In vitro culture method for antibody-expressing cells

Abstract

The present invention relates to an in vitro method for culturing one or more antibody-expressing cells. The method comprises culturing one or more antibody-expressing cells obtained from peripheral blood in the presence of IL-2, IL-21, and non-cell surface-presented CD40 stimulants and in the absence of feeder cells. Furthermore, this specification provides a method for producing antibodies, including the step of culturing one or more antibody-expressing cells according to the method of the present invention, novel CD40 stimulants and their use, and cell culture media.

Description

[Technical Field]

[0001] The present invention relates to an in vitro method for culturing one or more antibody-expressing cells. The method comprises culturing one or more antibody-expressing cells obtained from peripheral blood in the presence of IL-2, IL-21, and non-cell surface-presented CD40 stimulants and in the absence of feeder cells. Furthermore, this specification provides a method for producing antibodies, including the step of culturing one or more antibody-expressing cells according to the method of the present invention, novel CD40 stimulants and their use, and cell culture media. [Background technology]

[0002] The development of monoclonal antibodies is generally based on immunization of animals with the desired target antigen. Standard species include mice, rats, and hamsters, as well as rabbits, which produce hybridomas by fusing spleen cells with myeloma cells (Milstein and Kohler). In the case of rabbits, both hybridoma (Spieker-Polet H, Sethupathi P, Yam PC, Knight KL. Proc Natl Acad Sci USA. 1995;92(20):9348-9352) and B cell cloning (Seeber et al, PLoS One. 2014 Feb 4;9(2):e86184.doi:10.1371 / journal.pone.0086184) can be used. B cell cloning requires ex vivo culture and proliferation of primary antibody-expressing cells isolated from peripheral blood. Methods for culturing B cells in connection with B cell cloning are described, for example, in International Publications 2011 / 147903, 2013 / 076139, 2017 / 167714, 2018 / 122147, 2018 / 210896, and 2019 / 105864.

[0003] Efficient culture and proliferation of primary antibody-expressing cells obtained from peripheral blood is typically performed using a feeder cell-based system in the presence of conditioned cell supernatant, for example, with EL4-B5 cells (Zubler et al, 1985, J Immunol 1 June 1985;134(6):3662-3668). The conditioned cell supernatant is usually derived from a thymocyte line (thymocyte supernatant (TSN)) (Steenbakkers et al, Mol Biol Rep. 1994 Mar;19(2):125-34). This type of culture using feeder cells and TSN is commonly employed to efficiently proliferate primary antibody-expressing cells obtained from peripheral blood in a single-cell form.

[0004] However, culture systems using feeder cells and TSNs have certain drawbacks. Firstly, feeder cells need to be irradiated to avoid overgrowth of antibody-expressing cells. This irradiation is a cumbersome and time-consuming process and requires specialized and expensive equipment. Secondly, the presence of feeder cells can hinder the use of modern single-cell technologies such as droplet-based systems, scale-down processes, and high-throughput systems, due to the fact that a mixture of feeder cells and antibody-expressing cells is always required. Irradiated feeder cells also complicate the use of automated systems, as these cells have a shorter lifespan, requiring more complex automated processes to handle them. Finally, using cell-derived conditioned media such as feeder cells and TSNs is prone to lot-to-lot variability and therefore requires complex and time-consuming preparation processes with a high need for quality control.

[0005] Considering the above, there is a strong need to address the aforementioned shortcomings of ex vivo culture of primary antibody-expressing cells and thereby improve B cell cloning techniques and antibody production processes.

[0006] These drawbacks are addressed by the present invention as defined by the claims and the following disclosures. [Overview of the project]

[0007] According to a first aspect, the present invention relates to an in vitro method for culturing one or more antibody-expressing cells obtained from peripheral blood. The method comprises culturing one or more antibody-expressing cells in the presence of IL-2, IL-21, and a non-cell surface-presented CD40 stimulant (e.g., a soluble CD40 stimulant).

[0008] Using the culture method of the present invention, one or more antibody-expressing cells obtained from peripheral blood can be proliferated; that is, one or more antibody-expressing cells can divide during culture and increase in number.

[0009] Therefore, the method of the first embodiment may be an in vitro method for growing one or more antibody-expressing cells obtained from peripheral blood, the method comprising growing one or more antibody-expressing cells by culturing one or more antibody-expressing cells in the presence of IL-2, IL-21 and a non-cell surface-presenting CD40 stimulant.

[0010] As demonstrated by the accompanying examples, the inventors have surprisingly found that antibody-expressing cells, particularly antibody-secreting cells (e.g., plasmablasts) obtained from peripheral blood (e.g., rabbit peripheral blood), can be cultured and proliferated using the method according to the first embodiment, even in the absence of feeder cells (e.g., EL4B5) and conditioned cell supernatant (e.g., TSN), without losing their ability to produce antibodies. In particular, it has been demonstrated that antibody-expressing cells obtained from peripheral blood can be proliferated at least as efficiently as in the presence of feeder cells and TSN by adding IL-2, IL-21, and a non-cell surface-presented CD40 stimulant (e.g., soluble CD40-L) to the culture medium. Very surprisingly, the accompanying examples also demonstrate that even a single antibody-expressing cell obtained from peripheral blood can be successfully cultured and efficiently proliferated using the method of the present invention, even in the absence of feeder cells and TSN, and consequently in the absence of cell-cell contact. Cell-cell contact was thought to be important for efficient proliferation (see Zuber et al. 1985, mentioned above).

[0011] The surprising discovery that commonly used feeder cells and TSN can be replaced without substantial loss of culture and proliferation capabilities resolves the drawbacks associated with feeder cells and cell-derived conditioned media (e.g., TSN), as described above. Thus, the culture process becomes easier to handle, more robust, and consumes less time and resources. Another important advantage is that smaller culture vessels can be used in the absence of feeder cells, enabling the use of more highly multiplexed systems, such as 1536-well plates or 384-well plates instead of 96-well plates. In other words, it becomes easier to apply automated and high-throughput techniques. Similarly, the applicability of automated systems becomes possible because these approaches can be significantly hindered by the requirements of feeder cells.

[0012] In the method of the first aspect, the culturing step is performed in the absence of feeder cells, particularly in the absence of feeder cells for culturing antibody-expressing cells (e.g., EL4B5 cells frequently used for this purpose). In other words, the culturing step preferably does not include co-culturing with feeder cells, particularly feeder cells for culturing antibody-expressing cells such as EL4B5. Other exemplary but non-limiting examples of feeder cells for culturing antibody-secreting cells are 3T3 cells (J. Huang et al., 2013, Cells. Nat. Protoc. 8, 1907-1915) and CD40-L-expressing mammalian cells as described in WO 2013076139.

[0013] Furthermore, in the method of the first aspect, the culturing step is performed especially in the absence of conditioned cell supernatant such as thymocyte supernatant (TSN). "Conditioned cell supernatant" relates to a culture supernatant obtained by culturing cells (e.g., plasmablasts) that secrete cytokines that stimulate the survival and proliferation of antibody-expressing cells obtained from peripheral blood in a medium for a defined time. The culture supernatant is removed from cells (e.g., plasmablasts) that secrete cytokines that stimulate the survival and proliferation of antibody-expressing cells obtained from peripheral blood after the defined time, and contains cytokines and substances that promote the survival and proliferation of antibody-expressing cells ex vivo. A preferred example of conditioned cell supernatant is TSN. TSN can be produced as described in Steenbakkers et al, Mol Biol Rep. 1994 Mar;19(2):l25-34 (which is incorporated herein in its entirety). Briefly, TSN can be obtained as follows: thymocytes obtained from 4- to 6-week-old rabbits are cultured for 24 hours at a cell density of about 2×10 7 cells / ml in the presence of 5 μg / ml PHA-M (phytohemagglutinin M) and 50 ng / ml PMA (phorbol myristate acetate). This culture can be performed in RPMI 1640 supplemented with the following medium: 10% FCS, 0.29 g / l glutamine, 2 mM Na-Pyruvat, 5 mM Hepes and 2.5 μM ss-mercaptoethanol. Subsequently, the supernatant (=TSN) is obtained by centrifugation and sterile filtration.

[0014] In embodiments of the method of the first aspect, the culturing of antibody-expressing cells obtained from peripheral blood is performed in the presence of IL-2, IL-21 and IL-15. As shown in the attached examples, the further presence of IL-15 further promotes the proliferation of antibody-expressing cells.

[0015] In embodiments of the method of the first aspect, the culturing of antibody-expressing cells obtained from peripheral blood is performed in the presence of IL-2, IL-21 and B cell activating factor (BAFF). BAFF is soluble BAFF especially in the context of the present invention. As shown in the attached examples, the further presence of BAFF further promotes the proliferation of antibody-expressing cells.

[0016] In a particularly preferred embodiment, antibody-expressing cells obtained from peripheral blood are cultured in the presence of IL-2, IL-15, IL-21, and BAFF. As shown in the accompanying examples, this combination of cytokines promotes the proliferation of antibody-expressing cells, which is the best of all the conditions tested herein.

[0017] In various embodiments, one or all of IL-2, IL-15, IL-21, and BAFF may be recombinantly produced. Recombinantly produced IL-2, IL-15, IL-21, and / or soluble BAFF may be commercially available, as discussed in the appendix examples below.

[0018] IL-2, IL-21, IL-15, and / or BAFF used herein may be of the same or different species as the antibody-expressing cells, insofar as they have the corresponding culture and proliferation activity for the antibody-expressing cells described herein. As demonstrated in the accompanying examples, human IL-2, human IL-21, human IL-15, and human BAFF have been successfully used in conjunction with rabbit antibody-expressing cells. In one embodiment, the antibody-expressing cells obtained from peripheral blood are rabbit antibody-expressing cells, and one of IL-2, IL-21, IL-15, and BAFF is independently selected from human or rabbit. In one embodiment, the antibody-expressing cells are rabbit antibody-expressing cells obtained from peripheral blood, and each of IL-2, IL-21, IL-15, and BAFF is human. In one embodiment, the antibody-expressing cells are rabbit antibody-expressing cells obtained from peripheral blood, and each of IL-2, IL-21, IL-15, and BAFF is rabbit.

[0019] In the attached examples, it was found that certain compounds (e.g., cytokines) may be detrimental to the culture and especially the proliferation of antibody-expressing cells. Therefore, in the embodiments, the culture of one or more antibody-expressing cells is preferably carried out in the absence of one or more selected from inducible costimulators (ICOS), 4-1BB (also known as CD137), phorbol myristate acetate (PMA), and IL-4.

[0020] In some embodiments, one or more antibody-expressing cells are primary. As used herein, “primary” means that the cells are obtained directly from previously obtained peripheral blood or previously obtained peripheral blood mononuclear cells (PBMCs) for ex vivo culture, i.e., without further modification (such as immortalization by any means). In preferred embodiments, primary means that the antibody-expressing cells have not been frozen after being obtained from peripheral blood. Therefore, one or more antibody-expressing cells do not need to be immortalized. In some embodiments, primary antibody-expressing cells may be obtained 24 hours or less, particularly 12 hours or less, particularly 8 hours or less, particularly 4 hours or less, and particularly 1 hour or less before the start of culture.

[0021] As demonstrated in the attached examples, the culture step of the method according to the first embodiment can efficiently grow antibody-expressing cells with small cell numbers, and even in single-cell culture systems (i.e., in the latter case, even if there is no initial cell-to-cell contact). This allows the use of small-volume culture systems (e.g., 1536-well plates or 384-well plates) that are highly suitable for high-throughput screening.

[0022] One or more antibody-expressing cells may be 100 or fewer antibody-expressing cells, preferably 75 or fewer antibody-secreting cells, more preferably 50 or fewer antibody-expressing cells, and even more preferably 20 or fewer antibody-expressing cells during culture. In a particularly preferred embodiment, one or more antibody-expressing cells may be a single antibody-expressing cell. This single antibody-expressing cell may be cultured in a single-cell form and may be proliferated during culture. Single-cell culture and proliferation of antibody-expressing cells are very important for B-cell sorting methods used for screening antibodies against antigens of interest. Having a single cell allows for easy and clear determination of the coding sequence of the antibody produced by the single antibody-expressing cell.

[0023] In various embodiments, the culture volume of the method according to the first embodiment may be up to 190 μl, up to 100 μl, preferably up to 70 μl. In certain embodiments, the volume may be up to 10 μl. Since this method does not require feeder cells, the ability to use such small amounts for culture is a particular advantage of the method of the present invention. In particular, the method of the present invention can be used with 384-well, 1536-well plates and droplet techniques, which is difficult or even impossible when using feeder cells.

[0024] One or more antibody-expressing cells used herein are obtained from peripheral blood. "Obtained from peripheral blood" includes not only cells obtained directly from fresh, previously isolated peripheral blood (i.e., primary cells) but also cells derived from previously obtained and stored (e.g., frozen) peripheral blood mononuclear cells (PBMCs). In certain embodiments, the antibody-expressing cells used in the method according to the first aspect of the present invention are obtained directly from peripheral blood, i.e., blood or cryopreservation of PBMCs is not involved.

[0025] In some embodiments, the method according to the first embodiment may include ex vivo isolation of one or more antibody-expressing cells from peripheral blood and / or previously isolated PBMCs. "Isolating one or more antibody-expressing cells" means that the cells are concentrated to be substantially free of non-antibody-expressing cells. In some embodiments, "substantially free of non-antibody-expressing cells" means that 20% or less, preferably 10% or less, and most preferably 5% or less of the cells obtained by isolation are non-antibody-expressing cells. In some embodiments, "isolating" or "isolated" may be referred to as "concentrating" or "concentrated," respectively.

[0026] Ex vivo isolation of one or more antibody-expressing cells from peripheral blood (i.e., obtaining antibody-expressing cells from peripheral blood) may include (i) isolating PBMCs from peripheral blood and (ii) isolating one or more antibody-expressing cells from the isolated PBMCs. In various embodiments, PBMCs may be obtained and one or more antibody-expressing cells may be isolated from the PBMCs.

[0027] Peripheral blood mononuclear cells (PBMCs) are any peripheral blood cells that have a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not have a nucleus, and granulocytes (neutrophils, basophils, and eosinophils) have a multilobed nucleus. Therefore, PBMCs consist essentially of lymphocytes and monocytes, i.e., lymphocytes and monocytes concentrated from previously obtained peripheral blood. As will be understood by those skilled in the art, PBMCs are substantially free of other cells, although they may contain small amounts of other cells, as the methods used to isolate PBMCs are virtually impossible to obtain 100% purity. In various embodiments, PBMCs consist of at least 80%, preferably at least 85%, more preferably 90%, still more preferably 95%, and most preferably at least 99% mononuclear peripheral blood cells (i.e., lymphocytes and monocytes).

[0028] Peripheral blood is the blood that circulates throughout the entire body. Cellular components that can be isolated from human peripheral blood include red blood cells, white blood cells, and platelets.

[0029] In connection with the present invention, antibody-expressing cells are obtained ex vivo from peripheral blood. In certain embodiments, peripheral blood is obtained from a previously immunized animal (e.g., rabbit, rat, mouse, sheep, or hamster) that has been immunized with the antigen of interest. In some embodiments, peripheral blood may be obtained from 4 days after immunization to 15 days after immunization or the most recent booster immunization. In one embodiment, peripheral blood may be obtained from 4 days to a maximum of 9 days after immunization or the most recent booster immunization.

[0030] Methods for isolating PBMCs from peripheral blood samples are well known in the art. These cells can be extracted from blood, for example, using Ficol (a hydrophilic polysaccharide for separating layers of blood) and gradient centrifugation, thereby separating the blood into an uppermost layer of plasma, followed by a layer of PBMCs, and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and red blood cells. A non-limiting example of such a method is described in Kleiveland CR (2015) Scan. J. Clin. Lab. Invest. 1968, 21 (Suppl. 97): 77-89, which is incorporated herein by reference in its entirety. In an exemplary method, PBMCs may be obtained from peripheral EDTA blood as described below: EDTA blood is diluted 1:1 with PBS (e.g., Gibco, 10010-015), and one or more Leucoseps (商標)The solution may be applied to a tube (e.g., Greiner bio-one, 227288) in a volume of, for example, 20-30 ml. The tube may then be centrifuged, for example, at 800 g for 15 minutes without terminal pause. The interface formed within the tube may then be collected in a collection tube (e.g., Falcon, 352075) containing PBS (e.g., 130 ml), and the cells may be pelleted by centrifugation (e.g., 15 minutes). The cells may then be resuspended in solution or culture medium. To determine the cell count, for example, a C-Chip can be used. (商標) Fuchs-Rosenthal Counting Chambers (NanoEnTek, DHC-F01) and Trypan Blue may be used.

