Monoclonal antibody isolation

Abstract

The present invention relates to the production and isolation of monoclonal antibodies, in particular (but not limited to) novel feeder cell lines for culturing B cells that produce monoclonal antibodies. The invention also relates to application of the feeder cell line in culturing B cells generating monoclonal antibodies and in separating the B cells from a cell culture medium. The invention also relates to a method for culturing and isolating B cells producing monoclonal antibodies and a method for isolating monoclonal antibodies.

Description

Technical Field

[0001] The present invention relates to the production and separation of monoclonal antibodies, in particular (but not limited to) a novel feeder cell line for culturing B cells producing monoclonal antibodies. The present invention also relates to the use of feeder cell lines in culturing B cells producing monoclonal antibodies and in separating B cells from cell culture media. The present invention also relates to methods for culturing and isolating B cells producing monoclonal antibodies and methods for isolating monoclonal antibodies. Background Art

[0002] Epstein-Barr virus (EBV) is a B-lymphotropic gammaherpes virus that asymptomatically and persistently infects more than 90% of humans. In vitro, EBV induces the persistent proliferation (transformation and "immortality") of human B cells to generate lymphoblastoid cell lines (LCLs). EBV-infected LCLs derived from memory B cells with specificity for an antigen produce antibodies against that antigen. This EBV property has been exploited in the past (1) to isolate human monoclonal antibodies (MABs).

[0003] However, the current methods of using EBV to obtain MABs have some defects. First, the efficiency of EBV in transforming B cells is relatively low. This has been solved to a certain extent by adding the 24-mer oligodeoxynucleotide CpG ODN2006 to the culture medium of EBV-infected cells. CpG ODN 2006, as a TLR9 agonist, increased the transformation efficiency from about 2% to about 30% (2). Secondly, it is difficult to cultivate monoclonal cultures to produce MABs. The isolated individual LCL cells do not proliferate, which means that the culture is first grown in an oligoclonal manner, and several cells with different specificities are added to the same culture well. After two weeks of growth, the antibody production of the culture supernatant is evaluated, and the positive culture is subcloned again by limiting dilution. After a few weeks, the new subclone culture is further evaluated to test clonality, antibody production and specificity (3). In addition, the current method also has the problem of low yield of cultivation and subculture and evaluation of growth or clonality.

[0004] Given the above-mentioned issues associated with the classical EBV approach, the current preferred option for isolating MABs is the alternative single-cell molecular cloning approach (4). This approach involves single-cell sorting of antigen-specific B cells by FACS, followed by molecular cloning of individual cells to determine the antibody sequence of each cell. The sequence information is used to construct plasmid expression vectors for producing antibodies in separate cells dedicated to protein (antibody) expression. After the antibodies are produced by these specialized protein-expressing cells, the specificity, affinity, and neutralizing properties of the antibodies can be evaluated. This is a relatively high-yield method that can reduce the time required to obtain MABs by weeks and nearly double the overall efficiency compared to the classical EBV approach (5).

[0005] However, there are also many disadvantages in single cell molecular cloning and producing antibodies from mammalian cells dedicated to expression. First, compared with cultivating LCLs that produce antibodies infected by EBV, the cost of obtaining sequence information, performing molecular cloning, and expressing antibodies in separate cells is significantly higher. Secondly, obtaining antibody sequence information from a single cell is challenging and can affect efficiency. Finally, before the antibody is expressed from a plasmid vector in a cell line dedicated to expression, its specificity, affinity, and neutralization cannot be tested.

[0006] Therefore, there is a need to provide an improved platform for isolating human monoclonal antibodies with increased efficiency and high-yield capabilities. Summary of the invention

[0007] As described in the examples, the inventors have developed a new platform for separating human monoclonal antibodies from human peripheral blood mononuclear cells (PBMC). Specifically, the inventors have developed a novel feeder cell line, which is called Amalthea, and surprisingly proved that this feeder cell line can be used to support the effective overgrowth (outgrowth) of B cells producing antibodies infected with recombinant EBV. The inventors have proved that Amalthea feeder cells can support monoclonal cell cultures of cells producing antibodies, and the MAB produced in the culture medium can be used immediately for a variety of assays, thereby significantly saving time and reducing costs. In addition, the platform is compatible with fluorescence activated cell sorting (FACS) for selecting B cells with specificity for target antigens, thereby greatly improving yield.

