Isolation of monoclonal antibodies

Abstract

The present invention relates to the production and isolation of monoclonal antibodies, and particularly, but not exclusively, to novel feeder cell lines for culturing monoclonal antibody-producing B cells. The invention also extends to the use of said feeder cell lines in culturing monoclonal antibody-producing B cells and isolating said B cells from cell culture medium. The invention further extends to methods of culturing and isolating monoclonal antibody-producing B cells, and methods of isolating monoclonal antibodies.

Description

[Technical Field]

[0001] The present invention relates to the production and isolation of monoclonal antibodies, and particularly, but not exclusively, to novel feeder cell lines for culturing monoclonal antibody-producing B cells. The invention also extends to the use of said feeder cell lines in culturing monoclonal antibody-producing B cells and isolating said B cells from cell culture medium. The invention further extends to methods of culturing and isolating monoclonal antibody-producing B cells and methods of isolating monoclonal antibodies. [Background technology]

[0002] Epstein-Barr virus (EBV) is a B-lymphotropic gamma (γ) herpesvirus that asymptomatically and persistently infects over 90% of humans. In vitro, EBV induces continuous proliferation (transformation and "immortalization") of human B cells to generate lymphoblastoid cell lines (LCLs). EBV-infected LCLs, derived from antigen-specific memory B cells, produce antibodies against these antigens. This characteristic of EBV has previously been exploited to isolate human monoclonal antibodies (MABs) (1).

[0003] However, the current approach of using EBV to generate MABs has several drawbacks. First, the efficiency of B cell transformation by EBV is relatively low. This has been addressed to some extent by adding the 24-base-long oligodeoxynucleotide CpG ODN2006 to the culture medium of EBV-infected cells. CpG ODN2006 acts as a TLR9 agonist, increasing transformation efficiency from approximately 2% to approximately 30% (2). Second, difficulties exist in expanding monoclonal cultures to produce MABs. Isolated single LCL cells do not proliferate, which means that cultures are initially expanded oligoclonally, with several cells of different specificities being added to the same culture well. After two weeks of growth, culture supernatants are evaluated for antibody production, and positive cultures are subcloned again by limiting dilution. After several weeks, the new subcloned cultures are further evaluated to test for clonality, antibody production, and specificity (3). Additionally, current approaches suffer from low throughput for expanding cultures and subcultures to assess proliferation or clonality.

[0004] Given the challenges associated with the classical EBV method, an alternative method called single-cell molecular cloning is currently the preferred option for isolating MABs (4). This method involves single-cell sorting of antigen-specific B cells by FACS followed by molecular cloning from the single cells, which determines the antibody sequence of each cell. The sequence information is used to construct plasmid expression vectors for antibody production in separate cells specialized for protein (antibody) expression. After antibody production from these specialized cells, antibodies can be evaluated for specificity, affinity, and neutralization. This is a relatively high-throughput method that reduces the time required to obtain MABs by several weeks compared to the classical EBV method, and the overall efficiency is nearly doubled (5).

[0005] However, there are several drawbacks associated with single-cell molecular cloning and antibody production from specialized mammalian cells. First, obtaining sequence information, performing molecular cloning, and expressing antibodies in separate cells is significantly more expensive than culturing EBV-infected, antibody-producing LCLs. Second, obtaining antibody sequence information from a single cell is difficult, compromising efficiency. Finally, testing antibodies for specificity, affinity, and neutralization is not possible prior to expression from a plasmid vector in specialized cell lines. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Steinitz, M., Klein, G., Koskimies, S. and Makel, O. (1977) Nature, 269, 420-422. [Non-patent document 2] Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, MR, Murphy, BR, Rappuoli, R. and Lanzavecchia, A. (2004) Nat Med, 10, 871-875. [Non-patent document 3] Steinitz, M. (2014) Methods Mol Biol, 1060, 111-122 [Non-patent document 4] Tiller, T., Meffre, E., Yurasov, S., Tsuiji, M., Nussenzweig, MC and Wardemann, H. (2008) J Immunol Methods, 329, 112-124 [Non-patent document 5] Ehlers, AM, den Hartog Jager, CF, Kardol-Hoefnagel, T., Katsburg, MMD, Knulst, AC and Otten, HG (2021) Front Immunol, 12, 660037 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need to provide an improved platform for isolating human monoclonal antibodies that has high efficiency and high throughput capabilities. [Means for solving the problem]

[0008] As described in the Examples, we have developed a new platform for isolating human monoclonal antibodies from human peripheral blood mononuclear cells (PBMCs). Specifically, we developed a new feeder cell line called Amalthea and surprisingly demonstrated that this feeder cell line can be used to support the efficient outgrowth of antibody-producing B cells infected with recombinant EBV. We demonstrated that Amalthea feeders can support monoclonal cell culture of antibody-producing cells, and the MABs produced in the culture medium can be directly used in multiple assays, significantly saving time and reducing costs. Additionally, this platform is compatible with fluorescence-activated cell sorting (FACS), which is used to select B cells specific to an antigen of interest, significantly increasing throughput.

[0009] Therefore, according to a first aspect of the present invention there is provided a feeder cell line for culturing monoclonal antibody-producing B cells, comprising: Mega CD40 Ligand (mega CD40L) or a variant or fragment thereof, CD23, or a variant or fragment thereof, and / or Fluorescent proteins not expressed by B cells A feeder cell line expressing the

[0010] According to a second aspect of the present invention there is provided the use of a feeder cell line in culturing monoclonal antibody-producing B cells, comprising: Mega CD40 Ligand (Mega CD40L), or variants or fragments thereof, and / or CD23, or its variants or fragments A feeder cell line expressing the

[0011] According to a third aspect of the present invention there is provided the use of a feeder cell line in isolating monoclonal antibody-producing B cells, wherein the feeder cell line expresses a fluorescent protein that is not expressed by B cells.

[0012] According to a fourth aspect of the present invention, there is provided the use of a feeder cell line in isolating monoclonal antibody-producing B cells, wherein the feeder cell line does not express a drug selection marker expressed by B cells.

[0013] According to a fifth aspect of the present invention, there is provided a method for culturing monoclonal antibody-producing B cells, comprising the steps of: (i) B cells, Mega CD40 Ligand (Mega CD40L), or variants or fragments thereof, and / or CD23, or its variants or fragments contacting the cell with a feeder cell line expressing (ii) culturing the B cells and feeder cell line under conditions to support the proliferation of monoclonal antibody-producing B cells; A method is provided that includes:

[0014] According to a sixth aspect of the present invention there is provided a method for isolating monoclonal antibody-producing B cells from a cell culture medium, comprising the steps of: (i) contacting B cells with a feeder cell line that expresses a fluorescent protein that is not expressed by B cells; (ii) identifying a feeder cell line that expresses a fluorescent protein to enable the isolation of monoclonal antibody-producing B cells; A method is provided that includes:

[0015] According to a seventh aspect of the present invention there is provided a method for isolating monoclonal antibody-producing B cells from a cell culture medium, comprising the steps of: (i) contacting B cells with a feeder cell line, wherein the feeder cell line does not express a drug selection marker expressed by B cells; (ii) culturing the B cells and feeder cell line in the presence of a drug selection marker to allow for the isolation of monoclonal antibody-producing B cells; A method is provided that includes:

[0016] Preferably, the methods of the fifth, sixth and seventh aspects include the step of isolating the monoclonal antibody from the monoclonal antibody-producing B cells.

[0017] Thus, according to an eighth aspect of the present invention there is provided a method for isolating a monoclonal antibody from monoclonal antibody-producing B cells, comprising the steps of: (i) B cells, Mega CD40 Ligand (Mega CD40L), or a variant or fragment thereof, CD23, or a variant or fragment thereof, and / or Fluorescent proteins not expressed by B cells contacting the cell with a feeder cell line expressing (ii) culturing the B cells and feeder cell line under conditions to support the proliferation of monoclonal antibody-producing B cells; (iii) isolating monoclonal antibodies from the monoclonal antibody-producing B cells; A method is provided that includes:

[0018] As described in Examples 2 and 3, we demonstrated that modifying feeder cell lines to express mega CD40L and / or CD23 resulted in feeder cell lines capable of significantly increasing the number of monoclonal antibody-producing B cells grown. For example, we demonstrated that these feeder cell lines increased the efficiency of viral-mediated transformation / immortalization of B cells by more than fourfold. In addition, using our new feeder cell lines, it is now possible for the first time to support monoclonal growth of virus-infected cells without subcloning steps and serial dilutions, saving weeks and significantly increasing efficiency compared to the classical EBV method for monoclonal antibody isolation. Furthermore, the resulting monoclonal culture supernatants contain antibodies that can be evaluated without the need for molecular cloning of antibody sequences, as required by the currently most preferred method. This reduces the cost per evaluated antibody by 50- to 100-fold compared to the currently preferred single-cell molecular cloning method.