[0031] Obtaining / isolating one or more antibody-expressing cells from peripheral blood and / or PBMCs may involve isolating / enriching cells expressing membrane-bound antibodies (B cell receptors) (or multiple copies thereof). For example, enrichment methods such as MACS-based techniques or FACS-based cell sorting may be used to enrich antibody-expressing cells by selecting / enriching cells expressing surface antibodies such as subtype IgG, IgA, or IgM (e.g., IgG). For example, IgG-positive antibody-expressing cells may be isolated by staining the cells with a dye-labeled anti-IgG antibody and sorting the IgG-positive cells. To obtain antibody-expressing cells, the cells may further be stained with 7-aminoactinomycin D (7-AAD), a dye that can distinguish viable cells from apoptotic cells. In preferred embodiments, the intermediate cell size (FSC) is determined by analysis by flow cytometry. int Cells having ) may be selected to obtain antibody-expressing cells. In various embodiments, IgG is moderately positive (IgG int Cells that are ) can be selected as antibody-expressing cells. In various embodiments, antibody-expressing cells obtained from peripheral blood are plasmablasts. Plasmablasts are analyzed by flow cytometry and intermediate staining for IgG (IgG) is performed in PBMCs. int ) and intermediate cell size (FSC) int) are present and can be selected accordingly. Plasmablasts are distinguished from memory B cells in that they have lower IgG expression and are larger in size (FSC). In some embodiments, antibody-expressing cells include plasmablasts but not memory B cells. In some embodiments, antibody-expressing cells are enriched with respect to plasmablasts compared to memory B cells.

[0032] In flow cytometry, light scattered by cells can be measured by two photodetectors: forward scattering (FSC), which detects scattering along the laser path, and side scattering (SSC), which measures scattering at a 90-degree angle to the laser.

[0033] FSC intensity is proportional to the cell diameter and is mainly due to light diffraction around the cell. FSC signals can be used for cell identification by size. SSC, on the other hand, originates from light refracted or reflected at the interface between the laser and intracellular structures such as granules and nuclei. SSC provides information about the internal complexity (i.e., granularity) of the cell.

[0034] In some embodiments, the antibody-expressing cells cultured in connection with the present invention are plasmablasts. In some embodiments, the antibody-expressing cells cultured in connection with the present invention are not memory B cells.

[0035] Antibody-expressing cells (e.g., plasmablasts) are isolated, and the cells can be further selected / enriched for those that bind to each antigen of interest on the cell surface (i.e., antigen-specific antibody-expressing cells). Optionally, antigen-specific antibody-expressing cells can be selected so as not to bind to one or more potential cross-reactive structures. Antigen-specific antibody-expressing cells are preferably obtained from peripheral blood obtained from previously immunized animals (e.g., rabbits, mice, rats, sheep, hamsters), particularly rabbits, that have been immunized with the antigen of interest.

[0036] Antigen-specific antibody-expressing cells (such as plasmablasts) can be enriched from peripheral blood / PBMC via binding to an antigen of interest, for example, using MACS or FACS-based techniques (e.g., via staining with a dye-labeled antigen of interest). Additionally, the cells can be stained with an antibody against the antibody class of interest (e.g., IgG, IgA, or IgM) so that antibody expression can be selected by FACS sorting. For example, IgG-positive antibody-expressing cells can be isolated by staining the cells with a dye-labeled anti-IgG antibody and sorting for IgG-positive cells. The cells may further be stained with 7-aminoactinomycin D (7-AAD), a dye that can discriminate viable cells from apoptotic cells. In a preferred embodiment, cells having an intermediate cell size (FSC int ) are selected by analysis with a flow cytometer. In embodiments, cells that are moderately positive for IgG (IgG int ) can be selected by analysis with a flow cytometer. Plasmablasts typically have intermediate staining for IgG (IgG int ) and an intermediate cell size (FSC int ) and can be selected accordingly. Plasmablasts are distinguished from memory B cells in that they have lower IgG expression and are larger in size (FSC). In embodiments, the antibody-expressing cells cultured in the context of the present invention are plasmablasts. In embodiments, the antibody-expressing cells cultured in the context of the present invention are not memory B cells.

[0037] Thus, in embodiments, the antibody-expressing cells are intermediate-sized (FSC intThese are cells that can be obtained from or are obtained from peripheral blood (e.g., PBMCs obtained from peripheral blood) by enriching cells having IgG. In certain embodiments, the antibody-expressing cells are IgG-expressing cells, more preferably intermediate IgG-expressing cells (most preferably plasmablasts). In some embodiments, the antibody-expressing cells may be IgG-positive. In some embodiments, the antibody-expressing cells may be IgM-positive. In some embodiments, the antibody-expressing cells may be IgA-positive. "IgX" (i.e., IgG, IgM, or IgA) positivity means that the cells express "IgX" on their cell surface and optionally secrete it. Therefore, in some embodiments, the antibody-expressing cells express and / or secrete IgG, IgM, or IgA.

[0038] Additionally or alternatively, isolating or enriching one or more antibody-expressing cells from peripheral blood and / or PBMCs may involve isolating / enriching cells expressing one or more surface markers. Such surface markers may be selected from the group consisting of CD19 and CD43 or combinations thereof, in particular. In other words, obtaining antibody-expressing cells (e.g., antibody-secreting cells such as plasmablasts) may involve selecting cells by labeling them with surface markers, such cells being CD19-positive and / or CD43-positive. Plasmablasts are known to express CD19 and CD43 on their cell surface.

[0039] In certain embodiments of the method according to the first aspect, one or more antibody-expressing cells may include or consist of antigen-binding antibody-expressing cells. As used herein, “antigen-binding antibody-expressing cells” are cells that express antibodies that bind to a specific antigen of interest. Accordingly, “antigen-specific antibody-expressing cells” are cells that each express antibodies that bind to the same antigen of interest. Thus, in various embodiments, one or more antibody-expressing cells may each independently express antibodies against the same antigen of interest.

[0040] In certain embodiments of the method according to the first aspect, one or more antibody-expressing cells may include or consist of antigen-specific antibody-expressing cells. As used herein, “antigen-specific antibody-expressing cells” are cells that express antibodies that specifically bind to a particular antigen of interest. Thus, “antigen-specific antibody-expressing cells” are cells that each express an antibody that specifically binds to the same antigen of interest. Therefore, in various embodiments, one or more antibody-expressing cells may each independently express antibodies against the same antigen of interest.

[0041] Isolating one or more antigen-specific antibody-expressing cells from peripheral blood and / or PBMCs may involve selecting / enriching cells that express an antibody (or multiple copies thereof) that binds to (in some embodiments specifically binds to) the antigen of interest. Enrichment / selection may occur before or after the isolation or enrichment of antibody-expressing cells from peripheral blood or PBMCs. Selecting antigen-specific antibody-expressing cells may further involve removing cells that interact with potentially cross-reactive compounds.

[0042] An exemplary method for selecting antigen-specific cells may include providing beads to which the antigen of interest is attached, binding cells expressing antibodies that bind to the antigen of interest to the beads, separating the beads from free cells, and releasing the antigen-specific antibody-expressing cells from the beads. Optionally, the beads may be magnetic beads. For example, MACS technology may be used. Alternatively, cells may be stained with a labeled antigen of interest, and the stained cells may be selected, for example, by FACS sorting. For example, the label may be a fluorescent dye, and sorting may be achieved by FACS. Exemplary methods are also described in the accompanying examples.

[0043] In various embodiments, one or more antibody-expressing cells can be obtained by a method comprising (i) isolating PBMCs from a obtained peripheral blood sample, and (ii) isolating one or more antibody-expressing cells, preferably one or more antibody-expressing cells bound to the antigen of interest, from the isolated PBMCs. Each step may be as in any of its embodiments defined above. Optionally, a step of enriching cells expressing one or more surface markers of antibody-expressing cells may be performed before or after step ii) (see above).

[0044] In various embodiments, the method of the first embodiment may include (i) isolating PBMCs from the obtained peripheral blood sample, and (ii) isolating one or more antibody-expressing cells, preferably one or more antibody-expressing cells bound to the antigen of interest, from the isolated PBMCs. Each step may be as in any of the embodiments defined above.

[0045] In various embodiments, the antibody-expressing cells used in connection with the present invention are i) Isolating PBMCs from peripheral blood, ii) From the PBMC, a) IgG positive (e.g., IgG int ) and b) preferably, cells having an intermediate cell size (FSCint) are isolated using flow cytometry such as FACS sorting. These are cells obtained, or that can be obtained, from peripheral blood via isolation methods that include [specific components / methods].

[0046] In some embodiments, in ii), the cells may have intermediate IgG expression. In some embodiments, the cells isolated in ii) are selected to be negative in apoptosis staining (e.g., 7AAD staining), i.e., the cells are non-apoptotic. In some embodiments, CD19-positive cells and / or CD43-positive cells may be selected.

[0047] In various embodiments, the antibody-expressing cells used in connection with the present invention express antibodies that specifically bind to the antigen of interest, i.e., antigen-specific antibody-expressing cells. i) Isolating PBMCs from peripheral blood, ii) Isolating cells from PBMCs that bind to the antigen of interest on their cell surface (e.g., via MACS or flow cytometry using the labeled antigen of interest), iii) a) IgG positive (e.g., IgG int ) and b) preferably, intermediate cell size (FSC) is determined using flow cytometry such as FACS sorting. int To isolate cells that have ) and It can be obtained from peripheral blood via isolation methods including [specific method].

[0048] In some embodiments, in iii), the cells may have intermediate IgG expression. In some embodiments, the cells isolated in iii) are selected to be negative in apoptosis staining (e.g., 7AAD staining), i.e., the cells are non-apoptotic. In some embodiments, CD19-positive cells and / or CD43-positive cells may be selected.

[0049] In various embodiments, the antibody-expressing cells obtained from peripheral blood used herein are B cells obtained from peripheral blood having an intermediate cell size and intermediate IgG expression. Preferably, these B cells are antigen-specific, i.e., enriched with cells that bind to a specific antigen of interest.

[0050] In a specific embodiment, the antibody-expressing cells obtained from peripheral blood are plasmablasts obtained from peripheral blood (e.g., primary plasmablasts such as rabbit plasmablasts). The plasmablasts are isolated as PBMCs and sorted using FACS to determine intermediate IgG expression and intermediate cell size (FSC). int It can be obtained or obtained by enriching viable cells having ).

[0051] In certain embodiments, plasmablasts obtained from peripheral blood are antigen-specific plasmablasts, i.e., plasmablasts that express antibodies against the same antigen of interest. Such plasmablasts can be obtained by selecting plasmablasts that bind to the antigen of interest. In particular, enrichment for antigen binding can be performed at the PBMC level followed by plasmablast isolation, or after the plasmablasts have been isolated from the PBMCs. Plasmablasts can be obtained from peripheral blood by i) isolating PBMCs, and ii) isolating plasmablasts from said PBMCs, a) being IgG-positive (having intermediate IgG staining when FACS is used), and preferably having an intermediate cell size (FSCint) using FACS sorting. Corresponding methods are disclosed in the attached examples, and an exemplary flow cytometry blot showing a typical gating strategy for obtaining plasmablasts is shown in Figure 9.

[0052] One or more antibody-expressing cells can be cultured in a culture vessel. For example, when using single-cell culture, a single antibody-expressing cell may be cultured in a well of a 96-well culture plate or a 384-well plate.

[0053] As used herein, antibody-expressing cells obtained from peripheral blood may be or include antibody-secreting cells present in peripheral blood. These antibody-secreting cells are characterized by secreting antibodies into culture medium. Antibody-secreting cells that can be obtained from peripheral blood are preferably plasmablasts in the context of the present invention. Therefore, in various embodiments, antibody-expressing cells obtained from peripheral blood as used herein may be or include plasmablasts.

[0054] Antibody-secreting cells can be obtained by: (i) isolating antibody-expressing cells by any of the above methods (including any combination of the concentration steps); (ii) culturing the antibody-expressing cells (for example, using the method according to the first aspect of the present invention); and (iii) identifying the antibody-secreting cells by evaluating the presence of each class of antibody (e.g., IgG, IgM, or IgA) in the culture medium.

[0055] As described above, the method of the first aspect of the present invention includes culturing antibody-expressing cells in the presence of a non-cell surface-presented CD40 stimulant.

[0056] "Non-cell surface-presented CD40 stimulants" means that the CD40 stimulant is not presented by cells, particularly on the cell surface. Previously published culture methods for antibody-expressing cells, especially peripheral blood-derived B cells, have typically used feeder cells that express CD40-L on their cell surface in conjunction with cytokine mixtures.

[0057] The inventors have surprisingly found that, particularly by using non-cell-presenting CD-40 stimulants such as soluble CD-40 ligands, it is possible to culture and proliferate antibody-expressing cells obtained from peripheral blood in the absence of feeder cells. This means that in a single-cell culture system, antibody-expressing cells (e.g., plasmablasts) obtained from peripheral blood can be cultured and proliferated without any cell-to-cell contact or feeder cells. This provides much greater flexibility for culture, including the possibility of reducing the volume of culture vessels and / or using fully automated screening techniques.

[0058] A "non-cell-presenting CD40 stimulant" may be a soluble CD40 stimulant or a CD40 stimulant coated on a surface (e.g., the inner surface of a culture vessel), where the surface is not a cell surface. A "soluble CD40 stimulant" means that the CD40 stimulant is not present on the cell surface or artificial surface, and is soluble in the culture medium used.

[0059] A "surface-coated CD40 stimulant" is a CD40 stimulant, such as a CD40 ligand, bound (covalently or noncovalently) to a surface other than the cell surface. For example, a surface-coated CD40 stimulant may be a CD40 stimulant coated on the inner surface of a culture vessel used for culturing antibody-expressing cells.

[0060] A non-limiting example of a soluble CD40 stimulant is an antibody that mimics the binding of CD40 ligand (CD40-L) to CD40 and induces a signaling cascade triggered by the interaction of CD40-L and CD40 in CD40-expressing cells. Non-limiting examples of such antibodies are mAb G28.5 (Ledbetter JA et al., Circ Shock. 1994 Oct;44(2):67-72. PMID:7743602) and mAb 89 (Bjorck P et al., Immunology. 1994 Nov;83(3):430-7. PMID:7530692). Other non-limiting examples of soluble CD40 stimulants are soluble CD40 ligand constructs (e.g., MegaCD40-L from Enzo, see below: E. Marasco, et al.; Eur. J. Immunol. 47, 131 (2017), application: human PBMC culture) which are known in the prior art or newly provided herein. In preferred embodiments, soluble CD40-L (construct) used herein and / or in the accompanying examples is used. Preferably, the soluble CD40 ligand is recombinantly produced.

[0061] In a particularly preferred embodiment of the present invention, the soluble CD40 stimulant is a soluble CD40 ligand (i.e., CD40-L). The “soluble CD40 ligand” is a non-membrane-immobilized CD40-L protein or fragment thereof that is soluble in culture medium and can bind to and stimulate CD40 in antibody-expressing cells, i.e., mimics the naturally occurring interaction between CD40-L and CD40. Typically, the soluble CD40 ligand is recombinantly expressed.

[0062] Non-cell-presenting CD40 stimulants used herein (e.g., soluble CD40-L) may contain a CD40L domain.

[0063] In various embodiments, a non-cell-presented CD40 stimulant (e.g., soluble CD40-L) may include the CD40-L domain of SEQ ID NO: 1 (e.g., the 18kDa CD40-L domain), or an amino acid sequence having at least 75%, 80%, 90%, 95%, or 99% sequence identity thereto. This CD40-L domain has been shown to be sufficient for CD40 stimulation and is conserved to more than 75% sequence identity between rabbits and humans, mice, or rats. The sequence of SEQ ID NO: 1 is a rabbit sequence. Therefore, in various embodiments, a non-cell-presented CD40 stimulant (e.g., soluble CD40-L) may include the sequence of SEQ ID NO: 1 or a corresponding sequence derived from another species (e.g., human, mouse, rat, hamster, or sheep). In various embodiments, a CD40 stimulant (e.g., soluble CD40-L) may include any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a sequence having at least 75% sequence identity thereto. In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may contain any one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 80% sequence identity with it. In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may contain any one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 85% sequence identity with it. In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may contain any one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 90% sequence identity with it. In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may contain any one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 95% sequence identity with it. In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 99% sequence identity thereto.

[0064] In some embodiments, the CD40 stimulant (e.g., soluble CD40-L) includes, for example, the CD40-L domain of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a sequence having the sequence identity defined above but not containing any other CD40-L specific domains or sequences. As demonstrated in the accompanying examples, the use of the 18kDa CD40-L domain promoted the proliferation of antibody-expressing cells, particularly plasmablasts, more effectively than the use of the entire CD40-L extracellular domain (see SEQ ID NO: 7 for the corresponding rabbit sequence). Therefore, in some embodiments, the CD40 stimulant (e.g., soluble CD40-L) used herein does not contain any other CD40-L extracellular domain sequences other than the CD40-L 18kDa domain (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or any sequence having the sequence identity defined above). The CD40-L extracellular domain has the amino acid sequence shown in SEQ ID NO: 7.

[0065] In some embodiments, the CD40 stimulants used herein (e.g., soluble CD40-L) may include the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or variants thereof having CD40-L activity with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0066] The CD40 stimulating function of CD40 stimulants, particularly soluble CD40-L, is enhanced by the multimerization of the CD40-L domain. As demonstrated in the attached examples, hexameric soluble CD40-L constructs surprisingly promote the culture and proliferation of antibody-expressing cells with better efficacy than trimer soluble CD40-L constructs. Therefore, in various embodiments, the CD40-L construct is at least a trimer or more than a trimer. In even more preferred embodiments, the CD40-L construct is at least a tetramer, more preferably at least a hexamer, or a hexamer.

[0067] To achieve multimerization of CD40 stimulants (e.g., soluble CD40-L), the CD40 stimulant (e.g., soluble CD40-L) may contain a multimerizing domain, particularly an artificially introduced multimerizing domain (i.e., a multimerizing domain introduced by genetic engineering). Multimerizing domains are well known in the art.

