antibodies bound to non-B cells and IgM positive B cells. A magnet was used to isolate and remove the magnetic beads-labeled non-B cells and IgM positive B cells. The remaining fraction of cells not bound to the magnetic beads contained the memory B cell population, and in the third step, this fraction was incubated with anti-human IgG antibody microbeads (Miltenyi, Bergisch Gladbach, Germany) for 40 min at 4° C. Finally, in the fourth step, the microbead-bound cells were applied to a magnetic column. Surface IgG positive cells were retained in the column, while surface IgG negative cells passed through the column and were collected for further processing.The surface IgG positive cells, which represent memory cells, were allowed to drain or eluted from the column and then analyzed by FACS for surface expression of B220, IgG, and IgM. Memory cells express high levels of B220 and IgG on their surface.

[0077] An exemplary FACS plot from the FACS analysis is shown in FIG. 3A. Table 1 shows an overview of the FACS analysis before, during, and after the enrichment procedure to enrich for IgG+ memory B cells. As shown in Table 1, the percentage of B220 positive cells increased significantly from 27.2% to 55.9% after the four-step enrichment procedure. B220 is a cell surface marker for B cells (see, e.g., Khodadadi et al., Front Immunol 10:Article 721(2019);doi:10.3389 / fimmu.2019.00721). After enrichment, as shown in Table 1, only 8.8% of the B220 positive cells were IgM positive, whereas 33.4% of the B220 positive cells were IgG positive. As shown in Table 1, the percentage of IgG+ cells of the total live cells increased from less than 1% to more than 15%. Similarly, a significant proportion (28.1%) of IgG+ B cells were recovered by this process (Table 1). The ratio of IgG+ to IgM+ cells increased from about 0.006 to about 3.8 (Table 1). This ratio increased by more than 600-fold due to the enrichment process, as 99.96% of the IgM+ B cells were removed by the enrichment process.

[0078] [Table 1]

[0079] The flow-through fraction containing surface IgG negative cells including CD138 / TACI double positive plasma cells was enriched by a separation procedure to rescue IgG secreting but not surface IgG positive plasma cells by a direct B cell discovery platform. FACS was performed to analyze the expression of B cell markers to assess the enrichment of IgM negative plasma cells. An exemplary FACS plot from FACS analysis of the cells is shown in FIG. 3B. This enriched fraction contains a high affinity IgG secreting plasma cell population and is applied to direct B cell discovery technology for antigen binding secretion assay.

[0080] Bulk Culture The enriched population containing IgG+ memory B cells obtained by the four-step enrichment process was bulk cultured in T175 flasks at 625 B220 positive B cells / ml in EPMI medium supplemented with FBS, gamma-irradiated CD40L-expressing EL4B5 feeder cell line, rabbit T cell supernatant, and human IgG cross-linked microbeads. The volume of the bulk culture is generally about 200 mL. The culture was incubated at 37° C. and 5% CO2 for 6 days. After bulk culture, the cells were harvested and counted. The number after bulk culture was compared to the number of input B cells (the number of B cells used to seed the bulk culture) to calculate the number of cell divisions that occurred during bulk culture. Based on this count, it was determined that B cells underwent 9.8 cell divisions in the bulk culture to obtain the expanded population.

[0081] cell fusion The fusion partner P3 myeloma cells were expanded and harvested at logarithmic growth phase. The expanded population was combined with P3 myeloma cells at a B cell to P3 myeloma cell ratio of approximately 1:2.6. The cell mixture was washed twice with hypotonic Electro Cell Fusion (ECF) buffer and 2×10 6 The cells were resuspended to a density of 150×10 cells / ml. Electrical cell fusion was performed using a fusion chamber for high throughput fusion. In this experiment, 150×10 cells were 6 391 × 10 B cells were fused in 18 fusion events. 6Each fusion consisted of a 40 second 60v prealignment followed by 3 x 30 μsec pulses of 800V each.