[0019] Furthermore, by modifying feeder cell lines to express a fluorescent protein that is not expressed by antibody-producing B cells, as described in Example 4, the present inventors demonstrated that it is possible to easily distinguish feeder cells from B cells without any additional staining steps that would result in the loss of the cells of interest.

[0020] In one embodiment, the feeder cell line expresses (i) megaCD40L, or a variant or fragment thereof, and (ii) CD23, or a variant or fragment thereof. Optionally, the feeder cell line also expresses (iii) a fluorescent protein that is not expressed by B cells.

[0021] In another embodiment, the feeder cell line expresses (i) megaCD40L, or a variant or fragment thereof, and (ii) a fluorescent protein that is not expressed by B cells. Optionally, the feeder cell line also expresses CD23, or a variant or fragment thereof.

[0022] In another embodiment, the feeder cell line expresses (i) CD23, or a variant or fragment thereof, and (ii) a fluorescent protein that is not expressed by B cells. Optionally, the feeder cell line also expresses mega CD40L, or a variant or fragment thereof.

[0023] Alternatively, in another embodiment, the feeder cell line expresses (i) mega CD40L, or a variant or fragment thereof, (ii) CD23, or a variant or fragment thereof, and (iii) a fluorescent protein that is not expressed by B cells.

[0024] As used herein, the term "feeder cell line" can refer to a line of cells used in culture of target cells (i.e., B cells) to support the survival and / or growth of those target cells, for example, by producing various growth factors. In addition, the term "feeder cell line" can encompass cells engineered to express specific growth factors or proteins, such as megaCD40L and / or CD23.

[0025] The feeder cell line may be any cell line capable of supporting the growth of monoclonal antibody-producing B cells. For example, the feeder cell line may be selected from the group consisting of an osteosarcoma cell line, a mesenchymal cell line, an epithelial cell line, a lymphoblastoid cell line, a neuronal cell line, and / or an endothelial cell line. Most preferably, the feeder cell line is an osteosarcoma cell line.

[0026] In one embodiment, the feeder cell line may be selected from the group consisting of U2OS, MRC5, lymphoblastoid cell line (LCL), H1299, MCF7, HEK293, 3T3, Caco-2, and / or HeLa. U2OS is a cell line with epithelial morphology derived from bone tissue of an osteosarcoma patient. MRC5 is a diploid cell line composed of human fibroblasts derived from lung tissue. H1299 is a human non-small cell lung carcinoma cell line derived from lymph nodes. MCF7 is an epithelial cell line derived from human breast cancer cells. HEK293 is a cell line with epithelial morphology derived from human embryonic kidney. 3T3 is a fibroblast cell line isolated from a mouse embryo. Caco-2 is an epithelial cell line derived from colon cancer. HeLA is an immortal cell line originally isolated from cervical cancer. Most preferably, the feeder cell line is U2OS. Advantageously, this cell line is adherent and forms a feeder monolayer for LCL cells (ie, monoclonal antibody-producing B cells).

[0027] In one embodiment, the feeder cell line is irradiated. Preferably, the feeder cell line is irradiated before contacting B cells with the feeder cell line. Advantageously, irradiation stops the growth of the feeder cells and prevents them from taking over the culture.

[0028] In one embodiment, the feeder cell line is 137 Preferably, the feeder cell line is irradiated with a radiation dose of at least 5 Gy, at least 10 Gy, at least 15 Gy, at least 20 Gy, or at least 25 Gy. 137 More preferably, the feeder cell line is irradiated using a Cs gamma irradiator with a radiation dose of at least 30 Gy. 137 It is irradiated using a Cs gamma ray irradiator.

[0029] In one embodiment, the feeder cell line is irradiated using an X-ray irradiator. Alternatively, in another embodiment, the feeder cell line is not irradiated. Alternatively, the feeder cell line may be transiently treated with a cell cycle inhibitor. In one embodiment, the cell cycle inhibitor is mitomycin C. Alternatively, in another embodiment, the feeder cell line is not irradiated and is not treated with a cell cycle inhibitor.

[0030] CD40 is a TNF superfamily type II transmembrane protein. CD40 ligand (CD40L) is the physiological ligand that binds to CD40 on the surface of B cells and provides activation and survival signals for LCL growth (6, 7). Mega-CD40L consists of two trimers of CD40L, and advantageously, mega-CD40L is more stable and mitogenic than physiological CD40L (7).

[0031] One embodiment of the polypeptide sequence of mega CD40L is represented herein as SEQ ID NO: 1, as follows: [ka]

[0032] Preferably, therefore, mega CD40L comprises an amino acid sequence substantially as shown in SEQ ID NO: 1, or a fragment or variant thereof.

[0033] In one embodiment, the nucleotide sequence encoding mega CD40L is represented herein as SEQ ID NO:2, as follows: [ka]

[0034] Preferably, therefore, mega CD40L is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 2, or a fragment or variant thereof.

[0035] Mega-CD40L is commercially available as a soluble protein added to culture medium and has previously been used to enhance LCL growth (8, 9). However, the feeder cell lines of the present invention express mega-CD40L directly in the culture medium, which advantageously creates a microenvironment with unusually high concentrations of the more active recombinant mega-CD40L to quiesce growing cells. For example, the inventors surprisingly discovered that the feeder cell lines of the present invention can achieve 4- to 7-fold higher concentrations of mega-CD40L in the culture medium microenvironment compared to those typically added as a soluble protein (8, 9) (see Figure 2). Furthermore, the inventors believe that the concentration of mega-CD40L is even higher in the microenvironment close to the cell surface of secretory feeders, where growing LCL cells (i.e., monoclonal antibody-producing B cells) become quiescent.

[0036] As described herein, monoclonal antibody-producing B cells are cultured with a feeder cell line in a cell culture medium. Thus, mega-CD40L expressed by the feeder cell line is directly expressed in the cell culture medium, resulting in a specific concentration of mega-CD40L in the cell culture medium. Advantageously, the feeder cell line according to the present invention expresses high concentrations of mega-CD40L, or variants or fragments thereof, which support the expansion of monoclonal antibody-producing B cells.

[0037] Thus, in preferred embodiments, the feeder cell line expresses at least 1 ng / ml, at least 2 ng / ml, at least 3 ng / ml, at least 4 ng / ml, or at least 5 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium. More preferably, the feeder cell line expresses at least 10 ng / ml, at least 20 ng / ml, at least 30 ng / ml, at least 40 ng / ml, or at least 50 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium. Even more preferably, the feeder cell line expresses at least 51 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium.

[0038] In another preferred embodiment, the feeder cell line expresses at least 60 ng / ml, at least 70 ng / ml, at least 80 ng / ml, at least 90 ng / ml, or at least 100 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium. More preferably, the feeder cell line expresses at least 101 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium.

[0039] In another preferred embodiment, the feeder cell line expresses at least 110 ng / ml, at least 120 ng / ml, at least 130 ng / ml, at least 140 ng / ml, at least 150 ng / ml, at least 160 ng / ml, at least 170 ng / ml, at least 180 ng / ml, at least 190 ng / ml, or at least 200 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium. Even more preferably, the feeder cell line expresses at least 220 ng / ml, at least 240 ng / ml, at least 260 ng / ml, at least 280 ng / ml, or at least 300 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium. Even more preferably, the feeder cell line expresses at least 320 ng / ml, at least 340 ng / ml, at least 360 ng / ml, at least 380 ng / ml, or at least 400 ng / ml of mega CD40L, or a variant or fragment thereof, in the cell culture medium.

[0040] CD23 is a low-affinity IgE receptor present on the B cell membrane and upregulated by IL-4. CD23 is a 45 kDa type II membrane glycoprotein isoform, i.e., there are two isoforms, CD23a and CD23b.

[0041] Thus, in one embodiment, the CD23 or fragment or variant thereof expressed by the feeder cell line is CD23a or CD23b.