[0008] Therefore, a first aspect of the present invention provides a feeder cell line for culturing B cells producing monoclonal antibodies, the feeder cell line expressing:

[0009] - mega CD40 ligand (mega CD40L) or a variant or fragment thereof;

[0010] -CD23 or a variant or fragment thereof; and / or

[0011] - Fluorescent proteins not expressed by B cells.

[0012] A second aspect of the present invention provides a use of a feeder cell line in culturing B cells producing monoclonal antibodies, wherein the feeder cell line expresses:

[0013] - mega CD40 ligand (mega CD40L) or a variant or fragment thereof; and / or

[0014] -CD23 or a variant or fragment thereof.

[0015] The third aspect of the present invention provides use of a feeder cell line in isolating B cells producing monoclonal antibodies, wherein the feeder cell line expresses a fluorescent protein that is not expressed by B cells.

[0016] A fourth aspect of the present invention provides use of a feeder cell line in isolating B cells producing monoclonal antibodies, wherein the feeder cell line does not express a drug selection marker expressed by B cells.

[0017] A fifth aspect of the present invention provides a method for culturing B cells that produce monoclonal antibodies, the method comprising:

[0018] (i) contacting the B cells with a feeder cell line expressing:

[0019] - mega CD40 ligand (mega CD40L) or a variant or fragment thereof, and / or

[0020] -CD23 or a variant or fragment thereof; and

[0021] (ii) culturing the B cells and feeder cell line under conditions that support the growth of monoclonal antibody-producing B cells.

[0022] A sixth aspect of the present invention provides a method for isolating B cells producing monoclonal antibodies from a cell culture medium, the method comprising:

[0023] (i) contacting the B cells with a feeder cell line that expresses a fluorescent protein that is not expressed by the B cells; and

[0024] (ii) Identification of feeder cell lines expressing fluorescent proteins to isolate B cells producing monoclonal antibodies.

[0025] A seventh aspect of the present invention provides a method for isolating B cells producing monoclonal antibodies from a cell culture medium, the method comprising:

[0026] (i) contacting the B cells with a feeder cell line that does not express the drug selection marker expressed by the B cells; and

[0027] (ii) Culturing B cells with a feeder cell line in the presence of a drug selection marker to isolate B cells that produce monoclonal antibodies.

[0028] Preferably, the methods of the fifth, sixth and seventh aspects comprise the step of isolating the monoclonal antibody from B cells producing the monoclonal antibody.

[0029] Therefore, the eighth aspect of the present invention provides a method for isolating a monoclonal antibody from a B cell producing the monoclonal antibody, the method comprising:

[0030] (i) contacting the B cells with a feeder cell line expressing:

[0031] - mega CD40 ligand (mega CD40L) or a variant or fragment thereof,

[0032] -CD23 or a variant or fragment thereof, and / or

[0033] - Fluorescent proteins not expressed by B cells;

[0034] (ii) culturing the B cells and feeder cell line under conditions that support the growth of B cells that produce the monoclonal antibody; and

[0035] (iii) isolating monoclonal antibodies from B cells producing the monoclonal antibodies.

[0036] As described in Examples 2 and 3, the inventors have demonstrated that modifying feeder cell lines to express mega CD40L and / or CD23 can result in feeder cell lines that can significantly increase the number of overgrown B cells producing monoclonal antibodies. For example, the inventors have demonstrated that their feeder cell lines increase the efficiency of virus-mediated B cell transformation / immortality by more than 4 times. In addition, using the inventors' novel feeder cell lines, it is currently possible for the first time to support monoclonal overgrowth of virus-infected cells without the need for subcloning steps and serial dilutions, saving weeks and greatly improving efficiency compared to the classical EBV method for monoclonal antibody isolation. In addition, the supernatant of the resulting monoclonal culture contains antibodies that can be evaluated without the need for molecular cloning of the antibody sequence, while the most popular method currently requires molecular cloning of the antibody sequence. This reduces the cost of evaluating each antibody by 50 to 100 times compared to the currently preferred single-cell molecular cloning method.

[0037] Furthermore, as described in Example 4, the inventors have also demonstrated that by modifying the feeder cell line to express a fluorescent protein that is not expressed by antibody-producing B cells, feeder cells and B cells can be easily distinguished without any additional staining steps that would result in loss of target cells.