[0082] Post-fusion culture and N2 storage After cell fusion, cells were immediately submerged in 270 ml of DMEM medium with FBS, washed once, and placed into 3 x 200 ml of T175 bulk culture in DMEM with hypoxanthine azaserine (HA) to remove unfused myeloma cells. After 3 days of post-fusion culture, the hybridoma pool was harvested, washed, and frozen in 12 aliquots in 90% newborn calf serum (NCS) & 10% DMSO. After 24 hours at -80°C, the vials were transferred to liquid nitrogen for long-term storage. One vial was thawed and plated in a low density 96-well plate to assess clonal growth in DMEM selection medium. From this, the fusion efficiency and the complexity of the pool were calculated. Here, the total complexity after fusion was calculated to be 205,882 unique hybrids, and the fusion efficiency was calculated to be 0.140%.

[0083] Evaluation of fusion efficiency in hybrid pools & calculation of complexity The maximum number of unique clones in the hybrid pool (coined "complexity") is a calculation that estimates what proportion of the enriched bulk cultured memory B cell population will be immortalized in the hybrid pool. Combining complexity with FSCS-informed recovery of memory B cells in the enrichment step allows for an approximation of the in vivo immune repertoire captured in the ENG event. In this working example, 28% of the total IgG+ memory B cells from the in vivo repertoire were recovered after enrichment and were placed in the B cell culture and EHC. The fusion captured 60% of this fraction. Overall, the process captured (immortalized) 17% (28%*60%) of the IgG+ memory B cell repertoire from pooled B cells of 7 animals.

[0084] The complexity calculation also guides the depth of interrogation of the hybrid pool: exceeding the calculated complexity is generally avoided when seeding a hybrid pool, as this increases the likelihood of repeated interrogation of clonal copies, but makes it increasingly unlikely to identify novel unique clones.

[0085] Complexity calculations were based on three measurements and four assumptions: the measurements / variables were (1) increasing input number of B cells to the culture; (2) division in prefusion culture based on counts on day 6; and (3) the number of clones viable on a known amount of low density plating from the resulting hybrid pool. The assumptions were: (1) each enriched B cell subjected to bulk culture is a unique clone; (2) each enriched B cell subjected to bulk culture survives 6 days in culture; (3) all enriched B cells subjected to bulk culture divide at the same rate; and (4) 50% of the fused cells are lost during freeze-thaw of the hybridoma pool.

[0086] Deviations from these assumptions decrease the complexity of the hybrid pool, with the exception of freeze-thawing, which may decrease or increase the complexity.

[0087] The complexity of the hybrid pool cannot exceed the input number of B cells into the B cell culture.

[0088] Complexity of hybrid pool = (viable clones after fusion before freezing in culture) / (2^ divisions in postfusion culture)

[0089] Viable clones after fusion and culture but before freezing = (number of viable clones at low density plating / 50% loss during freezing) / percentage of total hybrid pool assessed for growth

[0090] Divisions in postfusion culture = 1 division in the first 24 hours, then 1 division every 16 hours = 1 + (days in postfusion culture - 1) * (24 / 16)

[0091] Division in B cell culture = LOG(viable cells on day 6 / input B cells on day 0) / LOG(2)

[0092] Clonal copies after B cell culture = 2^ divisions in culture

[0093] Clonal copies after fusion = clonal B cell copies at day 6 of culture * fusion efficiency %

[0094] Fusion efficiency % = complexity / B cells on day 6 of fusion

[0095] In cases where the post-fusion clonal copy number is greater than 1 (all clones are represented multiple times), the equation for clonal copies frozen after post-fusion culture is adjusted to account for siblings.

[0096] Example 2 This example describes another example of generating hybridomas using the methods of the present disclosure.