[0042] CD23a and CD23b act as LCL growth factors, and both isoforms have the same effect because the same domain is presented on the surface of feeder cells (10, 11). CD23b is specifically expressed by LCLs, and only LCL cells that express CD23 are transformed. LCL transformation is enhanced at an early stage by proximity to other CD23-expressing LCLs (12, 13). However, previous methods have focused on CD23 soluble fragments shed from B cells (10) or on the intact protein, which is extracted by cell lysis and then used as an additive in cell culture (11).

[0043] In contrast, the feeder cell lines of the present invention were engineered to express full-length CD23b to enhance single-cell LCL cloning in the absence of other LCL cells. Advantageously, this facilitates efficient monoclonal growth after single-cell index sorting for the establishment of monoclonal LCL cultures. The inventors surprisingly found that CD23b-expressing feeder cell lines increased LCL growth (i.e., monoclonal antibody-producing B cells) by approximately 20%.

[0044] Therefore, most preferably, the feeder cell line expresses CD23b.

[0045] One embodiment of the polypeptide sequence of CD23b is represented herein as SEQ ID NO:3, as follows: [ka]

[0046] Preferably, therefore, CD23b comprises an amino acid sequence substantially as shown in SEQ ID NO: 3, or a variant or fragment thereof.

[0047] In one embodiment, the nucleotide sequence encoding CD23b is represented herein as SEQ ID NO:4, as follows: [ka]

[0048] Preferably, therefore, CD23b is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 4, or a fragment or variant thereof.

[0049] The feeder cell line may express CD23a.

[0050] One embodiment of the polypeptide sequence of CD23a is represented herein as SEQ ID NO: 5, as follows: [ka]

[0051] Preferably, therefore, CD23a comprises an amino acid sequence substantially as shown in SEQ ID NO: 5, or a variant or fragment thereof.

[0052] In one embodiment, the codon-optimized nucleotide sequence encoding CD23a is represented herein as SEQ ID NO:6, as follows: [ka]

[0053] Preferably, therefore, CD23a is encoded by a nucleotide sequence substantially as shown in SEQ ID NO: 6, or a fragment or variant thereof.

[0054] The present inventors have demonstrated that the expression of a fluorescent protein in a feeder cell line makes it easy to distinguish the feeder cells from LCLs (i.e., monoclonal antibody-producing B cells). Therefore, unlike prior art methods, by using the feeder cell line of the present invention, it is not necessary to stain new LCLs prior to FACs for single B cell cloning, even if non-recombinant EBV that does not express GFP is used. This is a significant advantage, considering that an additional staining step would result in the loss of the target B cells.

[0055] Thus, in one embodiment, the feeder cell line expresses a fluorescent protein that is not expressed by B cells.

[0056] In some embodiments, monoclonal antibody-producing B cells are cultured with Epstein-Barr virus (EBV).

[0057] The EBV may be selected from any variety capable of inducing continuous proliferation (transformation and "immortalization") of B cells. Preferably, therefore, the B cells are EBV-infected monoclonal antibody-producing B cells. In some embodiments, recombinant EBV is used. Most preferably, therefore, the B cells are recombinant EBV-infected monoclonal antibody-producing B cells.

[0058] In one embodiment, EBV expresses a drug selection marker. Preferably, EBV expresses the hygromycin resistance gene. This allows for hygromycin antibiotic selection of EBV-infected B cells (14). Advantageously, because EBV-infected B cells express a drug selection marker (e.g., the hygromycin resistance gene), it is possible to kill the feeder cells without affecting the EBV-infected antibody-producing B cells. This method would otherwise require the use of a drug selection marker to stop feeder cell proliferation. 137Cs γ-ray or X-ray irradiation must be used, which requires facilities that are not always available. Alternatively, feeder cells may have to be transiently treated with cell cycle inhibitors such as mitomycin C, which require extensive washing and residual contaminants that may affect the proliferation of monoclonal antibody-producing B cells.

[0059] In one embodiment, the nucleotide sequence of the hygromycin resistance gene is represented herein as SEQ ID NO:7, as follows: [ka]

[0060] Preferably, therefore, the hygromycin resistance gene comprises a nucleotide sequence substantially as shown in SEQ ID NO: 7, or a fragment or variant thereof.

[0061] EBV may be engineered so that its genome contains at least one, two, three, four, five, or six 3-kb BamHI W repeats, preferably with each repeat encoding a W promoter. Advantageously, this increases the transformation efficiency of infected B cells, and therefore, EBV clones showing expansion of the repeat region to 6.6 repeats were selected for use (15, 16).

[0062] EBV may be engineered to express a fluorescent protein, such that the fluorescent protein is expressed by infected B cells. Thus, in a preferred embodiment, the EBV expresses a fluorescent protein.

[0063] Thus, in one embodiment, the feeder cell line expresses a first fluorescent protein and the B cells express a second fluorescent protein, where the first and second fluorescent proteins are different. The first and second fluorescent proteins may be selected from any fluorescent protein known to those skilled in the art, such as green fluorescent protein (GFP) and its variants, mCherry, mNeonGreen, monomeric red fluorescent protein (mRFP), monomeric infrared fluorescent protein (mIFP), Venus, Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, and / or Enhanced Yellow Fluorescent Protein (EYFP).

[0064] Preferably, the first and second fluorescent proteins emit fluorescent signals at different wavelengths, so that the cell populations can be distinguished from each other.

[0065] Preferably, the EBV used to infect the B cells expresses green fluorescent protein (GFP). Even more preferably, the EBV used to infect the B cells expresses enhanced green fluorescent protein. Thus, in a preferred embodiment, the B cells express enhanced green fluorescent protein. This allows for rapid detection of a population of EBV-infected B cells.

[0066] One embodiment of the polypeptide sequence of an improved GFP is represented herein as SEQ ID NO: 8, as follows: [ka]

[0067] Preferably, therefore, the improved GFP comprises an amino acid sequence substantially as set forth in SEQ ID NO: 8, or a fragment or variant thereof.

[0068] In one embodiment, the nucleotide sequence encoding the improved GFP is represented herein as SEQ ID NO:9, as follows: [ka]

[0069] Preferably, therefore, the improved GFP is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 9, or a fragment or variant thereof.

[0070] In another preferred embodiment, the feeder cell line expresses mCherry. One embodiment of the polypeptide sequence of mCherry is represented herein as SEQ ID NO: 10, as follows: [ka]

[0071] Preferably, therefore, mCherry comprises an amino acid sequence substantially as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0072] In one embodiment, the nucleotide sequence encoding mCherry is represented herein as SEQ ID NO:11, as follows: [ka]

[0073] Preferably, therefore, mCherry is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO: 11, or a fragment or variant thereof.

[0074] In one embodiment, the feeder cell line does not express a drug selection marker. Preferably, the feeder cell line does not express a hygromycin resistance gene.

[0075] This is because EBV-infected B cells express drug selection markers (e.g., hygromycin resistance genes), which allow selection against feeder cells after initial LCL growth. Therefore, the use of drug selection markers advantageously eliminates the need for additional equipment. 137 This allows selection against feeder cells without the use of Cs γ-rays or X-rays, or without cell cycle-blocking drug treatments that may affect the proliferation of monoclonal antibody-producing B cells. In other words, this allows for the practical killing of feeder cells without affecting the growing LCL cells.

[0076] In one embodiment, the methods of the fifth to eighth aspects further comprise isolating B cells from a sample obtained from a subject, preferably before the B cells are infected with EBV. Preferably, the B cells are isolated from the sample before contacting the B cells with a feeder cell line (i.e., before step (i) of the methods of the fourth, fifth, sixth, and eighth aspects). Preferably, the method comprises isolating B cells specific for an antigen of interest. For example, the antigen may be an HIV antigen (see Example 8) or a SARS-CoV-2 antigen (see Example 9). As described in Examples 8 and 9, the inventors have demonstrated that the methods of the present invention can be effectively used to isolate MABs specific for HIV-1 Env immunogens, such as ConM and ConS, and SARS-CoV-2.

[0077] As used herein, the term "B cell" can refer to any type of B cell or derivative thereof that is capable of producing antibodies. For example, a B cell can be a B lymphocyte, a plasma B cell, an effector B cell, an activated B cell, or a memory B cell. B cells can be obtained from a human who has been immunized with an antigen or who has acquired an immune response to an antigen as a result of disease. Alternatively, B cells can be obtained from an immunized, naive human who has not previously been exposed to the antigen of interest.