[0068] Multimerizing domains resulting in different stoichiometry of multimerization are known in the art. In principle, any multimerizing domain resulting in at least a dimer, preferably at least a trimer, and more preferably at least a hexamer CD40 stimulant (e.g., soluble CD40-L) can be used. Further degrees of multimerization may occur by multimerization caused by the CD40-L domain itself, e.g., the 18kDa domain. The degree of multimerization of the CD40 stimulant (e.g., soluble CD40-L) can be analyzed by HPLC analysis (e.g., using SEC-MALS). Corresponding examples of such methods are employed and described in the appended examples. The multimerizing domain in the context of the present invention is selected so as to maintain the CD40-L activity of the fusion protein, i.e., so as not to adversely affect the activity that stimulates the CD40 receptor. Those skilled in the art are well aware of the multimerizing domains that can be selected. Exemplary but non-limiting multimerization domains include Fc domains (e.g., IgG antibodies), leucine zipper domains (LZs), and C4b domains. In some embodiments, Fc domains, particularly IgG Fc domains, particularly human IgG Fc domains, and particularly human IgG1 Fc domains, may be used as multimerization domains.

[0069] An exemplary C4b sequence that may be used in the context of the present invention is SEQ ID NO: 8, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 8, which, when used in a CD40 stimulant (e.g., soluble CD40-L), exhibits the same polymerization as SEQ ID NO: 8.

[0070] An exemplary leucine zipper sequence that may be used in the context of the present invention is SEQ ID NO: 9, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 9, which, when used in a CD40 stimulant (e.g., soluble CD40-L), exhibits the same degree of multimerization (e.g., trimer) as SEQ ID NO: 9. As demonstrated in the accompanying examples, this leucine zipper achieved trimerization of the CD40-L construct.

[0071] Another exemplary leucine zipper sequence that may be used in the context of the present invention is SEQ ID NO: 10, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 10, which, when used in a CD40 stimulant (e.g., soluble CD40-L), exhibits the same degree of multimerization (e.g., trimer) as SEQ ID NO: 10. As demonstrated in the accompanying examples, this leucine zipper achieved trimerization of the CD40-L construct.

[0072] An exemplary leucine zipper is also described in Burkhardt et al., Cancer Immunol Immunother. 2013 Feb;62(2):347-57.doi:10.1007 / s00262-012-1331-4.Epub 2012 Aug 25.

[0073] An exemplary IgG Fc sequence that may be used in the context of the present invention is SEQ ID NO: 11, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 11, which, when used in a CD40 stimulant (e.g., soluble CD40-L), exhibits the same polymerization (e.g., hexamer) as SEQ ID NO: 11.

[0074] Using the Fc domain (for example, human IgG1), a soluble hexamer CD40-L can be obtained, as demonstrated in the attached examples. Surprisingly, such a hexamer CD40-L showed excellent culture and proliferation activity in the culture of antibody-expressing cells, more specifically antibody-secreting cells such as plasmablasts.

[0075] In various embodiments, soluble CD40-L includes a linker amino acid sequence, such as a flexible amino acid linker sequence (e.g., serine-glycine linker, SEQ ID NO: 19 or 22), between the polymerizing domain and the domain having CD40-L activity (e.g., an external domain or an 18kDa domain).

[0076] In various embodiments, the CD40 stimulant (e.g., soluble CD40-L) may include affinity tags such as His tags, e.g., octa-His tags (used for purification). Such affinity tags are selected to facilitate purification without adversely affecting CD40 stimulating activity.

[0077] In various embodiments, a CD40 stimulant (e.g., soluble CD40-L) may be used that contains multiple copies (e.g., 3, 4, 5, 6, 7, or 8) of CD40-L domains fused to one another in a single polypeptide (e.g., the external domains or 18kDa fragments or variants thereof described above). The fusion protein may contain flexible linker sequences (e.g., serine-glycine linkers, SEQ ID NO: 19 or 22) between the copies of the CD40-L domains. An exemplary soluble CD40-L containing a linear fusion of CD40-L domains is disclosed in Merz, Christian et al. (Journal of Immunotherapy 41(9):p385-398, November / December 2018. | DOI:10.1097 / CJI.0000000000000246) and U.S. Patent Application Publication No. 20170327588.

[0078] In some embodiments, the CD40 stimulant used in the method of the present invention (e.g., soluble CD40-L) may comprise at least two copies of the amino acid sequence of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, either directly or fused via a linker sequence (e.g., SEQ ID NOs. 19 or 22), or a variant thereof having CD40-L activity with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

[0079] In some embodiments, the soluble CD40 stimulant used in the method of the present invention (e.g., soluble CD40-L) may comprise at least three copies of the amino acid sequence of SEQ ID NOs. 1, 2, 3, 4, 5, or 6, either directly or fused via a linker sequence (e.g., SEQ ID NOs. 19 or 22), or a variant thereof having CD40-L activity with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

[0080] In various embodiments, the soluble CD40 stimulants used herein (e.g., soluble CD40-L) are recombinantly expressed constructs, i.e., constructs expressed from genetically modified expression constructs that do not exist in nature. Molecular cloning techniques for such genetic manipulation are well known in the art.

[0081] In some embodiments, the CD40 stimulant used herein (e.g., soluble CD40-L) may comprise any one of SEQ ID NOs: 12-18, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may comprise any one of SEQ ID NOs: 12-18, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may consist of any one of SEQ ID NOs: 12-18, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may consist of any one of sequence numbers 12 to 18, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may include any one of sequence number 12, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may include any one of sequence number 12, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may include or consist of sequence numbers 12 to 18. In some embodiments, the soluble CD40-L includes or consists of sequence number 12.

[0082] In some embodiments, the CD40 stimulant used in the method of the present invention may be a CD40 stimulant according to the fourth aspect of the present invention or any of its embodiments (e.g., soluble CD40-L).

[0083] As used herein, CD40 stimulants (e.g., soluble CD40-L) or their variants possess CD40 stimulating activity (e.g., CD40-L activity); i.e., variants lacking CD40 stimulating activity (e.g., CD40-L activity) are excluded from the term. CD40 stimulating activity means the ability to stimulate CD40 signaling, which promotes the culture and proliferation of antibody-expressing cells, as shown herein, and optionally, antibody secretion in the absence of feeder cells. In various embodiments, CD40 stimulating activity (e.g., CD40-L activity) means that the ability to promote cell proliferation (in batch and / or single-cell settings) is at least as good as that of soluble CD40-L of SEQ ID NOs. 12, 13, 14, 15, 16, 17, or 18, preferably SEQ ID NO. 12, under otherwise identical experimental conditions. Assays for evaluating the ability to promote cell proliferation are described in Example 6. Briefly, antibody-expressing cells can be isolated by antigen panning, IgG-positive and 7-AAD-negative cells can be selected, and deposited in culture medium as batches (i.e., 50 cells per well) as described in Example 3. The cells can then be cultured in batches in BM1 medium in the presence of cytokines (IL-2, IL-15, IL-21, and BAFF) and the tested CD40 stimulant construct (e.g., soluble CD40-L). Proliferation is then evaluated by determining the cell count, for example, after 7 days.

[0084] Alternatively or additionally, CD40 stimulating activity can be tested by stimulating naive B cells (e.g., isolated from PBMCs via CD19-MACS from non-immunized animals) with the reagent of interest and analyzing B cell proliferation after 4-7 days of culture via CFSE dilution in a flow cytometer. For this purpose, naive B cells are labeled with CFSE after MACS-based purification (10 μM CFSE and 5 × 10⁶ cells / ml in RPMI medium without FCS) at 37°C for 5 minutes. The reaction is stopped in cold medium. After the washing step, CFSE-labeled B cells are cultured for 4-7 days in B cell medium containing various concentrations of CD40 stimulating reagent (5 × 10⁶ cells / ml in a 96-well plate). 5(Cells / well). B cell proliferation can be tracked by analyzing CFSE labeling in live cells (7-AAD negative) in a flow cytometer. Proliferated cells show a decrease in CFSE labeling due to dilution of the label after cell division. Unstimulated naive B cells do not proliferate in culture, while CD40-stimulated naive B cells show a proliferation rate of up to 40%.

[0085] The soluble CD40 stimulants used herein (e.g., soluble CD40-L) may be recombinantly expressed. Exemplary methods and expression systems are provided in the appended examples. The CD40 stimulant used in the context of the present invention is selected to have CD40-L activity with the antibody-expressing cells of the species used. In preferred embodiments, the CD40 stimulant has the same species origin as the cells. In specific embodiments, the antibody-expressing cells may be rabbit cells, and the CD40 stimulant may be any of the soluble CD40-L described above or elsewhere herein.

[0086] The antibody-expressing cells (e.g., antibody-secreting cells or plasmablasts) used in the context of the present invention may be of different origins. For example, the antibody-expressing cells may be rabbit, rat, mouse, human, goat, hamster, or sheep cells. In certain embodiments, the antibody-expressing cells may be rabbit or mouse cells. In particularly preferred embodiments, the antibody-expressing cells may be rabbit cells.

[0087] The IL-2 used in the context of the present invention may be of the same or different origin as the antibody-expressing cells used. For example, when rabbit antibody-expressing cells are used, the IL-2 may be rabbit IL-2, or IL-2 of a different origin, provided it is IL-2. As demonstrated in the accompanying examples, for example, human IL-2 (UniProt: P60568) can be used with rabbit antibody-expressing cells. In the case of IL-2 of a different origin, it is of course necessary to ensure that such IL-2 can stimulate the survival and proliferation of the antibody-expressing cells used. This can be done by using the method described in the accompanying examples. In certain embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-2 is rabbit or human IL-2. In other embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-2 is human IL-2. The IL-2 is preferably recombinant.

[0088] The IL-21 used in the context of the present invention may be of the same or different origin as the antibody-expressing cells used. For example, when rabbit antibody-expressing cells are used, the IL-21 may be rabbit IL-21, or IL-21 of a different origin, provided it is IL-21. As demonstrated in the accompanying examples, for example, human IL-21 (UniProt:Q9HBE4) can be used with rabbit antibody-expressing cells. In the case of IL-21 of a different origin, it is of course necessary to ensure that such IL-21 can stimulate the survival and proliferation of the antibody-expressing cells used. This can be done by using the method described in the accompanying examples. In certain embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-21 is rabbit or human IL-21. In other embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-21 is human IL-21. The IL-21 is preferably recombinant.

[0089] The IL-15 used in the context of the present invention may be of the same or different origin as the antibody-expressing cells used. For example, when rabbit antibody-expressing cells are used, the IL-15 may be rabbit IL-15, or IL-15 of a different origin, provided it is IL-15. As demonstrated in the accompanying examples, for example, human IL-15 (UniProt: P40933) can be used with rabbit antibody-expressing cells. In the case of IL-15 of a different origin, it is of course necessary to ensure that such IL-15 can stimulate the survival and proliferation of the antibody-expressing cells used. This can be done by using the method described in the accompanying examples. In certain embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-15 is rabbit or human IL-15. In other embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the IL-15 is human IL-15. The IL-15 is preferably recombinant (i.e., recombinantly expressed).

[0090] The BAFF used in this invention is soluble BAFF, i.e., the extracellular domain of BAFF having BAFF activity (in the case of human BAFF 134-285). The BAFF used in the context of this invention may be of the same or different origin as the antibody-expressing cells used. For example, when rabbit antibody-expressing cells are used, the BAFF may be rabbit BAFF, or BAFF from a different origin, provided that BAFF activity is provided for the antibody-expressing cells. As demonstrated in the accompanying examples, for example, human (soluble) BAFF (UniProt: Q9Y275; amino acids 134-285) can be used with rabbit antibody-expressing cells. In the case of BAFF from a different origin, it is of course necessary to confirm before use that such BAFF can stimulate the survival and proliferation of the antibody-expressing cells used. This can be done by using the method described in the accompanying examples. In certain embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the BAFF is rabbit or human BAFF. In other embodiments, the antigen-expressing cells are rabbit antigen-expressing cells, and the BAFF is human BAFF. The BAFF is preferably recombinant.

[0091] In one embodiment, all factors selected from IL-2, IL-21, IL-15, and BAFF used are of the same species origin. The species origin may be the same as or different from the antibody-expressing cell origin. In various embodiments, all factors selected from IL-2, IL-21, IL-15, and BAFF may be human (optionally, the antibody-expressing cell is a rabbit cell).

[0092] The concentrations of IL-2, IL-21, IL-15, BAFF, and / or CD-40L used in the context of this invention can be changed, and suitable concentrations can be determined by those skilled in the art using the information and experiments provided herein.

[0093] The appropriate concentration of IL-2 can be 0.1–20,000 IU / ml, particularly 10–10,000 IU / ml, particularly 20–1,000 IU / ml, and most notably 400–900 IU / ml.

[0094] The appropriate concentration of IL-21 can be 0.1 ng / ml to 150 ng / ml, especially 1 ng / ml to 50 ng / ml, and most specifically 5 ng / ml to 45 ng / ml.

[0095] The appropriate concentration of IL-15 can be 0.1–30 ng / ml, particularly 1–15 ng / ml.

[0096] The appropriate concentration of BAFF can be 0.1 ng / ml to 300 ng / ml, especially 1 ng / ml to 150 ng / ml, and most specifically 50 ng / ml to 150 ng / ml.

[0097] The appropriate concentration of the CD40 stimulant depends on the type of CD40 stimulant used. When using the CD40-L constructs provided herein, appropriate concentrations may range from 0.1 ng / ml to 20,000 ng / ml, particularly from 1 ng / ml to 10,000 ng / ml, and most specifically from 10 to 8,000 ng / ml.

[0098] In a second aspect, the present invention relates to a method for producing antibodies that specifically bind to or against an antigen of interest, the method comprising culturing one or more antigen-binding cells or antigen-specific antibody-expressing cells according to the method of the first aspect of the present invention or any embodiment thereof described herein.

[0099] In the context of other embodiments, such as the method according to the first aspect of the present invention, all definitions and embodiments disclosed herein shall apply with necessary modifications.

[0100] In some embodiments, the method of a second aspect of the present invention further comprises b) isolating or purifying antibodies from culture media and / or antibody-expressing cells. In some embodiments, the antibodies are purified to a purity of over 90%, preferably over 95%, and most preferably over 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr B 848:79-87 (2007).

[0101] Methods for isolating or purifying antibodies are well known in the art. Isolation and purification mean that the antibody is separated from other components and provided in an essentially pure form.

[0102] Isolation and / or purification of antibodies of interest may be performed by any conventional means, such as gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high-pressure liquid chromatography (HPLC), reversed-phase HPLC, or immunoprecipitation. These methods are well known in the art and are generally described, for example, in Sambrook, J & Russell, DW

[2001] (Cold Spring Harbor Laboratory, NY). Isolation and / or purification may also involve affinity chromatography using a column immobilized with the antigen of interest.

[0103] Antibody-expressing cells may be any antibody-secreting cells obtained from peripheral blood as described above or elsewhere in this specification in the context of the methods of the first aspect of the present invention, provided that the cells specifically bind to or express and / or secrete antibodies against the antigen of interest. In certain embodiments, the antibody-expressing cells used in the methods of the second aspect of the present invention are antibody-secreting cells that specifically bind to or secrete antibodies against the antigen of interest. In certain embodiments, the cells are plasmablasts (e.g., rabbit plasmablasts) that specifically bind to or secrete antibodies against the antigen of interest. Antibody-expressing cells may be obtained from peripheral blood obtained from animals pre-immunized with the antigen of interest.

[0104] In a third aspect, the present invention relates to a method for producing an antibody, (i) Proliferating antibody-expressing cells obtained from peripheral blood according to the method of the first aspect of the present invention or any embodiment thereof disclosed herein, (ii) Determining the sequences of at least the heavy chain variable domain (VH) and light chain variable domain (VL) of the antibody expressed by the antibody-expressing cells obtained in step a), Expressing antibodies in host cells that contain heavy chain variable domains and light chain variable domains encoded by the heavy chain variable domains and light chain variable domains determined in (iii)(ii), respectively, (iv) Isolating the expressed antibody, This includes methods.

[0105] Antibody-expressing cells may be any antibody-secreting cells obtained from peripheral blood as described above or elsewhere in this specification in the context of the methods of the first aspect of the present invention, provided that the cells specifically bind to or express and / or secrete antibodies against the antigen of interest. In certain embodiments, the antibody-expressing cells used in the methods of the second aspect of the present invention are antibody-secreting cells that specifically bind to or secrete antibodies against the antigen of interest. In certain embodiments, the cells are plasmablasts (e.g., rabbit plasmablasts) that specifically bind to or secrete antibodies against the antigen of interest. Antibody-expressing cells may be obtained from peripheral blood obtained from animals pre-immunized with the antigen of interest. In some embodiments, the peripheral blood may be obtained from 4 days after immunization to 15 days after immunization or the most recent booster immunization. In one embodiment, the peripheral blood may be obtained from 4 days to a maximum of 9 days after immunization or the most recent booster immunization.

[0106] Methods for determining the sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) are: a) Extracting total RNA from antibody-expressing cells, b) Perform cDNA synthesis / reverse transcription of extracted poly(A) mRNA, c) Perform PCR using a set of species-specific primers for amplification of the VH coding sequence and the VL coding sequence, d) Sequencing the amplified sequence, It may include.