[0097] Six XenoMouse® animals (four XenoMouse® G2-K (XMG2-K) animals capable of producing human antibodies with kappa light chains; and two XenoMouse® G4-KL (XMG4-KL) animals capable of producing human antibodies with either kappa or lambda light chains) were immunized with antigen X, an antigen different from antigen Z in Example 1. The animals were first immunized with antigen X expressed in CHO-S cells administered intraperitoneally (IP), then boosted twice weekly for six weeks. The mice were allowed to rest for 2.5 months. Four days after the final boost, spleens and draining lymph nodes (LNs) were removed from the immunized mice. As in Example 1, the removed spleens were pooled together and processed into SCS, and the removed draining LNs were pooled together and processed into SCS. Essentially as described in Example 1, one SCS from the spleen was depleted of RBCs and then the SCS from the LN were combined to obtain a pooled SCS for further processing.

[0098] In Example 1, a four-step B cell enrichment process was described, followed by bulk culture. The first and second steps of the B cell enrichment process were aimed at depleting IgM positive (IgM+) cells, and the last two steps were aimed at enriching surface IgG+ cells (by positive selection of surface IgG+ cells using anti-human IgG antibody-labeled magnetic beads). To evaluate the importance of the B cell enrichment process and bulk culture steps in hybridoma generation, the pooled SCS was divided into five groups (groups 1-5), and each group was subjected to its own protocol, which was varied by including or excluding IgG+ enrichment and by including or excluding bulk culture. The treatments of each of the five groups are outlined in Table 2.

[0099] [Table 2]

[0100] As shown in Table 2, groups 1 and 2 did not undergo any bulk culture. Group 1 did not undergo any further steps of the enrichment process and was considered "no cut", and group 2 underwent only the first two steps of the four-step enrichment process described in Example 1. Groups 3-5 were bulk cultured for 6 days, with only group 5 including both IgM+ cell depletion (achieved by the first two steps of the enrichment process described in Example 1) and surface IgG+ cell enrichment (achieved by the last two steps of the enrichment process described in Example 1), group 3 excluded both IgM+ cell depletion and surface IgG+ cell enrichment, and group 4 excluded surface IgG+ cell enrichment. IgM+ cell depletion for groups 2, 4, and 5 was performed essentially as described in Example 1. Surface IgG+ cell enrichment for group 5 was performed essentially as described in Example 1. A summary of the characteristics of each of groups 1 to 5 (before bulk culture) is shown in Table 3.

[0101] [Table 3]

[0102] As shown in Table 3, for groups 2, 4, and 5 (where at least a portion of the four-step enrichment process was performed), about 10% or less of the enriched population were IgM positive (IgM+) B cells. Similarly, for group 5 (where all steps of the enrichment process were performed), the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population was greater than 4, meaning that the number of IgG+ cells outnumbered the IgM+ cells.

[0103] Cells from groups 3-5 were bulk cultured essentially as described in Example 1 to obtain expanded populations. Cells from groups 1-5 were used for cell fusion, which was carried out as described in Example 1. After fusion, cells were cultured in DMEM containing HA for 3 days. After fusion, viable cells were counted and screened for secretion of antigen X-specific antibodies using FMAT fluorometric microvolume assay technology (FMAT™ 8100 HTS System).

[0104] The counting and characterization results are shown in Table 4.

[0105] [Table 4]

[0106] As shown in Table 4, groups 1 and 2 (not bulk cultured) generated the lowest number of hybridoma clones and had the lowest % capture of antigen+repertoire, confirming the importance of bulk culture. Among groups 3-5 (bulk cultured), groups 4 and 5 (IgM depleted before bulk culture) had the highest % hit frequency. When both IgM depletion and surface IgG enrichment were performed, the % hit frequency was almost doubled (groups 4 and 5). Taken together, these results demonstrate the advantage of bulk culturing the B cell enriched population before cell fusion. The relative % of antigen+repertoire captured by group 5 was calculated to be 31% (Table 4). Considering that some B cells are inevitably lost during purification and that germinal center B cells contain sister and overlapping specificities, the capture of 31% repertoire by group 5 is excellent and likely represents a complete compartment. In addition, interrogation of the entire repertoire is feasible, requiring only 109 96-well plates to interrogate the entire repertoire (with a hit frequency of 14.9%), which is not the case for the methods in group 3.