[0078] B cells may be isolated from any biological sample, for example, blood, bone marrow, spleen, or lymph nodes. Preferably, B cells are isolated from a blood sample. More preferably, B cells are isolated from peripheral blood mononuclear cells (PBMCs). The blood may be venous or arterial. The blood sample may be assayed immediately. Alternatively, the blood sample may be stored at a low temperature, for example, in a refrigerator, or even frozen, before performing the method. Alternatively, the blood sample may be stored at room temperature, for example, at 18-22°C, before performing the method. PBMCs or B cells isolated from bone marrow, spleen, or lymph nodes can be stored at -80°C.

[0079] A "subject" can be any person that has B cells.

[0080] B cells may be isolated from a biological sample using methods well known to those skilled in the art, such as fractionation using antibody-coated magnetic beads, magnetic-activated cell sorting (MACS), or fluorescence-activated cell sorting (FACS). Preferably, the method comprises isolating B cells by FACS. FACS may be used with any suitable panel of markers to select antigen-specific B cells. Preferably, cells are kept at 4°C before EBV infection.

[0081] The B cells may then be infected with EBV and immortalized, i.e., made to divide and proliferate indefinitely. Thus, in one embodiment, the method further comprises the step of infecting the B cells with EBV. Preferably, the B cells are infected with EBV before contacting them with the feeder cell line (i.e., before step (i) of the methods of the fifth to eighth aspects). Even more preferably, the B cells are infected with EBV after they have been isolated from a sample obtained from a subject. Preferably, the EBV is as described above.

[0082] In one embodiment, infecting B cells with EBV comprises contacting B cells with EBV-containing RPMI medium. Such medium may be supplemented with fetal bovine serum (FBS, e.g., 10%) and is preferably pre-cleared by centrifugation (e.g., at about 2000 × g) and preferably filtered (e.g., through a 0.45 μm filter). Preferably, B cells are contacted with the EBV-containing medium at a multiplicity of infection (MOI) of at least 10, at least 20, at least 30, or at least 40. More preferably, B cells are contacted with the EBV-containing medium at a multiplicity of infection (MOI) of at least 50. Preferably, the B cells and EBV-containing medium are incubated at about 37°C with 5% CO2. Even more preferably, the B cells and EBV-containing medium are incubated for at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours.

[0083] The method may further comprise the step of washing and resuspending the B cell and EBV mixture in Roswell Park Memorial Institute Medium (RPMI), preferably RPMI medium containing FBS, most preferably 20% FBS. Preferably, the B cell and EBV mixture is washed with RPMI before the B cells are contacted with the feeder cell line (i.e., before step (i) of the methods according to the fifth to eighth aspects). Preferably, the RPMI medium is RPMI 1640 medium, GlutaMAX™ Supplement (ThermoFisher, 61870143).

[0084] The method then comprises the step of contacting the B cells with a feeder cell line according to the present invention (i.e., step (i) of the fifth to eighth aspects). Preferably, the feeder cell line is as described above. The B cells and feeder cell line are then cultured under conditions to support the proliferation of monoclonal antibody-producing B cells (i.e., step (ii) of the fifth and eighth aspects). It will therefore be understood that the method produces a culture of monoclonal antibody-producing B cells. For example, the method may comprise contacting the B cells with RPMI medium, preferably RPMI medium containing FBS, more preferably 20% FBS.

[0085] The method may further comprise contacting the B cells with a CpG oligonucleotide, preferably after the B cells have been contacted with the feeder cell line (i.e., after step (i) of the fifth to eighth aspects). CpG is an unmethylated DNA oligonucleotide that acts as a polyclonal activator of B cells. Preferably, the CpG oligonucleotide is a nuclease-resistant phosphorothioate oligonucleotide. Preferably, the CpG oligonucleotide is CpG ODN2006. One embodiment of the nucleotide sequence of CpG ODN2006 is provided herein as SEQ ID NO: 12, as follows: [ka]

[0086] Preferably, therefore, the CpG oligonucleotide comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 12, or a fragment or variant thereof.

[0087] In one embodiment, the method comprises contacting B cells with at least 0.2 μg / ml, at least 0.4 μg / ml, at least 0.6 μg / ml, at least 0.8 μg / ml, or at least 1.0 μg / ml of CpG oligonucleotide. In another embodiment, the method comprises contacting B cells with at least 1.2 μg / ml, at least 1.4 μg / ml, at least 1.6 μg / ml, at least 1.8 μg / ml, or at least 2.0 μg / ml of CpG oligonucleotide. In a preferred embodiment, the method comprises contacting B cells with at least 2.1 μg / ml, at least 2.2 μg / ml, at least 2.3 μg / ml, or at least 2.4 μg / ml of CpG oligonucleotide. Most preferably, the method comprises contacting B cells with at least 2.5 μg / ml of CpG oligonucleotide.

[0088] In one embodiment, the method preferably comprises culturing the B cells for at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days, most preferably at least 7 days, after contacting the B cells with the feeder cell line (i.e. after step (i) of the methods of the fifth and eighth aspects).

[0089] The method may then comprise isolating the monoclonal antibody-producing B cells (i.e., step (ii) of the sixth and seventh aspects). Preferably, the methods of the fifth and eighth aspects also comprise a step of isolating the monoclonal antibody-producing B cells after step (ii). As described herein, feeder cell lines express a fluorescent protein that is not expressed by B cells. Thus, the method may comprise identifying a feeder cell line that expresses a fluorescent protein, to allow isolation of the monoclonal antibody-producing B cells. The method may comprise isolating the monoclonal antibody-producing B cells using FACS.

[0090] The method may further comprise culturing the monoclonal antibody-producing B cells in RPMI, preferably RPMI containing FBS, most preferably 20% FBS. Preferably, the monoclonal antibody-producing B cells are cultured in RPMI after isolating the monoclonal antibody-producing B cells, i.e., after step (ii) of the fifth embodiment. The monoclonal antibody-producing B cells may be cultured with a CpG oligonucleotide (preferably ODN2006) at a concentration of at least 2.0 μg / ml, at least 2.5 μg / ml, at least 3 μg / ml, at least 3.5 μg / ml, at least 4.0 μg / ml, or at least 4.5 μg / ml. More preferably, the monoclonal antibody-producing B cells are cultured with a CpG oligonucleotide (preferably ODN2006) at a concentration of at least 5.0 μg / ml. Preferably, the culture is fed with fresh medium twice a week. Even more preferably, the medium of the initial feeding contains only the CpG oligonucleotide (preferably ODN2006). This step results in an expanded monoclonal B cell culture.

[0091] The expanded monoclonal B cell cultures may then be evaluated for the presence of fluorescently labeled EBV-infected cells. This evaluation may be performed, for example, after one or two weeks by detecting the presence of fluorescently labeled EBV-infected cells under a fluorescent microscope. The expanded B cell cultures produce monoclonal antibodies, preferably capable of binding to a specific antigen. As used herein, the term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies contains antibodies that are substantially similar and bind to the same epitope, excluding variants that may normally arise during monoclonal antibody production. Such variants are usually present only in small amounts.

[0092] Thus, the method may comprise isolating the monoclonal antibody from the monoclonal antibody-producing B cell culture, i.e., step (iii) of the eighth aspect. Isolating the monoclonal antibody from the monoclonal antibody-producing B cell culture may comprise collecting, centrifuging, and / or filtering the cell culture medium to obtain a cell culture supernatant comprising the monoclonal antibody. The method may further comprise separating and purifying the monoclonal antibody from the cell culture supernatant.

[0093] Advantageously, the feeder cell lines of the present invention allow the culture of monoclonal antibody-producing B cells indefinitely, preferably to the point where sufficient antibody is available in the culture supernatant for multiple assays, without the need for molecular cloning and expression in a secondary specialized protein-expressing cell line.

[0094] In addition, the feeder cell lines of the present invention support monoclonal cultures to the point where they become self-sustaining indefinitely, far exceeding the efficiency of molecular cloning from a single cell as performed by current methods, and ideally to the point where there are thousands or even millions of identical antibody-producing cells in a single culture that can be harvested for efficient determination of the antibody sequence by molecular biological methods. Thus, in a preferred embodiment, the method involves obtaining the gene sequence of the monoclonal antibody from mRNA produced by B cells in the monoclonal culture (as described in Example 8).