[0107] Alternatively, a method for determining the sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) is: a) Extracting total RNA from antibody-expressing cells, b) Perform cDNA synthesis / reverse transcription of extracted poly(A) mRNA, c) Cloning the amplified cDNA into a plasmid, d) Sequencing the VH and VL sequences by sequencing the cDNA cloned into the plasmid, It may include.

[0108] In various embodiments, the sequencing of VH and VL may be performed as follows: a) The antibody-expressing cells grown in this step are lysed for total RNA isolation. Then, cDNA synthesis is performed, for example, by RT-PCR (real-time polymerase chain reaction) and fixed oligo-dT primers that bind to the poly(A) tail. Subsequently, the heavy and light chains are inserted into the target plasmid (e.g., expression plasmid) by T4 polymerase and SLIC (sequence and ligation-independent cloning) or PIPE (polymerase incomplete primer elongation) cloning using real-time heavy and light chain PCR with specific primers. The final step is transformation of E. coli with the plasmid to generate biomass and isolation of pDNA (plasmid DNA), as well as control digestion and gel separation. This plasmid is also used in combination with specific primers for sequencing.

[0109] For expression in host cells, the coding sequence of an antibody, including the sequenced VH and VL sequences, can be cloned into an expression vector, which can then be transfected into host cells. Cloning can be performed as described in the context of sequencing above, except that an expression plasmid / vector is used.

[0110] Host cells suitable for antibody expression include prokaryotic cells and eukaryotic cells (e.g., HEK cells or CHO cells).

[0111] Methods and strategies for sequencing the VH and VL domains from B cells are also described by Seeber S et al. (PLoS One. 2014 Feb 4;9(2):e86184.doi:10.1371 / journal.pone.0086184.PMID:24503933) and Rashidian, J. (Methods in Molecular Biology, vol 2070. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-1-4939-9853-1 23), both of which are incorporated herein in their entirety.

[0112] Embodiments of other aspects of the present invention can be applied to the method of the third aspect with necessary modifications.

[0113] In one embodiment, the antibody may be IgG, particularly rabbit IgG. In various embodiments, the antibody may be a humanized antibody or a chimeric antibody.

[0114] In one embodiment of the present invention, the method may include culturing multiple antibody-expressing cells in different culture vessels and selecting antibody-expressing cells that secrete antibodies that specifically bind to an antigen and / or epitope of interest. In other words, the method may be part of a B-cell cloning method for identifying antibodies that specifically bind to a target of interest.

[0115] In a fourth aspect, the present invention relates to CD40 stimulants (e.g., CD40-L), in particular to non-cell surface-presented CD40 ligands (CD40-L), and in particular to soluble CD40-L.

[0116] The CD40 stimulants relate to compounds (e.g., soluble CD40-L) that possess CD40 stimulating activity.

[0117] CD40 stimulating activity refers to the ability to stimulate CD40 signaling, which promotes the culture and proliferation of antibody-expressing cells, as well as optionally, antibody secretion in the absence of feeder cells, as described herein. In various embodiments, CD40-L activity means that the ability to promote cell proliferation (in batch and / or single-cell settings) is at least as good as that of soluble CD40-L of SEQ ID NOs. 12, 13, 14, 15, 16, 17, or 18, preferably SEQ ID NO. 12, under otherwise identical experimental conditions. Assays for evaluating the ability to promote cell proliferation are described in Example 6. Briefly, antibody-expressing cells may be isolated by antigen panning, IgG-positive and 7-AAD-negative cells may be selected, and they may be deposited in culture medium as batches (i.e., 50 cells per well) as described in Example 3. Subsequently, the cells can be cultured in batches in BM1 medium in the presence of cytokines (IL-2, IL-15, IL-21, and BAFF) and the tested soluble CD40-L construct. Then, for example, after 7 days, proliferation is evaluated by determining the cell count.

[0118] Alternatively or additionally, CD40 stimulating activity can be tested by stimulating naive B cells (e.g., isolated from PBMCs via CD19-MACS from non-immunized animals) with the reagent of interest and analyzing B cell proliferation after 4-7 days of culture via CFSE dilution in a flow cytometer. For this purpose, naive B cells are labeled with CFSE after MACS-based purification (10 μM CFSE and 5 × 10⁶ cells / ml in RPMI medium without FCS) at 37°C for 5 minutes. The reaction is stopped in cold medium. After the washing step, CFSE-labeled B cells are cultured for 4-7 days in B cell medium containing various concentrations of CD40 stimulating reagent (5 × 10⁶ cells / ml in a 96-well plate). 5 (Cells / well). B cell proliferation can be tracked by analyzing CFSE labeling in live cells (7-AAD negative) in a flow cytometer. Proliferated cells show a decrease in CFSE labeling due to dilution of the label after cell division. Unstimulated naive B cells do not proliferate in culture, while CD40-stimulated naive B cells show a proliferation rate of up to 40%.

[0119] A “non-cell-presented CD40 stimulant” can be a soluble CD40 stimulant or a CD40 stimulant coated on a surface (e.g., the inner surface of a culture vessel), where the surface is not a cell surface. A “soluble CD40 ligand” is a non-membrane-immobilized CD40-L protein or fragment thereof that is soluble in cell culture medium and can bind to and stimulate CD40 in antibody-expressing cells, i.e., mimics the naturally occurring interaction between CD40-L and CD40. A “surface-coated CD40 stimulant” is a CD40 stimulant, such as a CD40 ligand, bound (covalently or non-covalently) to a surface other than a cell surface. For example, a surface-coated CD40 stimulant may be a CD40 stimulant coated (e.g., covalently or non-covalently) on the inner surface of a culture vessel used to culture antibody-expressing cells.

[0120] A CD40 stimulant (e.g., soluble CD40-L) according to a fourth aspect of the present invention is characterized by comprising a CD40-L domain. The CD40-L domain may consist of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 75%, 80%, 90%, 95%, or 99% sequence identity thereto. This fragment has been shown to be sufficient for CD40 stimulation and is conserved to more than 75% sequence identity between rabbits and humans, mice, or rats. The sequence of SEQ ID NO: 1 is a rabbit sequence. Therefore, in various embodiments, the soluble CD40-L used may comprise the sequence of SEQ ID NO: 1 or a corresponding sequence derived from another species (e.g., human, mouse, rat, hamster, or sheep). In various embodiments, CD40-L may comprise any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a sequence having at least 75% sequence identity thereto. In various embodiments, CD40-L may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 80% sequence identity with it. In various embodiments, CD40-L may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 85% sequence identity with it. In various embodiments, CD40-L may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 90% sequence identity with it. In various embodiments, CD40-L may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 95% sequence identity with it. In various embodiments, CD40-L may include one of sequence numbers 1, 2, 3, 4, 5, or 6, or a sequence having at least 99% sequence identity with it.

[0121] In some embodiments, soluble CD40-L includes, for example, the soluble 18kDa-CD40-L domain of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a sequence having the sequence identity defined above but not containing any other CD40-L specific domains or sequences. As demonstrated in the accompanying examples, the use of the 18kDa CD40-L domain promoted the proliferation of antibody-expressing cells, particularly plasmablasts, more effectively than the use of the entire CD40-L extracellular domain (see SEQ ID NO: 7 for the corresponding rabbit sequence). Therefore, in some embodiments, the soluble CD40-L used herein does not contain any other CD40-L extracellular domain sequences other than the CD40-L 18kDa domain (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or any sequence having the sequence identity defined above).

[0122] In some embodiments, the soluble CD40-L used herein may include the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a variant thereof having CD40-L activity having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0123] The CD40 stimulant according to the fourth aspect of the present invention is typically recombinantly expressed.

[0124] In various embodiments, the CD40-L domain contained in the CD40 stimulant of the fourth embodiment has a molecular weight of approximately 18 kDa or 18 kDa.

[0125] The CD40-stimulating function of the CD40-L construct is enhanced by polymerization. As demonstrated in the attached examples, the hexameric soluble CD40-L construct surprisingly promotes the culture and proliferation of antibody-expressing cells with better efficacy than the trimer soluble CD40-L construct. Therefore, in various embodiments, the CD40-L construct is at least a trimer or more than a trimer. In even more preferred embodiments, the CD40-L construct is at least a tetramer, more preferably at least a hexamer, or a hexamer.

[0126] Therefore, the CD40 stimulant of the present invention (e.g., soluble CD40-L) may contain a multimerizing domain, in particular an artificially introduced multimerizing domain (i.e., a multimerizing domain introduced by genetic engineering).

[0127] Multimerization domains resulting in different stoichiometry of multimerization are known in the art. In principle, any multimerization domain that results in at least a trimer, and more preferably at least a hexamer CD40-L construct, can be used. Further degrees of multimerization may occur by multimerization caused by the CD40-L domain itself, e.g., the 18kDa domain. The degree of multimerization of the CD40-L construct can be analyzed by HPLC (e.g., using SEC-MALS) analysis. Corresponding examples of such methods are employed and described in the appended examples. In the context of the present invention, the multimerization domain is selected so as to maintain or improve the CD40-L activity of the fusion protein, i.e., so as not to adversely affect the activity that stimulates the CD40 receptor. Those skilled in the art are well aware of the multimerization domains that can be selected. Exemplary but non-limiting multimerization domains that may be included in a CD40 stimulant (e.g., soluble CD40-L) according to a fourth aspect of the present invention are Fc domains (e.g., IgG antibodies), leucine zipper domains (LZ), and C4b domains. In various embodiments, Fc domains, particularly IgG Fc domains, particularly human IgG Fc domains, and particularly human IgG1 Fc domains, may be used as multimerization domains.

[0128] An exemplary C4b sequence that may be used in the context of the present invention is Sequence ID No. 8, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with Sequence ID No. 8, which, when used in a CD40-L construct, exhibits the same polymerization as Sequence ID No. 8.

[0129] An exemplary leucine zipper sequence that may be used in the context of the present invention is SEQ ID NO: 9, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 9, which, when used in a CD40-L construct, exhibits the same degree of multimerization (e.g., trimer) as SEQ ID NO: 9. As demonstrated in the accompanying examples, this leucine zipper achieved trimerization of the CD40-L construct.

[0130] Another exemplary leucine zipper sequence that may be used in the context of the present invention is SEQ ID NO: 10, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 10, which, when used in a CD40-L construct, exhibits the same degree of multimerization (e.g., trimer) as SEQ ID NO: 10. As demonstrated in the accompanying examples, this leucine zipper achieved trimerization of the CD40-L construct.

[0131] An exemplary leucine zipper is also described in Burkhardt et al., Cancer Immunol Immunother. 2013 Feb;62(2):347-57.doi:10.1007 / s00262-012-1331-4.Epub 2012 Aug 25.

[0132] An exemplary IgG Fc sequence that may be used in the context of the present invention is SEQ ID NO: 11, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO: 11, which, when used in a CD40-L construct, exhibits the same polymerization (e.g., hexamer) as SEQ ID NO: 11.

[0133] Using the Fc domain (for example, human IgG1), a soluble hexamer CD40-L can be obtained, as demonstrated in the attached examples. Surprisingly, such a hexamer CD40-L showed excellent culture and proliferation activity in the culture of antibody-expressing cells, more specifically antibody-secreting cells such as plasmablasts.

[0134] In some embodiments, the CD40 stimulant of the fourth embodiment (e.g., soluble CD40-L) includes a linker amino acid sequence, such as a flexible amino acid linker sequence (e.g., serine-glycine linker, SEQ ID NO: 19 or 22), between the polymerizing domain and the domain having CD40-L activity (e.g., an external domain or an 18kDa domain).

[0135] In some embodiments, the CD40 stimulant of the fourth embodiment (e.g., soluble CD40-L) may include an affinity tag such as a His tag, for example, an octa-His tag (e.g., used for purification).

[0136] In various embodiments, the CD40 stimulant of the fourth aspect (e.g., soluble CD40-L) is a recombinantly expressed construct, i.e., a construct expressed from a genetically engineered expression construct that does not exist in nature. Molecular cloning techniques for such genetic engineering are well known in the art.

[0137] In some embodiments, the CD40 stimulant of the fourth embodiment (e.g., soluble CD40-L) may comprise any one of SEQ ID NOs: 12-18, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may comprise any one of SEQ ID NOs: 12-18, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may consist of any one of SEQ ID NOs: 12-18, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may consist of any one of sequence numbers 12 to 18, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may include any one of sequence number 12, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity. In some embodiments, the soluble CD40-L used herein may include any one of sequence number 12, or a variant thereof having at least 85% sequence identity. In some embodiments, the soluble CD40-L used herein may include or consist of sequence numbers 12 to 18. In some embodiments, the soluble CD40-L includes or consists of sequence number 12.

[0138] CD40 stimulants such as CD40-L (e.g., soluble CD40-L) or its variants provided according to a fourth aspect of the present invention have CD40 stimulating activity; i.e., variants lacking CD40-L activity are excluded by this term. CD40 stimulating activity means the ability to stimulate CD40 signaling, which promotes the culture and proliferation of antibody-expressing cells, as shown herein, and optionally, antibody secretion in the absence of feeder cells. In various embodiments, CD40-L activity means that the ability to promote cell proliferation (in batch and / or single-cell settings) is at least as good as that of soluble CD40-L of SEQ ID NO: 12, 13, 14, 15, 16, 17, or 18, preferably SEQ ID NO: 12, under otherwise identical experimental conditions. Assays for evaluating the ability to promote cell proliferation are described in Example 6. In short, antibody-expressing cells can be isolated by antigen panning, IgG-positive and 7-AAD-negative cells can be selected, and they can be deposited in culture medium as batches (i.e., 50 cells per well) as described in Example 3. Subsequently, cells can be cultured in batches in BM1 medium in the presence of cytokines (IL-2, IL-15, IL-21, and BAFF) and the tested soluble CD40-L construct. Then, after, for example, 7 days, proliferation is assessed by determining the cell number. Alternatively or additionally, CD40 stimulating activity can be tested by stimulating naive B cells (e.g., isolated from PBMCs via CD19-MACS from non-immunized animals) with the reagent of interest and analyzing B cell proliferation after 4-7 days of culture via CFSE dilution in a flow cytometer. For this purpose, naive B cells are labeled with CFSE after MACS-based purification (10 μM CFSE and 5 × 10⁶ cells / ml in RPMI medium without FCS) at 37°C for 5 minutes. The reaction is stopped in cold medium. After the washing step, CFSE-labeled B cells are cultured for 4-7 days in B cell medium containing various concentrations of CD40 stimulating reagent (5 × 10⁶ cells / ml in a 96-well plate). 5(Cells / well). B cell proliferation can be tracked by analyzing CFSE labeling in live cells (7-AAD negative) in a flow cytometer. Proliferated cells show a decrease in CFSE labeling due to dilution of the label after cell division. Unstimulated naive B cells do not proliferate in culture, while CD40-stimulated naive B cells show a proliferation rate of up to 40%.

[0139] CD40 stimulants of the fourth aspect of this disclosure, particularly (soluble) CD40-L, are typically recombinantly expressed.

[0140] Exemplary methods and expression systems are provided in the accompanying examples. Expression using eukaryotic expression systems such as HEK 293T cells may be used, as described in the accompanying examples. Exemplary methods for purifying the CD 40 stimulant of the present invention are also described in the accompanying examples.

[0141] In a fifth aspect, the present invention relates to the use of a CD40 stimulant (e.g., soluble CD40-L) according to a fourth aspect of the present invention for culturing and / or growing one or more antibody-expressing cells obtained ex vivo from peripheral blood (e.g., plasmablasts) in the absence of feeder cells.

[0142] In the context of the first, second, third, and fourth embodiments, the embodiments and definitions provided herein above shall apply with necessary modifications.

[0143] In various embodiments, use includes any of the method steps defined in the context of the first embodiment and its embodiments.

[0144] In certain embodiments, the culture and / or growth of the fifth embodiment of use is carried out in the presence of IL-2 and IL-21. Optionally, the culture and / or growth may be carried out in the further presence of IL-15 and / or BAFF.

[0145] The preferred concentration ranges for these cytokines are described in the context of the method of the first embodiment and in the attached examples.

[0146] In various embodiments, the culture and / or proliferation of one or more antibody-expressing cells is carried out in a single-cell form.

[0147] In various embodiments, one or more antibody-expressing cells obtained from peripheral blood are rabbit cells.

[0148] In various embodiments, one or more antibody-expressing cells obtained from peripheral blood are antibody-secreting cells, particularly plasmablasts. Methods for obtaining such cells are described herein in the context of the first embodiment described above and in the accompanying examples.

[0149] In various embodiments, the antibody-expressing cells are primary cells.

[0150] In a sixth embodiment, the present invention relates to the use of a CD40 stimulant (e.g., non-cell surface bound CD40-L) according to a fourth embodiment or any embodiment of the present invention for B cell cloning. The use preferably involves culturing primary B cells obtained from peripheral blood in the presence of a CD40 ligand (CD40-L) according to a fourth embodiment or any embodiment of the present invention. In some embodiments, the culture is carried out in the further presence of IL-2 and IL-21. Optionally, IL-15 or (soluble) BAFF may be present in the culture. Preferably, the culture is carried out in the absence of feeder cells. Preferably, the culture is carried out in the absence of a conditioned cell supernatant, such as thymocyte supernatant (TSN).

[0151] The embodiments and definitions disclosed in any of the above-described contexts shall apply with necessary modifications.