[0038] In one embodiment, the feeder cell line expresses: (i) mega CD40L or a variant or fragment thereof; and (ii) CD23 or a variant or fragment thereof. Optionally, the feeder cell line further expresses: (iii) a fluorescent protein not expressed by B cells.

[0039] In another embodiment, the feeder cell line expresses: (i) mega CD40L or a variant or fragment thereof; and (ii) a fluorescent protein not expressed by B cells. Optionally, the feeder cell line also expresses: CD23 or a variant or fragment thereof.

[0040] In another embodiment, the feeder cell line expresses: (i) CD23 or a variant or fragment thereof; and (ii) a fluorescent protein not expressed by B cells. Optionally, the feeder cell line further expresses: mega CD40L or a variant or fragment thereof.

[0041] 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.

[0042] As used herein, the term "feeder cell line" may refer to a cell line used to culture target cells (i.e., B cells) to support their survival and / or growth (e.g., by producing various growth factors to support their survival and / or growth). In addition, the term "feeder cell line" may include cells that have been engineered to express specific growth factors or proteins (e.g., mega CD40L and / or CD23).

[0043] The feeder cell line can be any cell line capable of supporting the growth of B cells producing monoclonal antibodies. For example, the feeder cell line can be selected from 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.

[0044] In one embodiment, the feeder cell line can be selected from U2OS, MRC5, lymphoblastoid cell line (LCL), H1299, MCF7, HEK293, 3T3, Caco-2 and / or HeLa. U2OS is a cell line with epithelial morphology, which is derived from bone tissue of osteosarcoma patients. MRC5 is a diploid cell line composed of human fibroblasts, which is derived from lung tissue. H1299 is a human non-small cell lung cancer 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, which is derived from human embryonic kidney. 3T3 is a fibroblast cell line isolated from mouse embryos. 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, the cell line is adherent and forms a feeder cell monolayer for LCL cells (ie monoclonal antibody producing B cells).

[0045] In one embodiment, the feeder cell line is irradiated. Preferably, the feeder cell line is irradiated before the B cells are contacted with the feeder cell line. Advantageously, irradiation retards the growth of the feeder cells and prevents them from becoming dominant in the culture.

[0046] In one embodiment, the feeder cell line is used 137Cs gamma ray irradiator for irradiation. Preferably, the feeder cell line is used 137 Cs gamma ray irradiator is used to irradiate at a dose of at least 5 Gy, at least 10 Gy, at least 15 Gy, at least 20 Gy or at least 25 Gy. More preferably, the feeder cell line is used 137 The Cs gamma-ray irradiator performs irradiation at an irradiation dose of at least 30 Gy.

[0047] 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 can be transiently treated with a cell cycle inhibitory drug. In one embodiment, the cell cycle inhibitory drug is mitomycin C. Alternatively, in another embodiment, the feeder cell line is not irradiated or treated with a cell cycle inhibitory drug.

[0048] CD40 is a type II transmembrane protein of the TNF superfamily. CD40 ligand (CD40L) is a physiological ligand that binds to CD40 on the surface of B cells and provides activation and survival signals for LCL outgrowth (6, 7). Mega CD40L consists of two CD40L trimers and is advantageously more stable and mitogenic than physiological CD40L (7).

[0049] One embodiment of the polypeptide sequence of mega CD40L is represented herein as SEQ ID No: 1, as follows:

[0050] MKANLLVLLCALAAADADYKDDDDKGPGQVQLHEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDGRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGELQGDQNPQIAA HVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0051] [SEQ ID No:1]

[0052] Therefore, preferably, the mega CD40L comprises an amino acid sequence substantially as shown in SEQ ID No: 1 or a fragment or variant thereof.