[0107] Example 3 This example describes a third instance of generating hybridomas using the methods of the present disclosure.

[0108] The disclosed method for generating hybridomas is used to generate hybridomas that produce high affinity antigen-specific antibodies. In this example, the antigen was a G protein-coupled receptor (GPCR). GPCRs constitute a therapeutically relevant target class that is notoriously difficult to target with antibodies (Hutchings CJ, Expert Opin. Biol. Ther. 2020, vol 20, No. 8, 925-935). Antibodies against this GPCR have been generated in the past, but none have been cross-reactive with both human and cynomolgus orthologues, much less have an affinity of at least 1 nM for each orthologue. Therefore, the goal was to generate hybridomas that secrete human / cynomolgus cross-reactive GPCR-specific antibodies that exhibit an antigen affinity of at least 1 nM for each of the human / cynomolgus orthologues.

[0109] Over 300 Xenomouse animals (e.g., XenoMouse® G2-K (XMG2-K) animals capable of producing human antibodies with kappa light chains) were immunized with GPCR antigens following an immunization campaign that included GPCR DNA immunization (by gene gun), peptides spanning the extracellular region of the GPCR, GPCR-transfected cells, and the extracellular domain of the GPCR fused to a human IgG-Fc portion. After immunization, serum was collected from each of the immunized animals and GPCR-specific antibody titers were assessed using standard methods (e.g., evaluation of polyclonal antibody binding by FACS analysis to GPCRs transiently expressed on 293T cells). Results of the antibody titer analysis revealed that 48 animals (15% of the total number immunized) exhibited sufficient levels of antigen-specific antibody titers, as determined by a bound GeoMean signal on GPCR-transfected cells that was at least 3-fold higher compared to the GeoMean signal on mock-transfected cells.

[0110] This example describes hybridomas generated from the secondary lymphoid organs of one of these 48 animals. Briefly, 4 days after the last immunization boost, the spleen and draining LN of the animal were harvested. An SCS was prepared from the spleen and another SCS was prepared from the LN. The SCS prepared from the spleen was depleted of RBCs as described in Example 1, and the RBC-depleted SCS was then combined with the SCS prepared from the LN. This combined SCS was subjected to only the first two steps of the four-step B cell enrichment process described in Example 1. IgM+ cell / non-B cell depletion resulted in a 99.5% reduction in viable cell count and a 99.8% reduction in IgM+ B cells. The IgG to IgM ratio was increased by 70-fold. After IgM+ cell / non-B cell depletion, the % of B220+ cells in the enriched population increased to 46.2%. Similarly, after IgM+ / non-B cell depletion, the % of IgG+ cells among B220+ cells increased to 7.5%. After IgM and non-B cell depletion, at least 10% of IgG+ cells were recovered.

[0111] Approximately 40,000 B cells were bulk cultured as described in Example 1, and this bulk culture process resulted in approximately 11 cell divisions. After bulk culture, the cells were subjected to cell fusion as in Example 1. After bulk culture and fusion of B cells, an estimated 40% of the cultured B cells were immortalized, and a hybrid pool with a complexity of 16,000 unique hybrids was generated. After fusion, the cells were cultured in DMEM containing HA to remove unfused myeloma cells. Hybridoma cells were then single-cell sorted in 384-well plates with soluble antigens that recapitulate specific regions of the GPCR of interest, or polyclonally plated in 96-well plates with 5 hybridoma clones per well. Hybridoma cells were cultured to produce enough antibodies to be detected by FACS-based screening. Screening was performed by FACS analysis, determining antibody binding in culture supernatants to 293T cells transiently transfected with GPCR, or by binding to cancer cell lines endogenously expressing GPCR. This is the same procedure as used in Example 1, except that in this example, 293T cells expressing GPCR antigen were used. Characterization of binding to human and cynomolgus orthologues was also performed. The affinity of the final selected molecules was determined by on-cell KinExa.