[0152] B cell cloning technology is a method for screening monoclonal antibodies from immunized animals and includes isolating primary B cells, screening B cells expressing an antibody of interest and obtaining the antibody therefrom, or determining the sequences of the expressed VH and VL domains. The antibody containing the sequenced VH and VL domains can then be recombinantly expressed using, for example, CHO cells or HEK cells. Alternatively, in principle, instead of sequencing, hybridoma cells can also be generated from isolated B cells expressing an antibody with the desired characteristics.

[0153] In a seventh embodiment, the present invention provides a cell culture medium comprising a non-cell surface bound CD40 stimulant (e.g., soluble CD40-L) according to a fourth embodiment or any embodiment of the present invention. In some embodiments, the cell culture medium further comprises IL-2 and / or IL-21. In some embodiments, the cell culture medium comprises IL-15. In some embodiments, the cell culture medium comprises soluble BAFF. In some embodiments, the cell culture medium comprises IL-2, IL-21, and IL-15. In some embodiments, the cell culture medium comprises IL-2, IL-21, and soluble BAFF. In some embodiments, the cell culture medium comprises IL-2, IL-21, IL-15, and soluble BAFF.

[0154] In various embodiments, the cell culture medium does not contain one or more selected from ICOS, 4-1BB, PMA, and IL-4. In specific embodiments, the cell culture medium does not contain ICOS, 4-1BB, PMA, and IL-4.

[0155] Otherwise, the cell culture medium may be configured as a B cell culture medium. In certain embodiments, the cell culture medium may be based on an RPMI medium such as RPMI 1640 medium (e.g., Gibco, 31870). In some embodiments, the cell culture medium may be based on an eRDF medium (Murakami H, 1989, Advances in Biotechnological Processes, Vol. 11, Monoclonal Antibodies: Production and Application. Alan R. Liss, New York, pp. 107-141). In certain embodiments, the medium may contain one or more or all of glutamine, pyruvate, β-mercaptoethanol, and a buffer (e.g., Hepes). In some embodiments, the cell culture medium may contain, for example, 10% or less or 5% fetal bovine serum (FCS). In certain embodiments, the medium may contain one or more or all of fetal bovine serum, glutamine, and mercaptoethanol. In some embodiments, the cell culture medium may contain Staphylococcus aureus cells (SAC).

[0156] The exemplary cell culture medium components are the components of BM1 and BM2 media described in the attached examples.

[0157] As demonstrated in the attached examples, the cell culture medium of the present invention can promote in vivo survival and proliferation of primary antibody-expressing cells obtained from peripheral blood (e.g., rabbit blood) in bulk or in single-cell form, even in the absence of feeder cells and conditioned cell supernatant, such as thymocyte supernatant (TSN).

[0158] Therefore, the cell culture medium of the present invention is a medium for ex vivo culture and / or proliferation of antibody-expressing cells (e.g., primary antibody-expressing cells, e.g., obtained from rabbits), particularly plasmablasts (e.g., obtained from rabbits, e.g., primary plasmablasts).

[0159] This invention also relates to the following:

[0160] Item 1: An in vitro method for culturing one or more antibody-expressing cells obtained from peripheral blood, wherein the method is This involves culturing one or more antibody-expressing cells in the presence of IL-2, IL-21, and a non-cell surface-presenting CD40 stimulant. The culture is performed in the absence of feeder cells. In vitro method.

[0161] Item 2: The method according to Item 1, wherein one or more antibody-expressing cells are cultured in the presence of IL-15 and / or BAFF.

[0162] Item 3: The method according to Item 1 or 2, wherein the culture of one or more antibody-expressing cells is carried out in the absence of conditioned cell supernatant, particularly thymocyte supernatant (TSN).

[0163] Item 4: The method according to any one of items 1-3, wherein the culture of one or more antibody-expressing cells is carried out in the absence of one or more selected from ICOS, 4-1BB, PMA, and IL-4.

[0164] Item 5: The method described in any one of items 1-4, wherein one or more antibody-expressing cells are primary.

[0165] Item 6: The method according to any one of items 1 to 5, wherein one or more antibody-expressing cells are 100 or fewer antibody-expressing cells, preferably 75 or fewer antibody-expressing cells, more preferably 50 or fewer antibody-expressing cells, and even more preferably 20 or fewer antibody-expressing cells.

[0166] Item 7: The method according to any one of items 1 to 6, wherein one or more antibody-expressing cells are a single antibody-expressing cell, and the culture is performed in a single-cell format.

[0167] Item 8: The method according to any one of items 1-7, wherein antibody-expressing cells express and / or secrete IgG, IgM, or IgA.

[0168] Item 9: The method according to any one of items 1 to 8, further comprising isolating one or more antibody-expressing cells from the obtained peripheral blood sample.

[0169] Item 10: The method according to Item 9, comprising isolating one or more antibody-expressing cells from peripheral blood, isolating PBMCs from the obtained peripheral blood sample, and isolating one or more antibody-expressing cells from the isolated PBMCs.

[0170] Item 11: The method according to Item 9 or 10, wherein one or more antibody-expressing cells are one or more antigen-specific antibody-expressing cells, and isolating one or more antibody-expressing cells further comprises isolating cells that express one or more antibodies bound to an antigen of interest.

[0171] Item 12: The method according to Item 10 or 11, wherein the isolation of one or more antibody-expressing cells from peripheral blood comprises isolating cells expressing membrane-bound antibodies, such as membrane-bound IgG, IgA, or IgM.

[0172] Item 13: A method according to any one of items 10-12, wherein isolation of one or more antibody-expressing cells from peripheral blood is i) Isolating PBMCs from peripheral blood, ii) From PBMC, preferably using flow cytometry, a) IgG int b) Intermediate cell size (FSC int To isolate cells that have ) and iii) Optionally, isolate antigen-specific antibody-expressing cells that bind to the antigen of interest on their cell surface from the PBMCs, Methods that include...

[0173] Item 14: The method according to any one of items 1 to 13, wherein one or more antibody-expressing cells are one or more antibody-secreting cells.

[0174] Item 15: The method according to any one of items 1 to 14, wherein one or more antibody-expressing cells contain or consist of one or more plasmablasts.

[0175] Item 16: One or more antibody-expressing cells contain or consist of one or more IgG-expressing cells, and one or more IgG-expressing cells are preferably evaluated by flow cytometry to show intermediate IgG expression (IgG int ) and intermediate cell size (FSC) int The method described in any one of items 1 to 15, having )

[0176] Item 17: The method according to Item 16, wherein one or more IgG-expressing cells express antibodies against the same antigen of interest.

[0177] Item 18: The method according to any one of items 1 to 17, wherein one or more antibody-expressing cells are CD19-positive and / or CD43-positive.

[0178] Item 19: A method described in any one of items 1 to 18, wherein one or more antibody-expressing cells, i) Isolating PBMCs from peripheral blood, ii) From PBMC, preferably using flow cytometry, a) IgG int b) Intermediate cell size (FSC int To isolate cells that have ) and iii) Optionally, isolate cells from PBMCs that bind to antigens of interest on their cell surface, A method by which one can obtain or obtain something.

[0179] Item 20: The method according to any one of items 1 to 19, wherein one or more antibody-expressing cells are from rabbits, rats, mice, humans, goats, hamsters, or sheep.

[0180] Item 21: The method according to any one of items 1 to 19, wherein the antibody-expressing cells are rabbit cells.

[0181] Item 22: The method according to any one of items 1 to 21, wherein the non-cell surface-presented CD40 stimulant is a soluble CD40 stimulant, in particular a soluble CD40 ligand (CD40-L).

[0182] Item 23: The method according to any one of items 1 to 22, wherein the non-cell surface-presented CD40 stimulant contains a soluble 18kDa-CD40-L domain.

[0183] Item 24: The method according to any one of items 1 to 23, wherein the non-cell surface-presented CD40 stimulant comprises the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a variant thereof having CD40-L activity having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0184] Item 25: The method according to any one of items 1 to 21, wherein the non-cell surface-presented CD40 stimulant is a soluble CD40 ligand (CD40-L).

[0185] Item 26: The method according to Item 25, wherein soluble CD40-L is at least a trimer, in particular at least a hexamer, in particular a hexamer.

[0186] Item 27: The method described in Item 25 or 26, wherein CD40-L contains a polymerizing domain.

[0187] Item 28: The method according to Item 27, wherein the polymerizing domain is selected from the group consisting of an Fc domain, a leucine zipper, and C4b.

[0188] Item 29: The method according to Item 27, wherein the multimerizing domain is an Fc domain, preferably an Fc domain derived from human IgG, preferably human IgG1, IgG2, IgG3 or IgG4; mouse IgG1, IgG2a, IgG2b or IgG3; or an Fc domain of rabbit IgG.

[0189] Item 30: The method according to Item 27, wherein the multimerizing domain is a human IgG1 Fc domain, and in various embodiments, is an Fc domain having the amino acid sequence of SEQ ID NO: 11, or a variant thereof that is multimerizable and has at least 80% sequence identity with SEQ ID NO: 11.

[0190] Item 31: The method according to Item 27, wherein the polymerizing domain comprises an amino acid sequence selected from SEQ ID NOs: 11, 8, 9, and 10, or any one of the above variants having at least 80% sequence identity and capable of polymerizing soluble CD40-L.

[0191] Item 32: The method according to any one of items 1 to 31, wherein one or more antibody-expressing cells are rabbit cells.

[0192] Item 33: The method according to Item 32, wherein IL-2, IL-21, and optionally IL-15 and / or BAFF are each independently rabbit or human proteins, preferably recombinant expression proteins.

[0193] Item 34: The method according to Item 32 or 33, wherein the CD40 stimulant is a rabbit CD40 stimulant.

[0194] Item 35: The method described in any one of items 1 to 34, wherein IL-15 is present at concentrations of 0.1 to 30 ng / ml, particularly 1 ng / ml to 15 ng / ml.

[0195] Item 36: The method according to any one of items 1 to 35, wherein IL-21 is present at concentrations of 0.1 to 150 ng / ml, particularly 1 ng / ml to 50 ng / ml, and most particularly 5 ng / ml to 45 ng / ml.

[0196] Item 37: The method according to any one of items 1 to 36, wherein BAFF is present at concentrations of 0.1 ng / ml to 300 ng / ml, particularly 1 ng / ml to 150 ng / ml, and most particularly 50 ng / ml to 150 ng / ml.

[0197] Item 38: The method according to any one of items 1 to 37, wherein IL2 is present in concentrations of 0.1 to 20,000 IU / ml, particularly 10 to 10,000 IU / ml, particularly 20 to 1,000 IU / ml, and most particularly 400 IU / ml to 900 IU / ml.

[0198] Item 39: The method according to any one of items 1 to 38, wherein the CD40 stimulant is present at concentrations of 0.1 ng / ml to 20,000 ng / ml, particularly 1 ng / ml to 10,000 ng / ml, and most particularly 10 to 8,000 ng / ml.

[0199] Item 40: A method for producing antibodies (e.g., monoclonal antibodies) against an antigen of interest, (i) Culturing one or more antibody-expressing cells obtained from peripheral blood in accordance with the method described in any one of items 1 to 39, wherein optionally, all of the antibody-expressing cells express antibodies against the same antigen of interest. (ii) Isolating antibodies from culture medium and / or antibody-expressing cells, Methods that include...

[0200] Item 41: The method according to Item 40, further comprising immortalizing one or more antibody-expressing cells.

[0201] Item 42: A method for producing antibodies (e.g., monoclonal antibodies), (i) Proliferate antibody-expressing cells obtained from peripheral blood using the culture method described in any one of items 1 to 39, (ii) Determining the sequences of at least the heavy chain variable domain (VH) and light chain variable domain (VL) of the antibody expressed by the antibody-expressing cells obtained by proliferation in step a), Expressing antibodies in host cells that contain the heavy chain variable domain and light chain variable domain encoded by the heavy chain variable domain sequence and light chain variable domain sequence determined in (iii)(ii), respectively, (iv) Isolating the expressed antibody, Methods that include...

[0202] Item 43: The method according to any one of items 40 to 42, wherein the antibody-expressing cells are antibody-expressing cells as described in any one of items 8 to 21.

[0203] Item 44: The method described in any one of items 40-43, wherein the antibody is IgG.

[0204] Item 45: The method described in any one of items 40-44, wherein the antibody is rabbit IgG.

[0205] Item 46: The method according to any one of items 40 to 45, wherein the antibody is specifically bound to an antigen and / or epitope of interest, and the method comprises selecting antibody-expressing cells based on the fact that the antibody expresses an antibody that is specifically bound to an antigen and / or epitope of interest.

[0206] Item 47: A non-cell surface-bound CD40 stimulant comprising a CD40-L domain containing or consisting of the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a variant thereof having CD40-L activity with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

[0207] Item 48: A CD40 stimulant as described in Item 47, wherein the CD40 stimulant is at least a trimer, in particular at least a hexamer, in particular a hexamer.

[0208] Item 49: A CD40 stimulant as described in Item 47 or 48, comprising a multimerizing domain.

[0209] Item 50: A CD40 stimulant as described in Item 49, wherein the multimerization domain is selected from the group consisting of an Fc domain, a leucine zipper, and C4b.

[0210] Item 51: A CD40 stimulant according to Item 49, wherein the multimerizing domain is an Fc domain, preferably an Fc domain derived from human IgG, preferably human IgG1, IgG2, IgG3 or IgG4; mouse IgG1, IgG2a, IgG2b or IgG3; or an Fc domain of rabbit IgG.

[0211] Item 52: A CD40 stimulant according to Item 49, wherein the multimerizing domain is a human IgG1 Fc domain, and in various embodiments, an Fc domain having the amino acid sequence of SEQ ID NO: 11, or a variant thereof that is multimerizable and has at least 80% sequence identity with SEQ ID NO: 11.

[0212] Item 53: A CD40 stimulant according to Item 49, comprising any one of the above variants having an amino acid sequence selected from SEQ ID NOs: 11, 8, 9, and 10, or having at least 80% sequence identity, and capable of polymerizing soluble CD40-L.

[0213] Item 54: A CD40 stimulant according to any one of items 49 to 53, wherein the CD40 stimulant comprises a linker, e.g., a linker amino acid sequence (e.g., SEQ ID NO: 19 or 22), between the CD40-L domain and the multimerization domain.

[0214] Item 55: A CD40 stimulant as described in any one of items 47-54, wherein the CD40 stimulant is a CD40-L construct, and in particular a soluble CD40-L construct.

[0215] Item 56: Use of a non-cell surface-bound CD40 stimulant described in any one of items 47-55 for culturing and / or growing one or more antibody-expressing cells obtained ex vivo from peripheral blood in the absence of feeder cells.

[0216] Item 57: Use as described in Item 56, where culture and / or growth are carried out in the presence of IL-2 and IL-21.

[0217] Item 58: Use as described in item 56 or 57, wherein culture and / or growth are carried out in the presence of IL-15 and / or BAFF.

[0218] Item 59: Use as described in any one of items 56-58, where one or more antibody-expressing cells are used in a single-cell format.

[0219] Item 60: Use as described in any one of items 56-58, wherein one or more antibody-expressing cells are as defined in any one of items 8-21.

[0220] Item 61: Use of a non-cell surface bound CD40 stimulant as described in any one of items 47-55, preferably in the absence of feeder cells, for B cell cloning techniques.

[0221] Item 62: A cell culture medium containing a non-cell surface bound CD40 stimulant, IL-2, and IL-21, as defined in any of items 47-55.

[0222] Item 63: The cell culture medium described in Item 62, further comprising IL-15.

[0223] Item 64: Cell culture medium as described in Item 62 or 63, further comprising BAFF (soluble form).

[0224] Item 65: A cell culture medium described in any one of items 62-64, which does not contain one or more selected from ICOS, 4-1BB, PMA, and IL-4.

[0225] Item 66: A cell culture medium according to any one of items 62-65, wherein the cell culture medium is a medium for culturing antibody-expressing cells (e.g., primary antibody-expressing cells, e.g., obtained from rabbits), in particular plasmablasts (e.g., obtained from rabbits, e.g., primary plasmablasts).

[0226] The following definitions apply to all aspects, embodiments, and items of the present invention disclosed herein.

[0227] In general, terms used herein should be given their usual, customary meanings to those skilled in the art, and should not be limited to any special or customized meanings unless otherwise indicated.

[0228] As used herein, the terms “have,” “comprise,” or “include,” or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms can refer to both situations in which the entity described in this context has no further features in addition to the features introduced by these terms, and situations in which one or more further features exist. For example, the expressions “A has B,” “A comprises B,” and “A includes B” can both refer to situations in which A has no other elements other than B (i.e., A consists solely and exclusively of B), and situations in which entity A has one or more further elements other than B, such as element C, elements C and D, or further elements. Furthermore, as will be understood by those skilled in the art, in one embodiment, the expressions “comprising a” and “comprising an” mean “comprising one or more,” that is, equivalent to “comprising at least one.” Thus, unless otherwise indicated, expressions relating to one of several items refer in one embodiment to at least one such item, and in further embodiments to multiple such items; for example, identifying “cell” refers to identifying at least one cell, and in one embodiment to identifying a number of cells.

[0229] Therefore, as used herein, the terms “one or more” or “at least one” mean that one or more of the items referred to following this term may or may be used. For example, when this term indicates that one or more cells are cultured, this may be understood as culturing one or more cells, i.e., two, three, four, five, or any other number. Depending on the item to which this term refers, a person skilled in the art will understand what upper limit the term may refer to, if there is one.

[0230] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated.