[0053] In one embodiment, the nucleotide sequence encoding mega CD40L is represented herein as SEQ ID No:2 and is as follows:

[0054] ATGAAGGCCAACCTGCTGGTGCTGCTGTGTGCTCTGGCTGCCGCCGATGCCGACTACAAGGACGACGATGACAAAGGCCCTGGACAGGTCCAGCTGCACGAGGATGATGTGACCACCACCGAAGAACTGGCCCCTGCTCTTGTGCCTCCTCCAAAGGGAACATGTGCCGGCTGGATGGCTGGAATCCCTGGACACCCAGGCCACAATGGCACACCTGGCAGAGATGGAAGAGATGGCACCCCAGGCGAGAAGGGCGAAAAAGGCGACGCTGGACTGCTGGGACCTAAAGGCGAAACTGGCGACGTGGGAATGACAGGCGCTGAGGGCCCTAGAGGCTTTCCTGGAACACCTGGAAGAAAGGGCGAGCCTGGCGAACTGCAGGGCGATCAGAATCCTCAGATTGCCGCTCACGTGATCAGCGAGGCCAGCAGCAAGACAACAAGCGTGCTGCAGTGGGCCGAGAAGGGGTACTACACCATGAGCAACAACCTGGTCACCCTGGAAAACGGCAAGCAGCTGACCGTGAAGAGACAGGGCCTGTACTACATCTACGCCCAAGTGACCTTCTGCAGCAACAGAGAGGCCAGCTCTCAGGCCCCTTTTATCGCCAGCCTGTGCCTGAAGTCCCCTGGCAGATTCGAGAGAATCCTGCTGAGAGCCGCCAACACACACAGCTCCGCCAAACCTTGTGGCCAGCAGTCTATTCACCTCGGCGGAGTGTTTGAGCTGCAGCCTGGCGCTAGCGTGTTCGTGAATGTGACAGACCCTAGCCAGGTGTCCCACGGCACCGGCTTTACATCTTTCGGCCTGCTGAAGCTGTGA

[0055] [SEQ ID No:2]

[0056] Therefore, preferably, the mega CD40L is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 2 or a fragment or variant thereof.

[0057] mega CD40L is a commercially available soluble protein that can be added to the culture medium and has been previously used to enhance the overgrowth of LCL (8,9). However, the feeder cell lines of the present invention express mega CD40L directly into the culture medium, which has the advantage of creating a microenvironment for the overgrowing cells in which they are exposed to extremely high concentrations of more active recombinant mega CD40L. For example, the inventors unexpectedly discovered that the feeder cell lines of the present invention can achieve 4-7 times higher mega CD40L concentrations in the culture medium microenvironment compared to conventional addition of soluble proteins (8,9) (see Figure 2 ). In addition, the inventors believe that the concentration of mega CD40L is even higher in the microenvironment close to the surface of secretory feeder cells, where the overgrowing LCL cells (i.e., B cells that produce monoclonal antibodies) are located.

[0058] As described herein, monoclonal antibody-producing B cells are cultured in a cell culture medium together with a feeder cell line. Thus, mega CD40L expressed by the feeder cell line is directly expressed into the cell culture medium, resulting in a specific concentration of mega CD40L in the cell culture medium. Advantageously, the feeder cell line of the present invention expresses a high concentration of mega CD40L or a variant or fragment thereof, supporting the growth of monoclonal antibody-producing B cells.

[0059] Thus, in a preferred embodiment, 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. Still 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.

[0060] 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.

[0061] 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. 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. Still 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.

[0062] CD23 is a low affinity IgE receptor present on the B cell membrane that is upregulated by IL-4. CD23 is a 45-kDa type II membrane glycoprotein isoform, ie, there are two isoforms (CD23a and CD23b).

[0063] Thus, in one embodiment, the CD23 or fragment or variant thereof expressed by the feeder cell line is CD23a or CD23b.

[0064] CD23a and CD23b act as LCL growth factors because they result in the presentation of identical domains on the surface of feeder cells, and both isoforms have the same effect (10,11). CD23b is specifically expressed by LCLs, and only LCL cells expressing CD23 are transformed. LCL transformation is enhanced at an early stage by proximity to other CD23-expressing LCLs (12,13). However, previous approaches have focused either on soluble fragments of CD23 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).

[0065] In contrast, in the absence of other LCL cells, the feeder cell lines of the present invention express full-length CD23 to enhance single-cell LCL cloning. Advantageously, this facilitates efficient monoclonal overgrowth after single-cell index sorting to establish monoclonal LCL cultures. The inventors unexpectedly discovered that feeder cell lines expressing CD23b increased LCL overgrowth (i.e., B cells producing monoclonal antibodies) by about 20%.

[0066] Therefore, most preferably, the feeder cell line expresses CD23b.