[0095] The method may be an in vitro or ex vivo method. Preferably, the method is an in vitro method.

[0096] As described herein, the feeder cell lines according to the present invention were obtained by transducing a starting cell line (preferably U2OS cells) with one or more expression vectors encoding megaCD40L, CD23b and fluorescent markers such as mCherry. Accordingly, the inventors have developed novel expression vectors that may be used to transduce the feeder cell lines according to the present invention.

[0097] Thus, in a ninth aspect there is provided a lentiviral vector substantially as shown in Figure 1A, Figure 1B or Figure 1C.

[0098] In a tenth aspect, there is provided a kit for culturing monoclonal antibody-producing B cells, comprising: (i) the following: (a) mega CD40 ligand (mega CD40L), or a variant or fragment thereof; (b) CD23, or a variant or fragment thereof, and / or (c) a fluorescent protein not expressed by the B cells A kit is provided that includes a feeder cell line expressing

[0099] Preferably, the kit is used to carry out the method of any one of the fifth to eighth aspects.

[0100] In one embodiment, the kit further comprises EBV. In another embodiment, the kit further comprises reagents for isolating B cells from a biological sample, the EBV and reagents being as defined in the previous aspect of the invention.

[0101] The kit may further include instructions for use and / or a receptacle for obtaining a biological sample from a subject.

[0102] It will be understood that the present invention also extends to any nucleic acid or peptide, or variant, derivative or analogue thereof, that substantially comprises the amino acid or nucleic acid sequence of any of the sequences referred to herein (including variants or fragments thereof). The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" can refer to a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences referred to herein, for example a sequence that has 40% identity to any of the sequences specified herein.

[0103] Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity to any of the referenced sequences. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity to any of the referenced sequences, more preferably at least 90% identity to any of the sequences referenced herein, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0104] Those skilled in the art will understand how to calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences. To calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The identity percentage of two sequences can vary depending on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented by different programs), or structural alignment from 3D comparison, and (ii) the parameters used by the alignment method, such as local versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as function form and constants.

[0105] After alignment, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identical portions may be divided by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of non-gap positions, or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage identity is also strongly dependent on length. Therefore, the shorter the sequence pair, the higher the expected sequence identity will be by chance.

[0106] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The commonly used multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity; for protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet; for DNA and protein alignments: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0107] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T) x 100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps, and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes (i) preparing a sequence alignment using the ClustalW program, for example, using an appropriate set of parameters as described above, and (ii) substituting the values of N and T into the following formula: sequence identity = (N / T) x 100.

[0108] Alternative methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from any of the sequences described herein by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids.

[0109] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or modified without substantially affecting the sequence of the protein encoded thereby to provide functional variants thereof. Suitable nucleotide variants are those having a sequence that is altered by the substitution of different codons that encode the same amino acid within the sequence, thus resulting in a silent (synonymous) change. Other suitable variants are those that contain all or portions of a sequence that have a homologous nucleotide sequence but are altered by the substitution of different codons that encode amino acids with side chains with similar biophysical properties to the amino acid being replaced, resulting in a conservative change. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It is therefore understood that which amino acids may be replaced with amino acids having similar biophysical properties, and one of skill in the art would know the nucleotide sequences encoding these amino acids.

[0110] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. [Brief explanation of the drawings]

[0111] For a better understanding of the present invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which:

[0112] [Figure 1(1)] Figure 1 shows maps of three different lentiviral vector embodiments used to clone transgenes for the "Amalthea" feeder cell line (i.e., a feeder cell line of the present invention). Figure 1A shows the lentiviral expression vector used to clone Mega CD40L by Gateway cloning, and Figure 1B shows the lentiviral expression vector used to clone CD23B by Gateway cloning. Custom vectors from Vector Builder contain puromycin (PURO - for Mega CD40L) and blasticidin (BLAST - for CD23b) selection markers. DNA fragments encoding transgenes with the required Gateway cloning regions were produced by ThermoFisher Scientific's GeneArt service. [Figure 1(2)]Figure 1 shows maps of three different lentiviral vector embodiments used to clone transgenes for the "Amalthea" feeder cell line (i.e., a feeder cell line according to the present invention). Figure 1C shows the lentiviral expression vector used to clone the red fluorescent protein mCherry by Gateway cloning. The custom vector from Vector Builder contains a neomycin (NEO - for mCherry) selection marker. The DNA fragment encoding the transgene with the required Gateway cloning region was produced by ThermoFisher Scientific's GeneArt service. [Figure 2] Figure 2 shows the expression of megaCD40L in cell culture media on feeder cells (i.e., MRC5, U2OS, and LCL). 10,000 cells were seeded into wells of a 96-well culture plate for each cell line, and megaCD40L was expressed after lentiviral transduction and puromycin selection. After two days, the supernatants were collected and analyzed by ELISA using the Quantikine Human CD40 Ligand Immunoassay from Bio-Techne (catalog number DCDL40) according to the manufacturer's instructions. [Figure 3] Figure 3 shows the ability of Amalthea feeders (i.e., the feeder cell line of the present invention) to improve B cell transformation efficiency. B cells were isolated and infected with GFP-expressing recombinant EBV. Infected cells were cocultured with allogeneic PBMCs or Amalthea cells as feeders. Flow cytometry was performed after 2 days. Dead cells were stained with Draq7 (AbCam ab109202), and EBV infection was assessed by GFP expression. The transformation potential of infected cells was assessed by CD23 expression by staining with anti-CD23-BV421 (Biolegend 338522) (13). [Figure 4]Figure 4 shows that the presence of CD23b on feeder cells increases LCL growth. B cells freshly infected with recombinant EBV were placed into wells of a 96-well culture plate containing irradiated feeder cells with or without CD23b expression. On average, two B cells were placed into each well, and after two weeks of cell culture, the number of wells with LCL growth was assessed microscopically. The percentage of wells with LCL growth was determined for each feeder. The average percentage and standard deviation from two experiments are shown. [Figure 5] Figure 5 shows that Amalthea feeders (i.e., feeder cell lines according to the present invention) stably express mCherry. After G418 selection, Amalthea feeders were confirmed to be mCherry-positive and therefore easily identifiable by flow cytometry. The GFP channel was free and available for GFP-positive recombinant EBV-infected cells as needed. [Figure 6] Figure 6 shows indexed FACS of EBV-infected cells for single B cell cloning. Co-cultures of EBV-infected LCLs and Amalthea feeders were centrifuged once at 400 g for 2 min and resuspended in RPMI containing 5% FBS. Live mCherry-negative cells were single-cell sorted into wells of a 96-well plate. The top panel shows the FACS gating strategy for the entire population, and the bottom panel shows the same gating strategy applied to indexed single-cell sorting in one 96-well plate. Cells were sorted only in the 60 inner wells of the plate. [Figure 7] Figure 7 shows fluorescence microscopy of growing monoclonal LCL cultures. GFP-expressing LCLs can be detected early after single-cell sorting (here, on day 4 after sorting), and GFP expression persists long-term (here, 21 days), ensuring detection of proliferation. Amalthea mCherry-positive feeders can be clearly visualized, and their status can be assessed during culture. [Figure 8]Figure 8 shows the FACS gating strategy used to sort HIV Env-specific B cells. Using PBMCs derived from the blood of volunteers in an HIV vaccine trial, live (Draq7-negative) B cells (CD19-positive) that were class-switched (IgM- and IgD-negative) and bound the HIV Env probes ConM GFP and / or ConS Scarlet were isolated. [Figure 9] Figure 9 shows data from ELISAs of supernatants from monoclonal LCLs with specificity for HIV-1 Env immunogens. A) Human IgG production was examined for supernatants from 326 monoclonal cultures of LCLs derived from cells selected for binding to the HIV Env immunogens, ConM and ConS. Points represent concentrations derived from antibody binding to anti-human antibodies and comparison with a known standard curve. Error bars represent standard deviations from three replicates. B) After ELISA, to determine binding of supernatant antibodies to the ConM SOSIP HIV Env immunogen, the ratio of ConM binding to IgG concentration was calculated to assess the affinity of the MAB for ConM. A value of 1 indicates an affinity for the immunogen as high as the affinity of the anti-human antibody for IgG in the supernatant. C) Same as (B), but for the ConS UFO HIV Env immunogen. [Figure 10] Figure 10 shows the FACS gating strategy used to sort SARS-CoV-2 spike-specific B cells. Using PBMCs derived from the blood of COVID-19 convalescent patients, live (Draq7-negative) B cells (CD19-positive) were isolated that were class-switched (IgM-negative), IgA-negative, and capable of binding to the probe as seen by staining with anti-MYC-AF488 antibody. [Figure 11]Figure 11 shows pseudovirus neutralization by supernatants from monoclonal LCL cultures. The ability of antibodies in the supernatants to neutralize luciferase-expressing SARS-CoV-2 pseudovirus and prevent infection of susceptible cells was assessed. Results from one 96-well plate are shown. Only the inner 60 wells of the plate were used for culture. Each bar represents the level of neutralization by supernatants from the corresponding well on the plate compared to the pseudovirus-only control. Neutralization levels were determined by luciferase expression assay. [Figure 12(1)] Figure 12 shows pseudovirus neutralization of purified antibodies against SARS-CoV2. Assay-validated MABs were purified and their neutralization potency was tested by detailed neutralization assays using the indicated serial dilutions. [Figure 12(2)] Table 1 summarizes LCL growth after index-based single-cell sorting. Wells were assessed for green LCL growth after two weeks of culture following index-based cell sorting using a fluorescent microscope. The number and average percentage of growth across different cell culture plates ± standard deviation are shown for two independent experiments. [Figure 12(3)] Table 2 summarizes antibody information for 12 antibodies that were found to have high and broad affinity for the HIV Env immunogen. [Figure 12(4)] Continued from Figure 12(3) (Table 2). [Example]