[0231] Concentration, quantity, and other numerical data may be expressed or presented in the form of “ranges” as specified herein. It should be understood that such range forms are used merely for convenience and conciseness, and therefore should be interpreted flexibly to include not only the numbers explicitly listed as boundaries of the range, but also all individual numbers or subranges contained within that range, as if each number and subrange were explicitly listed. For example, the numerical range “150mg to 600mg” should be interpreted to include not only the explicitly listed values ​​of 150mg to 600mg, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 150, 160, 170, 180, 190, ... 580, 590, 600mg, and subranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600. This same principle applies to ranges listing only a single number. Furthermore, such interpretations should apply regardless of the scope or characteristics described.

[0232] When used in relation to a number, the term "approximately" means to encompass a range of numbers that have a lower limit 5% less than the given number and an upper limit 5% greater than the given number.

[0233] As used herein, the term “variant” should be understood as a polypeptide or polynucleotide that differs from the polypeptide or polynucleotide from which it is derived by one or more changes in its length or sequence. The polypeptide or polynucleotide from which a polypeptide or polynucleotide variant is derived is also known as the parent polypeptide or polynucleotide. The term “variant” includes “fragments” or “derivatives” of the parent molecule. Typically, a “fragment” is smaller in length or size than the parent molecule, while a “derivative” exhibits one or more differences in their sequence compared to the parent molecule. Modified molecules such as post-translational modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids such as methylated DNA are also included, but are not limited to, mixtures of different molecules such as RNA-DNA hybrids. Typically, a variant is constructed artificially, preferably by genetic means, while the parent protein or polynucleotide is the wild-type protein or polynucleotide, or its consensus sequence. However, naturally occurring variants should be understood to be included in the term "variant" as used herein. Furthermore, variants usable in this invention may be derived from homologs, orthologues, or paralogs of the parent molecule, or artificially constructed variants, insofar as the variant exhibits at least one biological activity of the parent molecule, i.e., is functionally active.

[0234] The term "in vitro" is used to indicate that a process or method is performed outside of an organism, preferably in a body fluid, isolated tissue, organ, or cell. In vitro methods may also be referred to as ex vivo methods herein.

[0235] As used herein, the terms “cultivating” or “culturing” refer to promoting the survival of cells in a cell culture setting. This term includes maintaining cell number, but more specifically, proliferation (i.e., an increase in cell number).

[0236] Peripheral blood mononuclear cells (PBMCs) are any peripheral blood cells that have a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes, while red blood cells and platelets do not have a nucleus, and granulocytes (neutrophils, basophils, and eosinophils) have a multilobed nucleus. Therefore, PBMCs consist essentially of lymphocytes and monocytes, i.e., lymphocytes and monocytes concentrated from previously obtained peripheral blood. As will be understood by those skilled in the art, PBMCs are substantially free of other cells, although they may contain small amounts of other cells, as the methods used to isolate PBMCs are virtually impossible to obtain 100% purity. In various embodiments, PBMCs consist of at least 80%, preferably at least 85%, more preferably 90%, still more preferably 95%, and most preferably at least 99% mononuclear peripheral blood cells (i.e., lymphocytes and monocytes).

[0237] Peripheral blood is the blood that circulates throughout the entire body. Cellular components that can be isolated from human peripheral blood include red blood cells, white blood cells, and platelets.

[0238] As used herein, the term “antibody-expressing cells” obtained from peripheral blood refers to cells obtained from peripheral blood that express at least an antibody (e.g., IgG, IgM, or IgA, particularly IgG) as a B cell receptor on their cell surface. Antibody-expressing cells may be B cells. Antibody-expressing cells may further secrete antibodies (e.g., IgG, IgM, or IgA, particularly IgG), i.e., they may be antibody-secreting cells. In particularly preferred embodiments herein, antibody-expressing cells are plasmablasts.

[0239] As used herein, the term “antibody-secreting cells” refers to cells that secrete antibodies (e.g., IgG, IgM, or IgA, particularly IgG). In certain embodiments, antibody-secreting cells still express the corresponding B cell receptors to some extent on their surface, i.e., they can be stained for each antibody.

[0240] "Plasma blasts" were analyzed by flow cytometry and intermediate staining for IgG was performed in PMC (IgG int ) and intermediate cell size (FSC) int Plasmablasts have IgG expression and can be selected accordingly. Plasmablasts are distinguished from memory B cells in that they have lower IgG expression and are larger in size (FSC). Plasmablast secreted antibodies. Plasmablasts are known to express CD19 and CD43 on their cell surface (especially in rabbits).

[0241] The cultivation of one or more cells in the presence of the substance means that the substance is present in the culture medium. Exemplary basic cell culture media that can be used are provided herein, and specific examples thereof are disclosed in the attached examples.

[0242] A "non-cell-presenting CD40 stimulant" may be a soluble CD40 stimulant or a CD40 stimulant coated on a surface (e.g., the inner surface of a culture vessel), where the surface is not a cell surface.

[0243] "Soluble CD40 stimulant" means that the CD40 stimulant is not present on the cell surface or artificial surface, and is soluble in the culture medium used.

[0244] A "surface-coated CD40 stimulant" is a CD40 stimulant, such as a CD40 ligand, bound (covalently or noncovalently) to a surface other than the cell surface. For example, a surface-coated CD40 stimulant may be a CD40 stimulant coated on the inner surface of a culture vessel used for culturing antibody-expressing cells.

[0245] As used herein, "CD40 ligand (CD40-L)" refers to a molecule possessing CD40-L activity.

[0246] As used herein, the “CD40 ligand domain” is a domain derived from full-length CD40-L, which possesses CD40-L activity. Exemplary CD40-L domains are the extracellular domain and the 18kda CD40L domain of CD40-L as described herein.

[0247] As used herein, “feeder cells” are cells that assist in the culture and proliferation of antibody-expressing cells (e.g., antibody-secreting cells, particularly plasmablasts) ex vivo. While not bound by theory, feeder cells may promote the survival and / or proliferation of antibody-expressing cells through intercellular interactions and / or the production of cytokines and potentially other factors.

[0248] As used herein, the term “sequence identity %” in relation to the amino acid sequence and / or nucleic acid sequence or nucleic acid molecule of a polypeptide / peptide represents the number of identical amino acid or nucleic acid residue matches in two or more aligned sequences compared to the number of residues that make up the full length of the sequence being compared (or the portion being compared as a whole). Using the alignment of two or more sequences or subsequences, the percentage of identical residues may be determined by comparing the (sub)sequences on a comparison window or on a designated region measured using a sequence comparison algorithm known in the art, and aligning for the greatest match, or by manually aligning and visually inspecting the sequence. Non-exclusive examples of algorithms used for determining sequence identity include, for example, those based on the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res 25 (1997), 3389-3402), the CLUSTALW computer program (Thompson, Nucleic Acids Res. 2 (1994), 4673-4680), or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85 (1988), 2444). The FASTA algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in its calculations, although this can be manually corrected to avoid overestimating the sequence identity percentage. However, CLUSTALW does consider sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul et al., Nucl Acids Res., 25(1977), 3389) are also available.

[0249] The term "antibody" as used herein is used in its broadest sense and encompasses a wide range of antibody structures, including, but is not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, and antibody fragments insofar as they exhibit the desired antigen-binding activity.

[0250] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0251] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0252] The "class" or "type" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies exist in five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0253] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and / or bind to the same epitope, with the exception of possible variant antibodies, such as those containing spontaneously occurring mutations or arising during the production of a monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of methods, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such and other exemplary methods for producing monoclonal antibodies are described herein. In some embodiments, monoclonal antibodies may be recombinantly expressed using, for example, a eukaryotic expression system.

[0254] The term "specifically binds" and its grammatical equivalent are: an antibody that binds 10 -7 M or less, in one embodiment 10 -8 M~10 -13 M, in further embodiments, 10 -9 M~10 -13 The dissociation constant (K) of M D This means that the antibody binds to its target. This term means that the antibody does not specifically bind to other biomolecules in which it is present, i.e., 10 -6 M or more, in one embodiment 10 -6 Dissociation constant (K) from M to 1M D It is further used to demonstrate that it binds to other biomolecules.

[0255] The term “host cell” refers to a cell that can or is transfected with, for example, a nucleic acid encoding a heterologous polypeptide. The term “host cell” includes both prokaryotic cells used for plasmid proliferation and eukaryotic cells used for nucleic acid expression and production of the encoded polypeptide. In one embodiment, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is a CHO cell, optionally a CHO KI cell (ATCC CCE-61 or DSM ACC 110), or a CHO DG44 cell (also known as CHO-DHFR[-], DSM ACC 126), or a CHO XE99 cell, a CHO-T cell (see, e.g., Morgan, D., et al., Biochemistry 26(1987) 2959-2963), or a CHO-S cell, or a Super-CHO cell (Pak, SCO, et al., Cytotechnol. 22(1996) 139-146). If these cells are not adapted to growth in serum-free medium or suspension, adaptation should be performed before use in this method.

[0256] The elements of the present invention are described here. While these elements are listed as embodiments having specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments and features. In particular, an embodiment disclosed in the context of one embodiment can be applied to other embodiments with the necessary modifications. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context indicates otherwise.

[0257] All references cited herein are incorporated herein by reference with respect to their entirety and to the disclosures specifically referred to herein.

[0258] array Rabbit 18kDa CD40-L domain (Uniprot:G1SKP7, amino acids 108-261) capable of stimulating sequence number 1-CD40 EINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL Human 18kDa CD40-L domain (Uniprot:P29965, amino acids 108-261) that can stimulate sequence number 2-CD40: ENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Rat 18kDa CD40-L domain (Uniprot:Q9Z2V2, amino acids 108-260) capable of stimulating sequence number 3-CD40: EKSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVVLENGRQLTVKREGLYYVYTQVTFCSNREPLSQRPFIVSLWLKPSSGSERILLRAANTHSSSKLCEQQSIHLGGVFELQAGASVFVNVTEASQVIHGIGFSSIGLLKL Mouse 18kDa CD40-L domain (Uniprot:P27548, amino acids 108-260) capable of stimulating sequence number 4-CD40: ENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL Sheep 18kDa CD40-L domain (Uniprot:Q4TVR2, amino acids 108-261) that can stimulate sequence number 5-CD40: EKNFEMHKGDQEPQIAAHVISEASSKTTSVLQWAPKGYYTLSSNLVTLENGKQLAVKRQGFYYIYTQVTFCSNREALNQAPFIASLCLKSPSGSERILLRAANTHSSSKPCGQQSIHLGGVFELQQGASVFVNVTDPSQVSHGTGFTSFGLLKL Chinese hamster 18kDa CD40-L domain (Uniprot:A0A3L7H1M2, amino acids 108-260) that can stimulate sequence number 6-CD40: IKLNTEEKKENSYEMQKGDEDPQIAVHVVSEANSKTASVLQWAKKGYYYTMKNNLVMLESGKQLTIKRQGLYYVYTQVTFCSNQGPSSKDPFLVSLCLKSTSGSERILLRAANTHSSSKPCGQQSVHLGGVFELQEDSSLFVNVTDASQVIHGIGFTSFGLLKL SEQ ID NO: 7 - Rabbit CD40-L domain extracellular domain (external domain); UniProt: G1SKP7, amino acids 47-261 HRRLDKIEDERNLHEDFVFMKTIQRCNKGEGSLSLLNCKEIRSQFEGFVK DIMLNKEEPKKEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYY TMSNTLVTLENGKQLKVKRQGFYYYIYAQVTFCSNQEPSSQAPFIASLCLK SSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQ VNHGTGFTSFGLLKL Sequence ID 8 C4b multimerizing domain; UniProt: P04003; amino acids 540-597 WETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL Sequence ID 9 - GCN4pII heptad repeat modified from the wild-type dimer GCN4 repeat found in Saccharomyces cerevisiae. GDRMKQIEDKIEEILSKIYHIENEIARIKKLIGER SEQ ID NO: 10 - Wild-type dimer GCN4 leucine-zipper (P03069, amino acids 253-274) LEDKVEELLSKNYHLENEVARL Sequence ID No. 11 - Human IgG1 Fc fragment (P01857, amino acids 104-329) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGPRPPGPX1X2 In the formula, X1 is either no amino acid or G. In the formula, X2 is either amino acid-free or K (in certain embodiments, X1 and X2 are amino acid-free, i.e., the sequence terminates at position 242). Sequence ID 12-Fc-CD40-L 18kDa DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGPRPPGPEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL Sequence number 13-LZ-CD40-L EctoD GDRMKQIEDKIEEILSKIYHIENEIARIKKLIGERTSGGSGGTGGSGGTGGSHRRLDKIEDERNLHEDFVFMKTIQRCNKGEGSLSLLNCKEIRSQFEGFVKDIMLNKEEPKKEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL LZ-CD40-L EctoD with sequence number 14-N-terminal His tag HHHHHHHHGDRMKQIEDKIEEILSKIYHIENEIARIKKLIGERTSGGSGGTGGSGGTGGSHRRLDKIEDERNLHEDFVFMKTIQRCNKGEGSLSLLNCKEIRSQFEGFVKDIMLNKEEPKKEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL SEQ ID NO: 15-LZ-CD40-L 18 kDa domain LEDKVEELLSKNYHLENEVARLGGGSGGGSGGGEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL SEQ ID NO: 16-LZ-CD40-L 18 kDa domain with N-terminal His tag HHHHHHHHLEDKVEELLSKNYHLENEVARLGGGSGGGSGGGEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNVTDASQVNHGTGFTSFGLLKL SEQ ID NO: 17-C4b-CD40-L WETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKELGGGSGGGSGGGEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNV TDASQVNHGT GFTSFGLLKL C4b-CD40-L with SEQ ID NO: 18 - N-terminal His tag HHHHHHHHWETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKELGGGSGGGSGGGEINFEMQKGDQDPQIAAHLISEASSKSSSVLQWAKKGYYTMSNTLVTLENGKQLKVKRQGFYYIYAQVTFCSNQEPSSQAPFIASLCLKSSGGSERILLRAANARSSSKTCEQQSIHLGGVFELQADASVFVNV TDASQVNHGT GFTSFGLLKL SEQ ID NO: 19 - Flexible amino acid linker 1 TSGGSGGTGGSGGTGGS SEQ ID NO: 20 - IL-2 signal peptide MRRMQLLLLIALSLALVTNS SEQ ID NO: 21 - Ig light chain signal peptide MGWSCIILFLVATATGVHS SEQ ID NO: 22 - Flexible amino acid linker 2 GGGSGGGSGGG SEQ ID NO: 23 - EPO signal peptide MGVHECPAWLWLLLSLLSLPLGLPVLGA

Brief Description of the Drawings

[0259] The following diagrams are provided to aid in understanding the present invention, and its true scope is specified in the claims. It is understood that modifications can be made to the described procedures without departing from the spirit of the invention.

[0260] [Figure 1] The SEC-MALS chromatogram received from miniDAWN TREOS II, Optilab T.rEX Detector showed a molecular weight of 89 kDa for LZ-CD40-L (=external domain of CD40-L). This correlates with the trimer structure of LZ-CD40-L EctoD (SEQ ID NO: 14). SEC: Size exclusion chromatography, MALS: Multi-angle static light scattering. [Figure 2] The SEC-MALS chromatogram received from the miniDAWN TREOS II, Optilab T.rEX Detector showed a molecular weight of 300 kDa for Fc-CD40-L. This correlates with the hexamer structure of Fc-CD40-L (SEQ ID NO: 12). SEC: Size Exclusion Chromatography, MALS: Multi-Angle Static Light Scattering [Figure 3] The SEC-MALS chromatogram received from miniDAWN TREOS II, Optilab T.rEX Detector showed the molecular weight of the 63kDa LZ-CD40-L 18kDa domain (SEQ ID NO: 16). This correlates with the trimer structure of LZ-CD40-L (18kDa). SEC: Size exclusion chromatography, MALS: Multi-angle static light scattering. [Figure 4] Average proliferation of antibody-secreting cell batches after 7 days, evaluated by cell count in culture medium compositions #1-7 within BM1. The inventors conducted five experiments for culture medium conditions #1-4 and four experiments for #5-7. BM1: Basal medium 1; TSN: Thymocyte supernatant; Fc: Human IgG1 Fc (aa104-329); BAFF: Soluble B-cell activator; Cytokines: IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), BAFF (150 ng / ml), and hFc-CD40-L (7.5 μg / ml); "-" indicates which cytokine is deficient. [Figure 5] Correlation between mean proliferation and mean IgG production levels of B cell batch sorting after 7 days using media compositions #1-7 in BM1. (N=2) BM1: Basal medium 1; TSN: Thymocyte supernatant; Fc: Human IgG1 Fc (aa104-329); BAFF: Soluble B cell activator; Cytokines: IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), BAFF (150 ng / ml), hFc-CD40-L (7.5 μg / ml). ; "-" indicates which cytokine is deficient. [Figure 6] Mean proliferation of B cell batches sorted 7 days after using media compositions #8-12 in BM2. (N=3) BM2: Basal medium 2; TSN: Thymocyte supernatant; Fc: Human IgG1 Fc (aa104-329); BAFF: Soluble B cell activator; Cytokines: IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), BAFF (150 ng / ml), and hFc-CD40-L (7.5-2.5 μg / ml). [Figure 7] Comparison of the use of CD40-L EctoD and CD40-L 18kDa fragments in cultured BM1 cells (N=3) 7 days later: Basal medium 1; TSN: Thymocyte supernatant; Fc: Human IgG1 Fc (aa104~329); LZ: Leucine zipper (note that the LZ used has a different amino acid sequence); BAFF: Soluble B cell activator; Cytokines: IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), BAFF (150 ng / ml); EctoD: External domain (aa47~261); 18kDa: (aa108~261); 7.5 μg / ml CD40-L construct. [Figure 8]The mean antigen-specific ELISA hits in single-cell sorting experiments in BM1 and BM2 cells after 7 days of culture. (N=8) BM1: basal medium 1; BM2: basal medium 2; TSN: thymocyte supernatant; Fc: human IgG1 Fc (aa104~329); BAFF: B cell activator; cytokines: IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), and soluble BAFF (150 ng / ml); 18kDa: (aa108~261); hFc-CD40-L (7.5 μg / ml). [Figure 9] A representative gating strategy for isolating plasmablasts. This flow cytometry blot is representative of a gating strategy used to sort IgGintFSCint B cells (i.e., plasmablasts) from PBMCs previously selected for antigen binding via MACS. The cells shown are gated to live lymphocytes via 7-AAD and FSC / SSC. Memory B cells show high levels of cell surface IgG and are small in size, while plasmablasts show moderate levels of cell surface IgG and are of medium size. [Modes for carrying out the invention] [Examples]

[0261] The following examples are provided to aid in understanding the present invention, and its true scope is specified in the claims. It is understood that modifications can be made to the procedures described without departing from the spirit of the invention.