[0067] One embodiment of the polypeptide sequence of CD23b is represented herein as SEQ ID No: 3, as shown below:

[0068] MNPPSQEIEELPRRRCCRRGTQIVLLGLVTAALWAGLLTLLLLWHWDTTQSLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGFVCN TCPEKWINFQRKCYYFGKGTKQWVHARYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNLDLKGEFIWVDGSHVDYSNWAPGEPTSRSQGEDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESMGPDSRPDPDGRLPTPSAPLHS

[0069] [SEQ ID No:3]

[0070] Therefore, preferably, CD23b comprises an amino acid sequence substantially as shown in SEQ ID No: 3 or a variant or fragment thereof.

[0071] In one embodiment, the nucleotide sequence encoding CD23b is represented herein as SEQ ID No: 4, as shown below:

[0072] ATGAATCCTCCAAGCCAAGAGATCGAGGAACTGCCCCGCAGACGGTGCTGTAGAAGAGGCACACAGATCGTGCTGCTGGGCCTTGTGACAGCTGCTCTGTGGGCTGGACTGCTGACACTGCTGCTGCTGTGGCACTGGGATACCACACAGAGCCTGAAGCAGCTGGAAGAAAGGGCCGCCAGAAACGTGTCCCAGGTGTCCAAGAACCTGGAAAGCCACCACGGCGACCAGATGGCCCAGAAGTCTCAGAGCACCCAGATCAGCCAAGAGCTTGAAGAACTGAGAGCCGAGCAGCAGCGGCTGAAGTCCCAAGATCTGGAACTGAGCTGGAACCTGAACGGACTGCAGGCCGATCTGAGCAGCTTCAAGTCTCAAGAGCTGAACGAGAGAAACGAGGCCAGCGACCTGCTGGAACGGCTGAGAGAAGAAGTGACCAAGCTGCGGATGGAACTGCAGGTTTCCAGCGGCTTCGTGTGCAACACATGCCCCGAGAAGTGGATCAACTTCCAGCGGAAGTGCTACTACTTCGGCAAGGGCACCAAGCAGTGGGTGCACGCCAGATACGCCTGCGACGATATGGAAGGCCAGCTGGTGTCCATTCACAGCCCCGAGGAACAGGACTTCCTGACCAAACACGCCAGCCACACCGGCTCTTGGATCGGACTGAGAAACCTGGACCTGAAGGGCGAGTTCATCTGGGTGGACGGCAGCCACGTGGACTACTCTAATTGGGCTCCTGGCGAGCCCACCAGCAGATCTCAAGGCGAGGATTGCGTGATGATGAGAGGCAGCGGCAGATGGAACGACGCCTTCTGCGATAGAAAGCTCGGCGCCTGGGTTTGCGACAGACTGGCCACATGTACACCTCCAGCCTCTGAGGGAAGCGCCGAGTCTATGGGCCCTGACTCTAGACCCGATCCTGACGGCAGACTGCCTACACCTTCTGCTCCTCTGCACAGCTGA

[0073] [SEQ ID No:4]

[0074] Therefore, preferably, CD23b is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 4 or a fragment or variant thereof.

[0075] Feeder cell lines may express CD23a.

[0076] One embodiment of the polypeptide sequence of CD23a is represented herein as SEQ ID No: 5, as shown below:

[0077] MEEGQYSEIEELPRRRCCRRGTQIVLLGLVTAALWAGLLTLLLLWHWDTTQSLKQLEERAARNVSQVSKNLESHHGDQMAQKSQSTQISQELEELRAEQQRLKSQDLELSWNLNGLQADLSSFKSQELNERNEASDLLERLREEVTKLRMELQVSSGFVC NTCPEKWINFQRKCYYFGKGTKQWVHARYACDDMEGQLVSIHSPEEQDFLTKHASHTGSWIGLRNLDLKGEFIWVDGSHVDYSNWAPGEPTSRSQGEDCVMMRGSGRWNDAFCDRKLGAWVCDRLATCTPPASEGSAESMGPDSRPDPDGRLPTPSAPLHS

[0078] [SEQ ID No:5]

[0079] Therefore, preferably, CD23a comprises an amino acid sequence substantially as shown in SEQ ID No: 5 or a variant or fragment thereof.