[0112] Over the course of several months to years, hybridomas were generated using secondary lymphoid organs from the remaining animals (47 remaining animals) selected based on antibody titer analysis in a similar manner as described above. Over 4000 hybridoma clones producing GPCR-specific antibodies were identified across the 48 immunized animals. However, only one showed affinity above 1 nM for both human and cynomolgus GPCR orthologues. Collectively, these results demonstrate the superior deep repertoire mining power of the disclosed EHG method. Given the greatest challenge with GPCRs as a target class and the very challenging affinity design goal of <1 nM for both human and cynomolgus orthologues, it was expected that this antibody would be very rare. This method allowed us to explore the immune repertoire of 48 antigen-responsive animals, recover over 4000 binders, and identify a single clone that met the design goal.

[0113] All references cited herein (e.g., publications, patent applications, and patents) are herein incorporated by reference 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.

[0114] The use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (especially with respect to the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "including," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated.

[0115] 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.

[0116] 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 representative 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.

[0117] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present 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 generating hybridomas, comprising: a. preparing an enriched population of IgG positive (IgG+) memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein: i. about 10% or less of the enriched population are IgM positive (IgM+) B cells; and / or ii. the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 0.5, and optionally greater than 1 or greater than 2; b. Bulk culturing the enriched population to obtain an expanded population; c. fusing cells of the expanded population with myeloma cells to obtain hybridomas; The method includes:

2. (a) immunizing one or more non-human animals with an immunogen; (b) harvesting secondary lymphoid organs from the immunized non-human animal, optionally about 3 to about 5 days after immunization; (c) preparing a single cell suspension (SCS) from said secondary lymphoid organs isolated from each immunized non-human animal; and / or (d) preparing a pooled SCS by combining the SCS from the secondary lymphoid organs of multiple immunized non-human animals. The method of claim 1 further comprising:

3. 3. The method of claim 1 or 2, wherein the enriched population is prepared by depleting IgM+ cells and / or selecting for IgG+ cells.

4. The method of any one of claims 1 to 3, wherein the enriched population is prepared by negative selection of IgM+ cells and / or positive selection of IgG+ cells.

5. 5. The method of claim 4, wherein greater than 90% of IgM+ B cells are depleted during the negative selection and / or the positive selection, and optionally greater than 95% of IgM+ B cells are depleted during the negative selection and / or the positive selection.

6. 6. The method of claim 5, wherein greater than 98% of IgM+ B cells are depleted during said negative selection, and optionally greater than 99% of IgM+ B cells are depleted during said negative selection and said positive selection.

7. 7. The method of any one of claims 1 to 6, wherein the enriched population is prepared by removing the non-B cells, RBCs, IgM+ cells, or a combination thereof, from a single cell suspension prepared from the secondary lymphoid organ, optionally by removing the non-B cells, red blood cells (RBCs), IgM+ cells, or a combination thereof, using an antibody specific for one or more cell surface markers expressed by non-B cells, red blood cells (RBCs), or IgM+ cells.

8. 8. The method of claim 7, wherein the antibody is linked to biotin, and the method comprises the step of removing the non-B cells, RBCs, and / or IgM+ cells using streptavidin-labeled beads, optionally streptavidin-labeled magnetic beads.

9. 9. The method of claim 8, wherein greater than 98% of the IgM+ cells present in the single cell suspension are depleted.

10. 10. The method of any one of claims 1 to 9, wherein the enriched population is prepared by selecting surface IgG+ cells, optionally using anti-IgG antibody labeled beads, optionally anti-human IgG antibody labeled magnetic beads.

11. 11. The method of claim 10, wherein greater than 99% of the IgM+ cells present in the single cell suspension have been depleted and / or the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells has been increased by at least about 50-fold or at least about 100-fold.

12. 12. The method of any one of claims 1 to 11, comprising bulk culturing the enriched population of IgG+ cells with anti-human IgG antibody labeled beads and feeder cells in cell culture medium comprising rabbit T cell supernatant.