[0262] Example 1: Immunization of rabbits with antigen and isolation and deposition of primary antibody-expressing cells from peripheral blood obtained from the rabbits. Animal immunization In the following examples, isolated antibody-secreting cells obtained from peripheral blood of rabbits immunized with different immunogens were used (a total of 8 to 11 different immunogens, each used to obtain antibodies against completely unrelated target antigens). The data presented in the following examples are based on average data obtained from independently analyzed rabbit blood samples, including blood samples from independent experiments, i.e., blood samples from immunization with different immunogens.

[0263] For each immunization, 12-16 week old NZW (New Zealand White) rabbits were immunized with the respective immunogen. The animals were immunized weekly during the first month, and then monthly thereafter. The first immunization was performed using complete Freund's adjuvant (CFA), and subsequent immunizations were performed using incomplete Freund's adjuvant (IFA).

[0264] Isolation of antibody-expressing cells from peripheral blood obtained from antigen-immunized rabbits, and subsequent staining for FACS sorting and deposition. To isolate antigen-specific antibody-expressing cells, peripheral blood was obtained from each rabbit immunized with the immunogen of interest.

[0265] For the isolation of antibody-expressing cells, all working reagents were cooled to 4°C. Furthermore, all incubation steps were performed at 4°C. Unless otherwise specified, centrifugation was performed at 4°C and 600g for 10 minutes (min) with terminal pauses.

[0266] First, PBMCs (i.e., lymphocytes and monocytes) were isolated from rabbit EDTA blood. The rabbit EDTA blood was diluted 1:1 with PBS (Gibco, 10010-015) and 20-30 ml of Leucosep was added. (商標) The cells were distributed into tubes (Greiner bio-one, 227288). Centrifugation was performed at 800g for 15 minutes without terminal rest. After transferring the interphase cells from one animal to the other collection tube (Falcon, 352075) containing 130 ml of PBS, the cells were centrifuged for 15 minutes. To obtain an accurate cell count, the cell pellet was resuspended in 40 ml of PBS. Cell counts were obtained using C-Chip.(商標) Using Fuchs-Rosenthal Counting Chambers (NanoEnTek, DHC-F01), 90 μl of the sample was diluted 1:10 with trypan blue (Gibco, 15250061; working concentration: 0.08%) and then performed. The remaining cells were centrifuged in a SuperClear tube (VWR, 525-0156).

[0267] In the second step, antigen-specific antibody-expressing cells were concentrated from the pelleted PBMCs. To prepare PBMCs for concentrating antibody-expressing cells, the PBMC pellet was resuspended in 1 ml of FACS (Table 1) buffer containing biotin-conjugated antigen (antigen corresponding to each immunogen used for immunizing animals) per cell, and the mixture was incubated for 25 minutes to capture antibody-expressing cells. To wash the cells, the solution was filled to 40 ml with PBS and centrifuged. The pellet was resuspended in 1,0x10 7 per cell in 90 μl of labeled buffer and 10 μl of streptavidin microbeads (Miltenyi Biotec, 130-048-101). During the 15-minute incubation, the magnetic beads could bind to the biotin-conjugated antigen, and the antibody-expressing cells bound to it. The mixture was washed by filling the solution to 40 ml with PBS and centrifuging. For the concentration step, the pellet was resuspended in 1,0x10 7 per cell in 500 μl of MACS buffer. To separate the antibody-expressing cells from the remaining cells, the sample was passed through a magnetized LS column (Miltenyi Biotec, 130-042-401) and rinsed three times with 3 ml of MACS buffer. The antibody-expressing cells were eluted into a SuperClear tube (VWR, 525-0150) by washing the column with 5 ml of MACS buffer without a magnetic field. 8 Staining, sorting, and deposition of antibody-expressing cells (especially plasmablasts)

[0268] Next, the antigen-specific antibody-expressing cells obtained by the protocol detailed above were surface IgG by FACS sorting Next, the antigen-specific antibody-expressing cells obtained by the protocol detailed above were sorted for surface IgG by FACS +The cells were then stained with anti-rabbit IgG antibody and 7-AAD to allow for the selection of viable cells.

[0269] For this purpose, antibody-expressing cells were pelleted by centrifugation, transferred to round-bottom tubes (Falcon, 352054), and stained for 30 minutes at 4°C with 500 μl of FACS buffer containing 1 μg / ml of FITC-labeled monoclonal mouse anti-rabbit IgG antibody (Southern Biotech, 4090-02). In a final 5-minute centrifugation step, the cells were washed with 2 ml of PBS and resuspended in 500 μl of FACS buffer. Five minutes before sorting, 1 μl of 7-AAD was added per 100 μl of FACS buffer.

[0270] Next, the stained cells were sorted and deposited using a Sony Biotechnology SH-800S cell sorter. Specifically, plasmablasts were sorted into IgG int FSC int Cells were selected by gating. A representative gating strategy for depositing plasmablasts is shown in Figure 9.

[0271] Seeded cultures were prepared for a "batch sorting experiment" (i.e., an experiment in which 50 or 100 cells were deposited per well). For this purpose, 2600 IgG-positive and 7-AAD-negative cells were deposited in 2.6 ml of basal medium (37°C). 50 μl of seeded culture was transferred to each concentrated medium condition (1.36 ×) containing 140 μl. In this way, an initial plasmablast cell number of 50 cells was achieved at a final medium concentration of 190 μl / well.

[0272] For the "single-cell" experiment, plasmablasts were cultured in 190 μl / well (when feeder cells were used) or 70 μl / well (in the recombinant setting without feeder cells). The total volume was placed in a plate, and the plasmablasts were directly deposited into the wells using a Sony Biotechnology SH-800S cell sorter. [Table 1]

[0273] Example 2: Expression and purification of CD40 ligand construct For some of the experiments in the following examples, different recombinant CD40 ligand (CD40-L) constructs were expressed:

[0274] The first molecule, LZ-CD40-L (SEQ ID NO: 14, also known as LZ-CD40-L extracellular domain or LZ-CD40-L EctoD), is characterized by an octahistidine tag, a trimer leucine zipper GCN4pII heptad repeat (LZ) (Naito, MH (2013). Cancer Immunol Immunother (62), pp. 347-357). This is obtained by a modification of the wild-type dimer GCN4 repeat found in Saccharomyces cerevisiae (GDRMKQIEDKIEEILSKIYHIENEIARIKKLIGER; SEQ ID NO: 9), a flexible 17-amino acid linker (TSGGSGGTGGSGGTGGS; SEQ ID NO: 19), and the entire extracellular domain of rabbit CD40-L (G1SKP7, amino acids 47-261; SEQ ID NO: 7). The LZ-CD40-L construct was expressed by transient transfection using the FreeStyle 293 Expression System, following the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). For expression, the N-terminal IL-2 signal peptide (MRRMQLLLLIALSLALVTNS; SEQ ID NO: 20), which is cleaved during secretion, was used.

[0275] LZ-CD40-L was purified by His-Tag using a Ni-NTA column (Superflow, Qiagen) and concentrated using an Amicon® 10kDa (Merck) centrifuge. The final purification step was performed using a preparative Superdex® 200 column (GE Healthcare).

[0276] Analysis was performed using SEC (size exclusion chromatography) (Thermo Fischer, Ultimate 3000 UHPLC) and MALS (multi-angle static light scattering) detectors (miniDAWN TREOS II, Optilab T.rEX; Wyatt Technologies).

[0277] After purification, LZ-CD40-L exhibited a trimer structure with a molecular weight of 89 kDa (Figure 1, marked peaks).

[0278] The second molecule, Fc-CD40-L (SEQ ID NO: 12), also known as FC-CD40-L 18kDa, features a human IgG1-Fc fragment (SEQ ID NO: 11 (X2 is not an amino acid)) linked to an 18kDa rabbit CD40-L fragment (G1SKP7, aa108~261; SEQ ID NO: 1). For expression, an N-terminal fused Ig light chain signal peptide (MGWSCIILFLVATATGVHS; SEQ ID NO: 21) cleaved before secretion was used. The Fc-CD40-L construct was expressed by transient transfection using the FreeStyle 293 Expression System according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). 18kDa CD40-L was defined by sequence comparison with human CD40-L, and it was found that the corresponding definition of the 18kDa CD40-L fragment possesses CD40-L activity (Durandy et al., Journal of Biological Chemistry, Volume 270, Issue 13, Pages 7025-7028, 1995).

[0279] Fc-CD40-L 18kDa was purified via human IgG1 Fc-Tag using MabSelect® SuRe® (GE Healthcare) and concentrated using an Amicon® 10kDa (Merck) centrifuge. The second purification step was performed using a preparative Superdex® 200 column (GE Healthcare). Analysis was performed using SEC (size exclusion chromatography) (Thermo Fischer, Ultimate 3000 UHPLC) and a MALS (multi-angle static light scattering) detector (miniDAWN TREOS II, Optilab T.rEX; Wyatt Technologies).

[0280] After purification, Fc-CD40-L exhibited a hexamer structure with a molecular weight of 300 kDa (Figure 2, mark at the first peak).

[0281] The third molecule, LZ-CD40-L(18kDa), SEQ ID NO: 16, features a flexible GGGSGGGSGGGG (SEQ ID NO: 22) amino acid linker fused to an octahistidine tag, a GCN4 leucine-zipper (LZ) motif (P03069, amino acids 253-274), and an 18kDa rabbit CD40-L fragment (G1SKP7, amino acids 108-261; SEQ ID NO: 1). The construct was expressed with an N-terminal EPO signal peptide (MGVHECPAWLWLLLSLLSLPLGLPVLGA; SEQ ID NO: 23) cleaved during secretion. LZ-CD40-L(18kDa) was expressed by transient transfection using the FreeStyle 293 Expression System according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA).

[0282] LZ-CD40-L (18kDa) was purified by His-Tag using a Ni-NTA column (Superflow, Qiagen) and concentrated using an Amicon® 10kDa (Merck) centrifuge. The final purification step was performed using a preparative Superdex® 200 column (GE Healthcare).

[0283] Analysis was performed using SEC (size exclusion chromatography) (Thermo Fischer, Ultimate 3000 UHPLC) and MALS (multi-angle static light scattering) detectors (miniDAWN TREOS II, Optilab T.rEX; Wyatt Technologies).

[0284] After purification, LZ-CD40-L (18 kDa) exhibited a trimer structure with a molecular weight of 63 kDa (Figure 3, marked peaks).

[0285] The final working concentrations of LZ-CD40-L and LZ-CD40-L(18kDa) in the cell culture medium were 7.5 μg / ml, while Fc-CD40-L was used at final working concentrations of 2.5 to 7.5 μg / ml in the cell culture medium.

[0286] Example 3: Identification of recombinant factors for replacing TSNs and feeder cells in a culture system for antibody-expressing cells In the latest technologies, single-cell culture and proliferation systems for primary antibody-expressing cells, such as plasmablasts obtained from rabbit peripheral blood, typically use feeder cell-based systems (e.g., using EL4-B5 cells) and conditioned cell culture supernatants (typically thymocyte supernatant (TSN)) to achieve high proliferation efficiency of primary antibody-expressing cells (Seeber et al, PLoS One. 2014 Feb 4;9(2):e86184.doi:10.1371 / journal.pone.0086184).

[0287] As mentioned above, there are certain limitations to using conditioned cell culture supernatant and feeder cells to proliferate antibody-expressing cells.

[0288] The aim of this study was to explore the possibility of fully recombinant culture settings for antibody-expressing cells to address the limitations of TSN and feeder cell-based culture systems, which provide at least equivalent efficiency in the proliferation of antibody-expressing cells.

[0289] The inventors hypothesized that specific secreted cytokines in TSNs, as well as ligands or receptors provided to antibody-expressing cells in intercellular contact with feeder cells, are important factors for the proliferation of antibody-expressing cells in ex vivo culture. Therefore, the ability of numerous different culture conditions using different cytokine combinations and different feeder cell substitutions was analyzed in a “batch selection” pre-screening that evaluated the proliferation of cell pools of 100 cells per well (first DoE) or 50 cells per well (second DoE) after 7 days of culture. For pre-screening, different cytokines, ligands, and receptors were evaluated in two DoE (experimental design)-based experiments: (i) a first DoE to evaluate combinations of soluble factors for substituting TSNs without substituting feeder cells, and (ii) a second DoE to evaluate different combinations of factors for substituting TSNs and feeder cells.

[0290] The "batch sorting" experiment used for DoE was performed using 100 plasmablasts from peripheral blood per well (isolated according to Example 1).

[0291] The incubation period was 7 days at 37°C and 5.0% CO2.

[0292] Two different B-cell media were used for DoE batch sorting and growth media. The first medium (referred to as BM1), used for the first DoE experiment, was based on RPMI 1640 (Gibco, 31870) medium containing 5% fetal bovine serum (FCS; Gibco 16149), 292 μg / ml glutamine (Gibco, 10378), 2 mM pyruvate (Gibco, 11360), 24 μM β-mercaptoethanol (Gibco, 31350), and 10 mM HEPES (Gibco, 15630). The second medium (BM2), used for the second DoE, was based on eRDF medium (Murakami H, 1989, Advances in Biotechnological Processes, Vol. 11, Monoclonal Antibodies: Production and Application. Alan R. Liss, New York, pp. 107-141). This contained 5% fetal bovine serum (Gibco 16149), 877 μg / ml glutamine (Sigma Aldrich, G5792), and 24 μM β-mercaptoethanol (Gibco, 31350). Before initiating the culture of antibody-expressing cells, SAC (Staphylococcus aureus cells; Merck, 507858; 1:19.500) were added to BM1 and BM2.

[0293] The key finding from batch sorting DoE experiments was determining the number of antibody-expressing cells, particularly plasmablasts, in each well. To quantify antibody-expressing cell proliferation in batch sorting, a Sony Biotechnology SH-800S cell sorter was used to determine the cell count in each well after 7 days of culture. The parameter was BSC (side scatter, also called SSC) versus FSC (forward scatter) for separating EL4-B5 cells from antibody-expressing cells. Before performing the quantification step, 50 μl of supernatant (cell-free) from each culture was transferred to a new 96-well plate for IgG quantification (N=4). The remaining 140 μl of culture was used for cell counting without dilution.

[0294] The first DoE for TSN substitution was determined in 28 different combinations and 6 experimental replicates, using hIL-2 (0-1,000 IU / ml, Roche), hIL-4 (0-50 ng / ml, PeproTech, 200-04), hIL-5 (0-20 ng / ml, PeproTech, 200-05), hIL-6 (0-50 ng / ml, in-house production), hIL-10 (0-50 ng / ml, PeproTech, 200-10), hIL-12 (0-10 ng / ml, PeproTech, 200-12H), hIL-15 (0-15 ng / ml, PeproTech, 200-15), hIL-21 (0-20 ng / ml, PeproTech, 200-21), and hBAFF (0-150 ng / ml, R&D). The tests were performed using (Systems, 7537-BF-025), hAPRIL (growth-inducing ligand (0-150 ng / ml, R&D Systems, 5860-AP-010)), and hTNFα (tumor necrosis factor alpha (0-5 ng / ml, PeproTech, 300-01A)).