[0113] As described throughout, there are several drawbacks associated with current methods for isolating antigen-specific human monoclonal antibodies from B cells. Therefore, the inventors set out to test whether their modified feeder cell lines could be used to improve the efficiency and throughput of culture and isolation of monoclonal antibodies from B cells.

[0114] The present inventors used three lentiviral vectors to modify feeder cell lines to express mega-CD40L, CD23b, and mCherry fluorescent proteins (see Figure 1). The present inventors then tested whether mega-CD40L and CD23b expressed by the feeder cell lines could increase the transduction efficiency and growth of monoclonal antibody-producing B cells (Examples 2 and 3). The present inventors further set out to test whether fluorescent proteins different from those expressed by B cells could be used to distinguish the feeder cell lines from MAB-producing B cells (Example 4). Finally, the present inventors set out to demonstrate whether their feeder cell lines could be used in methods to isolate MABs specific to HIV and SARS-CoV-2 antigens (Examples 8 and 9).

[0115] Materials and Methods Generation of recombinant EBV for use in infecting B cells This is as described in reference (17). Briefly, a) HEK293 EBV producer cells are grown to confluence in RPMI medium containing 10% FBS. b) To produce recombinant EBV, producer cells are transfected with a plasmid expressing a transgene that induces the EBV lytic cycle and virus release. c) Recovering the recombinant EBV-containing supernatant. d) Viral titers are determined by an assay involving infection of susceptible lymphoma cells with serial dilutions of the harvested supernatant and counting of GFP-positive (green) cells. EBV stocks can be kept at 4°C for more than one year.

[0116] Lentiviral transduction of feeder cells Exogenous expression was achieved by lentiviral transduction of U2OS cells. MegaCD40L was cloned and lentiviral particles were generated using a lentiviral expression vector with a puromycin selection cassette (Figure 1A). 1 μg / ml puromycin was used for selection two days after transduction. Full-length CD23b expression was achieved by lentiviral transduction of U2OS cells already expressing MegaCD40L / Puro. CD23B was cloned and viral particles were generated using a lentiviral expression vector with a blasticidin selection cassette (Figure 1B). Blasticidin was added to the medium two days after lentiviral transduction at a concentration of 10 μg / ml. The feeder cell line also expresses the fluorescent protein mCherry for easy identification. This was facilitated by a custom lentiviral vector containing a neomycin resistance gene (Figure 1C). G418 was used at 250 μg / ml for selection two days after transduction.

[0117] Cultivation and use of Amalthea feeder cells a) Amalthea cells are adherent cells grown in RPMI medium containing 10% FBS. b) To support LCL growth, the day before, Amalthea cells are irradiated to stop their proliferation and prevent them from taking over the culture. A radiation dose of 30 Gy 137 Use a Cs gamma irradiator. Alternatively, an X-ray irradiator can be used. Alternatively, transient mitomycin C treatment can be used. c) After irradiation, count the cells and seed them into the cell culture vessel of choice. To form a confluent monolayer, use 8.3 x 10 cells. 4 cells / cm 2 Sow seeds in.

[0118] Method Protocol for MAB Isolation a) Viable, class-switched B cells specific for the antigen are selected using FACS with an appropriate panel of markers. Cells are kept at 4°C at all times. This is a process well established in the art and adapted to each specific antigen. b) The sorted cells are infected with recombinant EBV by mixing them with EBV-containing medium at an MOI of 50 and incubating at 37° C. for 3 hours. c) The cells are washed and resuspended in 0.5 ml of RPMI medium containing 20% fetal bovine serum (FBS). d) Add the resuspended cells to wells of a 48-well culture plate containing feeder cells that were irradiated the day before (this allows sufficient time for the cells to settle, adhere, and produce recombinant protein appropriate for feeder function) in 0.5 ml of RPMI medium containing 20% FBS. Add CpG ODN2006 (Invivogen tlr1-2006) to a final concentration of 2.5 μg / ml. e) Infected cells are grown in bulk for 7 days. f) After growing the infected cells (now LCLs) for 7 days, FACS is used to deposit single, live, mCherry-negative (non-feeder) cells into wells of a 96-well culture plate. Cell culture wells are filled with 2.5 x 10 cells in 50 μl of RPMI containing 20% FBS, prepared the day before. 4 Each culture is monoclonal at this stage. g) Add 50 μl of fresh RPMI containing 20% FBS and CpG ODN2006 at a concentration of 5 μg / ml to each well for a final concentration of 2.5 μg / ml h) Cultures are fed twice a week by changing 50 μl of medium. For the first feeding only, the fresh medium contains CpG ODN2006. Feeding is performed using a pipetting robot (Integra Viaflo96). i) After 2 weeks, monoclonal LCL cultures are assessed for proliferation by the presence of GFP-positive EBV-infected cells under a fluorescent microscope. Alternatively, a fluorescent plate reader can be used. j) MAB-containing supernatants are collected for evaluation of the MABs, and cells are harvested from grown cultures for genetic sequencing of the MABs produced. If larger amounts of monoclonal antibodies are required for molecular assays, monoclonal LCL cultures can be continuously expanded and antibody-containing supernatants harvested as needed.

[0119] Results and Discussion Example 1 – Lentiviral transduction of feeder cell lines To clone transgenes for the "Amalthea" feeder cell line (i.e., the feeder cell line of the present invention), we used three different lentiviral vectors, as shown in Figures 1A, 1B, and 1C. The first and second lentiviral expression vectors (Figures 1A and 1B) were used to clone megaCD40L and CD23B, respectively, into the feeder cell line to support the expansion of monoclonal antibody-producing B cells. For easy identification of the feeder cell line, the third lentiviral vector was used to clone the fluorescent protein mCherry.

[0120] Example 2 - Amalthea feeders increase transformation efficiency To test the B cell transduction efficiency using Amalthea feeders (i.e., the feeder cell line of the present invention), B cells were isolated from human blood samples using a Miltenyi B cell isolation kit (130-091-151). The B cells were infected with recombinant EBV by mixing them with a virus stock solution at a multiplicity of infection (MOI) of 50, incubating them at 37°C for 3 hours in 5% CO2, and then washing them with RPMI medium and resuspending them in RPMI supplemented with 20% fetal bovine serum (FBS). Half of the infected cells were transferred to 2.5 x 10 cells to act as feeders. 5 Place one half of the wells of a 24-well plate containing 1.75 x 10 irradiated (30 Gy) PBMCs and the other half containing 1.75 x 10 PBMCs as feeders. 5irradiated (30 Gy) Amalthea cells were placed in wells containing the cells. CpG ODN2006 (Invivogen tlr1-2006) was added to a final concentration of 2.5 μg / ml in all wells. Cyclosporin A was added to wells with allogeneic PBMCs at 1 μg / ml. Feeder cells were irradiated and seeded the day before infection.