13. The method of any one of claims 1 to 12, wherein: (A) the step of bulk culturing the enriched population is initiated at a seeding density of about 350 B220 positive B cells per mL to about 700 B220 positive B cells per mL, optionally about 600 B220 positive cells per mL to about 650 B220 positive cells per mL; (B) the enriched population is bulk cultured in a volume of at least about 25 mL and not more than about 2 L; and / or (C) the enriched population is bulk cultured in a volume of about 50 mL to about 500 mL, optionally about 100 mL to about 300 mL.

14. 14. The method of any one of claims 1 to 13, comprising bulk culturing the enriched population for at least about 4 days, at least about 5 days, or at least about 6 days.

15. The method of any one of claims 1 to 14, wherein: (A) the cells of the enriched population are subjected to at least about six or at least about seven cell divisions to produce the expanded population; (B) all B cells of the expanded population are used for fusion with myeloma cells and / or all cells of the expanded population are combined with myeloma cells; and / or (C) B cells are not selected for fusion with myeloma cells based on production of antibodies that bind to an antigen and / or B cells of the enriched or expanded population are not screened for production of antibodies that bind to an antigen.

16. 16. The method of any one of claims 1 to 15, wherein the step of screening for cells producing antigen-specific antibodies is performed only after the hybridomas are obtained and not before the hybridomas are obtained, or the step of screening for antigen-specific antibodies is performed only before harvesting the secondary lymphoid organs and after the hybridomas are obtained, optionally wherein the step of screening for antigen-specific antibodies before harvesting the secondary lymphoid organs comprises titer analysis of serum obtained from living immunized animals.

17. 1. A method for producing a hybridoma that produces an antigen-specific antibody, comprising: a. immunizing one or more non-human animals with an immunogen; b. Removing secondary lymphoid organs from the immunized non-human animal; c. Preparing a single cell suspension (SCS) from the secondary lymphoid organs isolated from each immunized non-human animal; d. preparing a pooled SCS by combining all the SCS prepared in (c); e. removing greater than 95% of IgM+ cells from said pooled SCS and / or positively selecting surface IgG+ cells from said pooled SCS to obtain an enriched population of IgG positive (IgG+) memory B cells, wherein: i. about 10% or less of the enriched population are IgM positive (IgM+) B cells; and / or ii. the ratio of the number of IgG+ memory B cells to the number of IgM+ B cells in the enriched population is greater than 0.5, and optionally greater than 1 or greater than 2; f. Bulk culturing the enriched population to obtain an expanded population; g. fusing cells of the expanded population with myeloma cells to obtain hybridomas; h. culturing a single hybridoma in an individual well and screening the supernatant of each well for antigen-specific antibody to identify hybridomas that produce antigen-specific antibodies; The method includes:

18. 1. A method for screening for hybridomas expressing an antigen-specific antibody, comprising: a. Producing hybridomas according to the method of any one of claims 1 to 17; b. culturing a single hybridoma in an individual well; c. screening the supernatants of each well for antigen-specific antibodies; The method includes:

19. 1. A method for screening for hybridomas expressing an antigen-specific antibody, comprising: a. preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals, wherein less than about 5% of the cells in the enriched population are IgM+ B cells; b. Bulk culturing the enriched population to obtain an expanded population; c. fusing cells of the expanded population with myeloma cells to obtain hybridomas; d. Culturing a single hybridoma in an individual well; e. screening the supernatant of each well for antigen-specific antibodies; The method includes:

20. 1. A method for producing an antigen-specific antibody, comprising: a. preparing an enriched population of IgG+ memory B cells from cells obtained from secondary lymphoid organs of one or more immunized non-human animals; b. Bulk culturing the enriched population to obtain an expanded population; c. fusing cells of the expanded population with myeloma cells to obtain hybridomas; d. Culturing a single hybridoma in an individual well; e. Screening the supernatant of each well for antigen-specific antibodies to identify hybridomas expressing antigen-specific antibodies; f. Expanding the culture of the hybridoma identified in (e) to produce antigen-specific antibodies; The method includes:

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