[0295] Table 2 shows all terms that were significant ("Prob>|t|" < 0.05) for this first DoE model. All terms that were not significant ("Prob>|t|" > 0.05) were excluded from the model. The strongest positive effects on the proliferation of antibody-secreting cells were found with IL-2, IL-15, IL-21, and BAFF. APRIL had a small positive effect, but its t-ratio was low at 2.70. IL-4 showed a negative effect on cell proliferation. [Table 2]

[0296] Secondary DoEs for feeder cells and TSN replacement: hIL-2 (0-10,000 IU / ml, in-house production), hIL-4 (0-50 ng / ml, PeproTech, 200-04), hIL-6 (0-100 ng / ml, in-house production), hIL-10 (0-100 ng / ml, PeproTech, 200-10), hIL-13 (0-50 ng / ml, PeproTech, AF-200-13), hIL-21 (0-50 ng / ml, PeproTech, 200-21), hBAFF soluble form (0-150 ng / ml, R&D Systems, 7537-BF-025), hAPRIL (0-150 ng / ml, R&D Systems, 5860-AP-010), hTNFα (0~100ng / ml, PeproTech, 300-01A), hOX40 (0~10.000ng / ml, R&D Systems, 3388-OX-050), hICOS (inducible costimulatory factor (0~10.000ng / ml, R&D Systems, 169-CS-050)), hCD28 (0~10.000ng / ml), hSema4D (0~10.000ng / ml, R&D Systems, 7470-S4-050), hCD27 (0~10.000ng / ml, R&D Systems, 382-CD-100), h4-1BB (0~10.000ng / ml, R&D The study was conducted using 32 different combinations and 6 experimental replicates with cells from Systems (838-4B-100), h4-1BBL (0-10,000 ng / ml, PeproTech, 310-11), rabbit CD40-L (0-10,000 ng / ml, Roche, LZ-CD40-L), and PMA (phorbol-12-myristate-13-acetate (0-5 ng / ml, Sigma Aldrich, 880134P)). In this DoE, cytokines were combined with cell surface proteins to investigate whether these proteins affect cell proliferation and cytokine combinations, as well as to confirm cytokines from the first DoE(i). Fifty plasmablasts were used per culture well in this DoE.

[0297] Similar to the first DoE, all terms in the second DoE that did not have a significant effect ("Prob>|t|">0,05) were excluded (Table 3). Compared to the first DoE, APRIL had no significant effect on cell proliferation. Furthermore, ICOS, 4-1BB, and PMA showed negative effects, which were significant. Only CD40-L showed a significant positive effect on the term for feeder cell replacement. In addition, the second DoE confirmed significant positive effects of IL-2, IL-21, and BAFF on cell proliferation. [Table 3]

[0298] Combining the results from the two DoE studies revealed that IL-2, IL-15, IL-21, and BAFF are promising factors for TSN replacement, and that soluble CD40 ligand is a candidate for feeder cell replacement.

[0299] Example 4: Further evaluation of recombinant factors for replacing TSNs and feeder cells in a batch sorting culture system of antibody-expressing cells using BM1 medium. Candidate TSN substitutions (IL-2, IL-15, IL-21, and BAFF) and feeder cell substitutions (CD40-L) identified in the DoE pre-screening of Example 3 were studied in more detail by increasing the sample volume in a "batch sorting experiment," and the proliferation of a cell pool of 50 cells per well was evaluated. Briefly, antibody-expressing cells were isolated from newly isolated PBMCs by antigen panning, and IgG-positive (IgG int SSC int Selected 7-AAD-negative cells were deposited in culture medium as batches (50 cells / well) as described in Example 3 above. Subsequently, the cells were cultured in batches in BM1 medium, in the presence of different cytokine combinations, and in the presence of feeder cells or soluble CD40-L constructs, as described in Example 3 above. The following combination of factors was used: [Table 4]

[0300] To quantify cell proliferation in BM1, seeded cultures containing 2,600 antibody-expressing cells in 2.6 ml were prepared for each experiment. 50 μl of seeded culture was transferred to each culture medium composition (Table 4), and the experiment was repeated three times. Five experiments were conducted for medium conditions #1-4, and four experiments were conducted for conditions #5-7.

[0301] The readout was determined by the cell count after 7 days of culture (see Example 3 above).

[0302] Figure 4 shows that there was no cell proliferation under the blank condition (i.e., BM1 medium only). The value for the reference condition of TSN+EL4-B5(#2) was set to 100%. Compared to the reference, cell proliferation in #3 using EL4-B5+ cytokines was approximately 75% higher, demonstrating that TSN can be efficiently replaced.

[0303] Cell proliferation in condition #4 was 1.4 times lower than in the experiment using cytokines + EL4-B5 (#3). Cell proliferation in medium condition #4 was 23% higher than in the reference condition (#1). When BAFF was absent from the cytokine composition (#5), cell proliferation was approximately 1.4 times lower than when all four cytokines were used (#4) and 85% of the reference (#2). Compared to condition #5, the reduction in proliferation when BAFF and IL-15 were not present (#6) was lower. Cell proliferation in #6 was 27% lower than the reference and approximately 1.7 times lower than when all cytokines were used (#4). When only IL-2 and IL-15 were used as cytokines (#7), there was a high reduction in proliferation. Cell proliferation was only 5% of the reference (#2). This data demonstrates that high proliferation efficiency can already be achieved with IL-2 and IL-21 alone. Efficiency is further improved by BAFF and / or IL-15.

[0304] To confirm that cultured cells are still capable of antibody secretion (i.e., the increase in cell number in Figure 4 corresponds to the proliferation of antibody-secreting cells), IgG levels in the cell supernatant were evaluated. Specifically, in four experiments, the IgG concentration (12IgG values ​​for each composition) was quantified from each replica of eight (#1-7) different culture medium compositions (Figure 5).

[0305] Rabbit IgG was quantified using an anti-rabbit specific IgG ELISA. The ELISA buffer contained 1.0% BSA (Roche, 3535240) and PBS (Gibco, 10010-015). All steps were performed at room temperature for 1 hour. The washing procedure included three washes with 300 μl of washing buffer. The washing buffer contained 0.9% NaCl (Merck, 1064045000) and 0.05% Tween 20 (Sigma Aldrich, 8.22184). A 96-well streptavidin precoated plate incubated with 500 ng / ml biotin-conjugated polyclonal goat anti-rabbit IgG (Sigma-Aldrich; B8895) was used. After the washing procedure, the plate was incubated with cell culture supernatant at different dilutions. The samples were diluted with ELISA buffer. After another washing step, the plates were incubated with polyclonal goat anti-rabbit IgG-Fc at a 1:12.500 dilution. This was followed by a final washing procedure and a 10-minute incubation with ABTS (Roche, 10102946001). The detection wavelength was 405 nm and the reference wavelength was 492 nm.

[0306] The cell proliferation and IgG levels of the reference conditions using TSN+EL4-B5(#2) were set to 100%. In all culture medium settings, IgG expression levels correlated well with proliferation levels, confirming that antibody-secreting cells proliferated and remained IgG-producing. This data confirmed the proliferation of antibody-secreting cells and demonstrated that cell count is a sufficient measure for evaluating culture.

[0307] The results described above and shown in Figures 4 and 5 confirm that primary antibody-expressing cells, particularly antibody-secreting cells such as primary plasmablasts, can be efficiently proliferated ex vivo in the absence of feeder cells and TSNs. The results support the idea that cell survival and proliferation can be efficiently triggered in the absence of feeder cells and TSNs by using a defined cytokine mixture containing at least IL-2 and IL-21, and soluble CD40-L (hexameric structure) containing 18kDa fragments of extracellular domains (aa108-261) with Fc tags.

[0308] Example 5: Evaluation of recombinant factors for replacing TSNs and feeder cells in a batch sorting culture system in BM2 medium. To confirm that the conditions for antibody-expressing cell proliferation identified in Example 1 are independent of the basal medium, the inventors used a second batch sorting experiment set conducted in BM2 medium. Three experiments were performed using BM2 medium and the medium compositions summarized in Table 5. [Table 5]

[0309] Figure 6 shows, as expected, that there was no cell proliferation under the blank condition after 7 days. The value for the reference condition TSN+EL4-B5(#9) was set to 100%. Compared to the reference cell proliferation in #10 using recombinant cytokines (IL-2, IL-15, IL-21, and BAFF) + 7.5 μg / ml Fc-CD40-L, the value was approximately 6% higher and at a comparable level.

[0310] This confirms that the culture settings identified in Examples 3 and 4 can also be used in other B cell culture media that do not contain feeder cells and TSNs.

[0311] Furthermore, the effect of Fc-CD40-L concentration on B cell proliferation was tested. At a 50% lower Fc-CD40-L (#11) concentration (5.0 μg / ml), cell proliferation was only 5% lower than reference #9 and approximately 1.1 times lower than #10 at 7.5 μg / ml. Further reducing Fc-CD40-L by 50% to 2.5 μg / ml resulted in a 27% decrease in B cell proliferation compared to reference #9. Compared to #10 at 7.5 μg / ml, cell proliferation at 2.5 μg / ml was approximately 1.4 times lower. Despite this small reduction, the results demonstrate that CD40-L can be used over a wide concentration range and that those skilled in the art can easily adapt the concentration as needed.

[0312] Example 6: Comparison of different soluble CD40-L constructs To confirm that the results of the above examples can be repeated using different types of CD40-L multimerized constructs and different CD40-L protein fragments, three independent experiments were performed using three different CD40-L constructs (see Figure 7). This was done in a "batch sorting experiment" where the growth of a 50-cell / well cell pool was evaluated after 7 days of culture. Briefly, antigen-specific plasmablasts were isolated by antigen panning, and IgG-positive cells (IgG int Selected 7-AAD-negative cells were deposited in batches (i.e., 50 cells per well) on culture medium as described in Example 3 above. Subsequently, the cells were cultured in batches in BM1 medium, in the presence of cytokines (IL-2, IL-15, IL-21, and BAFF) or TSN, and in the presence of feeder cells or soluble CD40-L constructs, as described in Example 3 above.

[0313] The first construct, consisting of hIgG1-Fc (aa104~329) linked to a rabbit CD40-L fragment (aa108~261), achieved the best proliferation of all three constructs (see Figure 7). It showed 43% more B cell proliferation than the reference (EL4-B5+TSN) and 32% higher B cell proliferation than the second-best construct, LZ-CD40-L (18kDa). The LZ-CD40-L EctoD combination, consisting of a trimer leucine zipper linked to the rabbit CD40-L external domain, achieved the lowest proliferation at 68% of the reference.

[0314] These results confirm that evaluated combinations of cytokines with different CD40-L fragments and different multimerization domains can replace standard procedures using EL4-B5 cells and TSNs. However, it is also demonstrated that the hexameric construct with hIgG1-Fc as the multimerization domain (i.e., resulting in a hexameric construct (see above)) and the 18kDa CD40-L fragment were the best among all soluble CD40-L constructs tested.

[0315] Example 7: Antibody-expressing cell proliferation after single-cell deposition To demonstrate that the conditions identified in batch sorting experiments (see Examples 3-5) can be successfully applied at the single-cell level, single-cell deposition experiments were conducted. A total of eight experiments were performed, and the results are shown as averages.

[0316] Specifically, antibody-expressing cells (especially plasmablasts) were isolated, and IgG-positive (int) and 7-AAD-negative cells were selected (as described in Example 1) and deposited as single cells. For recombination setup, the single antibody-expressing cells were deposited in 190 μl (EL4-B5 + TSN BM1, 96-well plate) medium / well and 70 μl (cytokines + Fc-CD40-L BM2, 384-well plate) / well using a Sony Biotechnology SH-800S cell sorter. The entire volume was placed in plates, and the single antibody-expressing cells were deposited in the wells.

[0317] Single-cell cultures were performed for 7 days in two settings. A reference setting (EL4-B5+TSN BM1) contained 1.0 ml of TSN and 105.263 cells / ml of 50 Gy-irradiated EL4-B5 cells in BM1 medium, while a recombinant setting (cytokines+Fc-CD40-L in BM2 medium) contained IL-2 (773 IU / ml), IL-15 (15 ng / ml), IL-21 (20 ng / ml), BAFF (150 ng / ml), and Fc-CD40-L (7.5 μg / ml). Both settings contained SAC1:19.500.

[0318] For each experiment, antibody-expressing cells of the reference setting were cultured in four 96-well plates (Thermo Fisher Scientific, 161093), and antibody-expressing cells of the recombinant setting were cultured in one 384-well plate (Corning, 3701). The culture period was 7 days at 37°C and 5.0% CO2. The use of 384-well plates was possible because no feeder cells were present. The advantage of the novel culture method described herein is that smaller wells and culture volumes can be used.

[0319] Antigen-specific ELISA was performed on each well to determine whether anti-antigen IgG-producing cells proliferated. This is considered an indirect measure of cell proliferation.

[0320] The ELISA buffer contained 1.0% BSA (Roche, 3535240001) and PBS (Gibco, 10010-015). All steps were incubated at room temperature for 1 hour. The washing procedure consisted of three washes with 300 μl of washing buffer. The washing buffer contained 0.9% NaCl (Merck, 1064045000) and 0.05% Tween 20 (Sigma Aldrich, 8.22184). A 96-well (Roche, 11989685001) or 384-well (Roche, 11974998) streptavidin pre-coated plate incubated with 200 ng / ml biotin-labeled specific antigen was used. The antigen was the same as that used in the panning step for antigen-specific B cell isolation. After the washing procedure, the plate was incubated with undiluted culture supernatant. After a separate washing procedure, the plates were incubated with peroxidase (POD)-labeled polyclonal goat anti-rabbit IgG-Fc at a 1:12.500 dilution. Dilution was then performed with ELISA buffer. Subsequently, a final washing procedure and a 10-minute incubation with ABTS (Roche, 10102946001) were followed. The detection wavelength was 405 nm and the reference wavelength was 492 nm.

[0321] On average across eight experiments using 384 wells per experiment, the reference setting with EL4-B5+TSN BM 1 showed 57 antigen-specific hits (Figure 8). Four selected cytokines (IL-2, IL-15, IL-21, and BAFF) + Fc-CD40-L revealed 54 hits (i.e., wells with cells producing antigen-specific antibodies).

[0322] This finding demonstrates that by using cytokines and CD40-L-Fc protein, it was possible to establish single-cell culture conditions for antibody-expressing cells without the need for feeder cells or conditioned cell supernatant.

Claims

1. An in vitro method for culturing one or more antibody-expressing cells obtained from peripheral blood, wherein the method is The process involves culturing one or more antibody-expressing cells in the presence of IL-2, IL-21, and a non-cell surface-presenting CD40 stimulant. The culture is performed in the absence of feeder cells. In vitro method.

2. The method according to claim 1, wherein the culture of the one or more antibody-expressing cells is carried out in the presence of IL-15 and / or BAFF.

3. The method according to claim 1 or 2, wherein the culture of the one or more antibody-expressing cells is carried out in the absence of conditioned cell supernatant, particularly thymocyte supernatant (TSN).

4. The method according to any one of claims 1 to 3, wherein the one or more antibody-expressing cells is a single antibody-expressing cell, and the culture is performed in a single-cell format.

5. The method according to any one of claims 1 to 4, wherein the one or more antibody-expressing cells are one or more antibody-secreting cells, particularly one or more plasmablasts, and / or the one or more antibody-expressing cells are primary cells.

6. A method according to any one of claims 1 to 5, wherein one or more antibody-expressing cells i) Isolating PBMCs from peripheral blood, ii) From the PBMC, preferably using flow cytometry, a) IgG ｉｎｔ b) Intermediate cell size (FSC) ｉｎｔ To isolate cells that have ) It can be obtained by a method including, or obtained by, iii) Optionally, the antibody-expressing cells express antibodies that specifically bind to the antigen of interest, i.e., antigen-specific antibody-expressing cells, and the method for obtaining these cells or obtaining these cells further includes isolating cells that bind to the antigen of interest from the PBMC. method.

7. The method according to any one of claims 1 to 6, wherein the antibody-expressing cells are rabbit cells.

8. The method according to any one of claims 1 to 7, wherein the non-cell surface-presented CD40 stimulant is a soluble CD40 stimulant, particularly a soluble CD40 ligand (CD40-L), particularly soluble CD40-L, and in particular the non-cell surface-presented CD40 stimulant comprises a CD40-L domain consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a variant thereof having CD40-L activity having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity therewith.

9. The method according to claims 1 to 8, wherein the non-cell surface-presented CD40 stimulant is at least a trimer, more particularly at least a hexamer, more particularly a hexamer.

10. The method according to claims 1 to 9, wherein the non-cell surface-presented CD40 stimulant comprises a multimerizing domain, and in the embodiment, the multimerizing domain is selected from the group consisting of an Fc domain, a leucine zipper, and C4b.

11. A method for producing antibodies against an antigen of interest, (i) Culturing one or more antibody-expressing cells obtained from peripheral blood according to the method of any one of claims 1 to 10, wherein all of the one or more antibody-expressing cells express antibodies against the same antigen of interest, (ii) Isolating the antibody from the culture medium and / or the antibody-expressing cells, Methods that include...

12. A method for producing antibodies, (i) Proliferating antibody-expressing cells obtained from peripheral blood using the culture method described in any one of claims 1 to 10, (ii) Determining the sequences of at least the heavy chain variable domain (VH) and light chain variable domain (VL) of the antibody expressed by the antibody-expressing cells obtained by the proliferation in step a), Expressing in host cells an antibody containing the heavy chain variable domain and the light chain variable domain encoded by the heavy chain variable domain sequence and the light chain variable domain sequence determined in (iii) and (iii), respectively, (iv) Isolating the expressed antibody, Methods that include...

13. (i) a CD40-L domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or a variant thereof having CD40-L activity with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto, and (ii) optionally a multimerization domain, comprising a non-cell surface bound CD40 ligand (CD40-L).

14. (i) Use of a non-cell surface bound CD40 ligand (CD40-L) according to claim 13 for culturing and / or growing one or more antibody-expressing cells obtained ex vivo from peripheral blood in the absence of feeder cells, or (ii) for a B cell cloning method.

15. A cell culture medium comprising the non-cell surface bound CD40 ligand (CD40-L) according to claim 13, IL-2, IL-21, and optionally IL-15 and / or BAFF.

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