[0121] Two days after infection, flow cytometry was performed to determine the percentage of infected (GFP-positive) and activated (CD23-positive) B cells. At 2 days postinfection, cells are activated (13) but have not yet begun to proliferate (18). As shown in Figure 3, the number of infected and activated B cells on the Amalthea feeder increased more than fourfold. Specifically, only 15% of live cells were infected and activated when supported by a PBMC feeder, compared with 62.8% when supported by an Amalthea feeder.

[0122] Example 3 - Amalthea feeders increase LCL growth through expression of CD23b Two 96-well culture plates were filled with 2.5 x 10 4The 96-well culture plates were prepared by seeding irradiated feeders into each well. The feeders in one culture plate were U2OS cells (Amalthea progenitor cells prior to lentiviral transduction for CD23b expression) exogenously expressing megaCD40L and mCherry. The feeders in the other culture plate were Amalthea cells further transduced with CD23b lentivirus. The next day, B cells were isolated and infected with recombinant EBV as described above. Viable cells were counted and serial dilutions were performed in RPMI medium containing 20% FBS. On average, two viable cells were placed into each well of a 96-well culture plate containing feeders with or without CD23b expression. RPMI medium was supplemented with CpG ODN2006 as described above. Cultures were grown for 2 weeks and fed on days 2, 6, and 12 by exchanging 50 μl of medium, again supplemented with CpG ODN2006 during the first feeding. On day 14, LCL proliferation was assessed by fluorescence microscopy, and the percentage of wells with proliferation was plotted (Figure 4). Across two experiments, the number of growing LCL cultures increased by approximately 20% when CD23b-expressing Amalthea feeders were used.

[0123] Example 4 - Amalthea feeders are easily distinguishable from MAB-producing B cells Amalthea feeders were stably transduced with lentivirus for expression of mCherry and neomycin resistance genes. Using 2 mg / ml G418 for selection, flow cytometry confirmed that nearly all cells were mCherry-positive (Figure 5). Because of mCherry expression in Amalthea cells, they could be easily distinguished from mCherry-negative LCLs. Therefore, even when using non-recombinant EBV, which does not express GFP, it is not necessary to stain new LCLs prior to FACS for single B cell cloning. This is a significant advantage, considering that an additional staining step would have resulted in the loss of the desired cells.

[0124] Example 5 - Amalthea feeders can be used without irradiation Amalthea feeders do not express the hygromycin resistance gene that recombinant EBV-infected LCLs express. This means that when using recombinant EBV, hygromycin can be used to kill feeder cells in co-cultures once the feeder cells are no longer needed, without affecting the antibody-producing, hygromycin-resistant LCL cells. This makes this platform flexible enough to potentially be used when irradiators are not available to terminate feeder cells and drug treatment is undesirable.

[0125] Example 6 - Amalthea feeders facilitate confirmed single B cell cloning Indexed single-cell FACS of B cells into wells of a 96-well plate was used for the first time for B cell culture cloning (LCL). Antigen-specific class-switched B cells were infected with recombinant EBV and plated at 1.75 × 10 cells per well of a 24-well plate. 5 Bulk culture was performed for the first 7 days on irradiated (30 Gy) Amalthea feeders seeded with 1000 cells the day before. After 7 days, cells from the initial bulk culture (new LCLs and Amalthea feeders) were single-cell sorted into wells of a 96-well plate. Live mCherry-negative cells were single-cell sorted into the inner 60 wells (Figure 6). To prevent evaporation from the culture wells, the outermost wells were filled with sterile PBS. Sorted cells were cultured in the 96-well plate format as described above for 2 weeks, and then the wells were assessed for LCL proliferation. In two independent experiments, 540 and 1500 cells were sorted into individual wells, and proliferation was observed in 198 and 273 wells, respectively (Table 1).

[0126] Example 7 – GFP-expressing LCLs can be easily visualized for growth assessment Single-cell sorted cultures can be tracked and evaluated throughout the growth period (Figure 7), facilitating simple and reliable identification of growing cultures and enabling forward planning without culture loss. The platform has the potential for automated growth detection using a fluorescent plate reader, which can significantly increase throughput potential.

[0127] Example 8 – MABs against HIV envelope proteins The following example demonstrates that the present invention is effective in isolating useful MABs. This method is very high throughput; after three weeks, supernatants containing antigen-specific monoclonal antibodies are available for multiple assays, shown here for ELISA and neutralization assays. This means that only assay-validated MABs are selected for sequence analysis and downstream applications, reducing cost and labor. Uniquely for the present invention, this is done directly from B cell cultures that are confirmed to be monoclonal from the start.

[0128] Blood samples were obtained from volunteers in an HIV vaccine trial. The immunogens used to vaccinate the volunteers were based on the HIV-1 envelope (Env) glycoprotein consensus sequence. Two immunogen variants, designated ConM SOSIP and ConS UFO, were used, representing two different strategies to stabilize soluble immunogens in a native-like conformation of HIV Env.

[0129] PBMCs were isolated by gradient centrifugation, and live, immunogen-specific, class-switched B cells were sorted by FACS. To select immunogen-specific B cells, two probes containing either the ConM SOSIP or ConS UFO protein sequences fused to either superfolder GFP (sfGFP) or mScarlet-I fluorescent protein (mScarlet-I), respectively, were generated. ConM-GFP and / or ConS-Scarlet-specific B cells were sorted as shown in Figure 8.

[0130] Sorted cells were infected with recombinant EBV and cocultured with Amalthea feeders for 7 days as described above to generate proliferative LCLs from antigen-specific B cells. LCL cells were then single-cell sorted into 96-well plates and cocultured with Amalthea feeders as described above, using the gating strategy shown in Figure 6.

[0131] 326 monoclonal LCL cultures were expanded, and supernatants and cells were harvested from all. The supernatants were tested by ELISA for the presence of human IgG antibodies; all were found to be positive at concentrations ranging from 100 to 10,000 ng / ml, with most at 1 to 2 μg / ml (Figure 9A). Separate ELISAs were performed on the same supernatants to assess specificity for ConM SOSIP and ConS UFO; the majority showed binding to at least one immunogen. The ratio of ConM SOSIP or ConS UFO binding to the IgG concentration in the supernatant provided a readout of antibody affinity for each immunogen (Figures 9B and 9C). Affinity information for each monoclonal antibody produced helped select the 12 best monoclonal antibodies for affinity level and breadth and determine their sequences for further study (Table 2). Antibody sequences were determined using techniques widely used in the field. Briefly, harvested LCL cells were frozen at -80°C in lysis buffer containing 0.01 M Tris pH 8.0 and Ribolock RNase inhibitor (ThermoFisher EO0381). cDNA was generated from cellular mRNA by reverse transcription using SuperScript IV Reverse Transcriptase (ThermoFisher 18090200) and random hexamers (ThermoFisher N8080127). MAB sequences were revealed by PCR amplification using a human antibody-specific primer pool (4) and Sanger sequencing of the PCR products.

[0132] Example 9 – Neutralization of MABs against SARS CoV2 Blood samples were obtained from convalescent COVID-19 patients, and PBMCs were isolated by gradient centrifugation. Soluble SARS-CoV-2 MYC-tagged protein was produced (19) and used as a probe to select antigen-specific B cells (Figure 10). Sorted SARS-CoV-2-specific B cells were infected with recombinant EBV, and monoclonal LCL cultures were generated as described above. Two weeks after single cell sorting into wells of 96-well plates, monoclonal culture supernatants were harvested from 75 96-well plates. These supernatants were used directly in a standard neutralization assay of SARS-CoV-2 pseudoviruses as described (19) (Figure 11). Several neutralizing supernatants with varying neutralizing potencies were identified. For the best neutralizers, antibody sequences were determined as described in Example 8, and more antibodies were produced and purified for more detailed studies. As an example, Figure 12 shows comprehensive neutralization testing of two of our isolated and purified antibodies compared to a control antibody isolated by others. Our D2S15 MAB neutralizes better than the other antibodies tested at higher dilutions.

[0133] Conclusion As explained throughout the Examples, the present inventors surprisingly discovered that their Amalthea feeder cell line increases the efficiency of EBV-mediated transformation / immortalization of B cells by more than four-fold compared to classical prior art EBV methods. In particular, the present inventors determined that modifying feeder cell lines to express mega CD40L and / or CD23 results in feeder cell lines that can significantly increase the number of monoclonal antibody-producing B cells grown.

[0134] In addition, using our new feeder cell line, it is now possible for the first time to support monoclonal growth of EBV-infected cells without a subcloning step, saving several weeks and significantly increasing efficiency compared to the classical EBV method for monoclonal antibody isolation. Furthermore, the resulting monoclonal culture supernatant contains antibodies that can be evaluated without the need for molecular cloning of the antibody sequence, which is currently required for the most preferred method. This reduces the cost per antibody evaluated by 50- to 100-fold compared to the currently preferred single-cell molecular cloning method.

[0135] Finally, by modifying a feeder cell line to express a fluorescent protein that is not expressed by B cells, we demonstrated that it is possible to distinguish feeder cells from B cells without any staining step that would result in the loss of the cells of interest.

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Claims

1. A feeder cell line for culturing monoclonal antibody-producing B cells, Mega CD 40 ligand (Mega CD 40L), or its variants or fragments, CD23, or its variety or fragment, and / or The fluorescent protein not expressed by the aforementioned B cells A feeder cell line that expresses [specific gene / phenomenon].

2. The feeder cell line according to claim 1, wherein the feeder cell line is selected from the group consisting of osteosarcoma cell lines, mesenchymal cell lines, epithelial cell lines, lymphoblastoid cell lines, neuron cell lines and endothelial cell lines, and preferably the feeder cell line is an osteosarcoma cell line.

3. The feeder cell line according to claim 1, wherein the feeder cell line is selected from the group consisting of U2OS, MRC5, lymphoblastoid cell line (LCL), H1299, MCF7, HEK293, 3T3, Caco-2, and HeLa, and preferably the feeder cell line is U2OS.

4. The feeder cell line is irradiated, preferably the feeder cell line １３７ The feeder cell line according to claim 1, wherein the feeder cell line is irradiated using a Cs gamma ray irradiator or an X-ray irradiator, or the feeder cell line is transiently treated with mitomycin C.

5. The feeder cell line according to claim 1, wherein the megaCD40L, or its variant or fragment, substantially comprises the amino acid sequence shown in SEQ ID NO: 1, or a fragment or variant thereof, and / or the megaCD40L, or its variant or fragment, substantially encodes the nucleotide sequence shown in SEQ ID NO: 2, or a fragment or variant thereof.

6. The aforementioned feeder cell line is (i) Mega CD 40L, or a variant or fragment thereof, in a cell culture medium containing at least 1 ng / ml, at least 2 ng / ml, at least 3 ng / ml, at least 4 ng / ml, or at least 5 ng / ml (ii) Mega CD 40L, or a variant or fragment thereof, in a cell culture medium containing at least 10 ng / ml, at least 20 ng / ml, at least 30 ng / ml, at least 40 ng / ml, or at least 50 ng / ml (iii) Cell culture medium containing at least 51 ng / ml of Mega CD 40L, or a variant or fragment thereof, and / or (iv) At least 10¹ ng / ml of Mega CD 40L, or a variant or fragment thereof, in the cell culture medium. A feeder cell line according to claim 1, which expresses [the specified expression].

7. The feeder cell line according to claim 1, wherein the CD23 or its fragment or variant expressed by the feeder cell line is CD23a or CD23b.

8. The feeder cell line according to claim 1, wherein the feeder cell line expresses CD23b, preferably CD23b comprises substantially the amino acid sequence shown in SEQ ID NO: 3, or a variant or fragment thereof, and / or CD23b is encoded by substantially the nucleotide sequence shown in SEQ ID NO: 4, or a fragment or variant thereof.

9. The feeder cell line according to claim 1, wherein the feeder cell line expresses CD23a, preferably CD23a comprises substantially the amino acid sequence shown in SEQ ID NO: 5, or a variant or fragment thereof, and / or CD23a is encoded by substantially the nucleotide sequence shown in SEQ ID NO: 6, or a fragment or variant thereof.

10. The feeder cell line according to claim 1, wherein the feeder cell line expresses a first fluorescent protein, and the B cells express a second fluorescent protein, and the first and second fluorescent proteins are different.

11. The feeder cell line according to claim 1, wherein the B cells express improved green fluorescent protein (GFP).

12. The feeder cell line according to claim 11, wherein the improved GFP comprises substantially the amino acid sequence shown in Sequence ID No. 8, or a fragment or variant thereof, and / or the improved GFP is encoded substantially by the nucleotide sequence shown in Sequence ID No. 9, or a fragment or variant thereof.

13. The feeder cell line according to claim 1, wherein the feeder cell line expresses mCherry.

14. The feeder cell line according to claim 13, wherein mCherry comprises substantially the amino acid sequence shown in SEQ ID NO: 10, or a fragment or variant thereof, and / or mCherry is encoded substantially by the nucleotide sequence shown in SEQ ID NO: 11, or a fragment or variant thereof.

15. The feeder cell line according to claim 1, wherein the monoclonal antibody-producing B cells are cultured using Epstein-Barr virus (EBV), and preferably the EBV is recombinant EBV.

16. The feeder cell line according to claim 15, wherein the recombinant EBV expresses a drug selection marker, and preferably the recombinant EBV expresses a hygromycin resistance gene.

17. The feeder cell line according to claim 16, wherein the hygromycin resistance gene substantially comprises the nucleotide sequence shown in Sequence ID No. 7, or a fragment or variant thereof.

18. Use of a feeder cell line according to any one of claims 1 to 17 in culturing monoclonal antibody-producing B cells.

19. Use of a feeder cell line according to any one of claims 1 to 17 in isolating monoclonal antibody-producing B cells.

20. A method for culturing monoclonal antibody-producing B cells, (i) A step of bringing B cells into contact with the feeder cell line described in claim 1, (ii) A step of culturing the B cells and the feeder cell line under conditions to support the proliferation of the monoclonal antibody-producing B cells. Methods that include...

21. A method for isolating monoclonal antibody-producing B cells from cell culture medium, (i) A step of bringing B cells into contact with the feeder cell line described in claim 1, (ii) A step of identifying the feeder cell line that expresses a fluorescent protein not expressed by the B cells in order to enable the isolation of the monoclonal antibody-producing B cells. Methods that include...

22. A method for isolating monoclonal antibody-producing B cells from cell culture medium, (i) A step of bringing B cells into contact with the feeder cell line described in claim 16, (ii) A step of culturing the B cells and the feeder cell line in the presence of a drug selection marker in order to enable the isolation of the monoclonal antibody-producing B cells. Methods that include...

23. A method for isolating monoclonal antibody-producing B cells from a cell culture medium, (i) A step of bringing B cells into contact with the feeder cell line described in claim 17, (ii) A step of culturing the B cells and the feeder cell line in the presence of a drug selection marker in order to enable the isolation of the monoclonal antibody-producing B cells. Methods that include...

24. The method according to any one of claims 20 to 23, further comprising the step of isolating a monoclonal antibody from the monoclonal antibody-producing B cells.

25. A method for isolating monoclonal antibodies from monoclonal antibody-producing B cells, (i) A step of bringing B cells into contact with the feeder cell line described in claim 1, (ii) A step of culturing the B cells and the feeder cell line under conditions to support the proliferation of the monoclonal antibody-producing B cells, (iii) A step of isolating monoclonal antibodies from the monoclonal antibody-producing B cells and Methods that include...

26. The method according to claim 20, 21, 22, 23, or 25, further comprising the step of isolating B cells from a sample obtained from a subject, wherein the B cells are preferably specific to the antigen of the target.

27. The method according to claim 20, 21, 22, 23, or 25, further comprising the step of contacting the B cells with a CpG oligonucleotide, wherein the CpG oligonucleotide is preferably a nuclease-resistant phosphorothioate oligonucleotide.

28. The method according to claim 27, wherein the CpG oligonucleotide substantially comprises the nucleotide sequence shown in Sequence ID No. 12, or a fragment or variant thereof.

29. A lentiviral vector essentially as shown in Figure 1A, Figure 1B, or Figure 1C.

30. A kit for culturing monoclonal antibody-producing B cells, (a) Mega CD 40 ligand (Mega CD 40 L), or a variety or fragment thereof, (b) CD23, or its variety or fragment, and / or (c) Fluorescent proteins not expressed by the B cells A kit containing a feeder cell line that expresses [specific cell type].

31. The kit according to claim 30 for carrying out the method of claim 20, claim 21, claim 22, claim 23, or claim 25, and / or for using a feeder cell line according to any one of claims 1 to 17.

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