[0080] In one embodiment, the codon-optimized nucleotide sequence encoding CD23a is represented herein as SEQ ID No: 6, as shown below:

[0081] atggaagagggccagtacagcgagatcgaggaactgcctcggcggagatgctgtagaagaggcacacagatcgtgctgctgggccttgtgacagctgctctgtgggctggactgctgacactgctgctgctgtggcactgggataccacacagagcctgaagcagctggaagaaagggccgccagaaacgtgtcccaggtgtccaagaacctggaaagccaccacggcgaccagatggcccagaagtctcagagcacccagatcagccaagagcttgaggaactgagagccgagcagcagagactgaagtcccaggacctggaactgagctggaacctgaatggactgcaggccgacctgagcagcttcaagtcccaagagctgaacgagagaaacgaggccagcgacctgctggaacggctgagagaagaagtgaccaagctgcggatggaactgcaggtttccagcggcttcgtgtgcaacacatgccccgagaagtggatcaacttccagcggaagtgctactacttcggcaagggcaccaagcagtgggtgcacgccagatacgcctgcgacgatatggaaggccagctggtgtccattcacagccccgaggaacaggacttcctgaccaaacacgccagccacaccggctcttggatcggcctgagaaacctggatctgaagggcgagttcatctgggtggacggcagccacgtggactactctaattgggctcctggcgagcccaccagcagatctcaaggcgaggattgcgtgatgatgagaggcagcggcagatggaacgacgccttctgcgatagaaagctcggcgcctgggtttgcgacagactggccacatgtacacctccagcctctgagggaagcgccgagtctatgggccctgactctagacccgatcctgacggcagactgcctacaccttctgctcctctgcacagctga

[0082] [SEQ ID No:6]

[0083] Therefore, preferably, CD23a is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 6 or a fragment or variant thereof.

[0084] The inventors have demonstrated that expressing fluorescent proteins in feeder cell lines allows feeder cells to be easily distinguished from LCLs (i.e., B cells producing monoclonal antibodies). Therefore, unlike prior art methods, by using the feeder cell lines of the present invention, even with non-recombinant, non-GFP-expressing EBV, there is no need to stain new LCLs prior to FACS for single B cell cloning. This has significant advantages, as additional staining steps can result in loss of target B cells.

[0085] Thus, in one embodiment, the feeder cell line expresses a fluorescent protein that is not expressed by B cells.

[0086] In some embodiments, the monoclonal antibody-producing B cells are cultured using Epstein-Barr virus (EBV).

[0087] EBV can be selected from any variant that can induce B cell continuous proliferation (conversion and " immortalization "). Therefore, B cell is preferably a B cell producing monoclonal antibody infected by EBV. In some embodiments, recombinant EBV is used. Therefore, B cell is most preferably a B cell producing monoclonal antibody infected by recombinant EBV.

[0088] In one embodiment, EBV expresses a drug selection marker. Preferably, EBV expresses a hygromycin resistance gene. This allows the hygromycin antibiotic to select B cells infected by EBV (14). Advantageously, because B cells infected by EBV express drug selection markers (e.g., hygromycin resistance gene), feeder cells can be killed without affecting antibody-producing B cells infected by EBV. Otherwise, it is necessary to use 137 Cs gamma or X-ray irradiation is used to arrest feeder cell growth, which requires equipment that is not always available. Alternatively, feeder cells must be transiently treated with cell cycle inhibitors such as mitomycin C, which requires extensive washing, and residual contaminants may affect the growth of monoclonal antibody-producing B cells.

[0089] In one embodiment, the nucleotide sequence of the hygromycin resistance gene is represented herein as SEQ ID No: 7, as shown below:

[0090]

[0091] [SEQ ID No:7]

[0092] Therefore, preferably, the hygromycin resistance gene comprises a nucleotide sequence substantially as shown in SEQ ID No: 7 or a fragment or variant thereof.

[0093] EBV can be engineered to have a genome containing at least 1, 2, 3, 4, 5 or 6 3 kb BamHI W repeats, preferably each encoding a W promoter. Advantageously, this will increase the efficiency of transformation of infected B cells. Therefore, an EBV clone with a repeat region expansion of 6.6 repeats was selected (15, 16).

[0094] EBV can be engineered to express a fluorescent protein so that the fluorescent protein is expressed by infected B cells. Thus, in a preferred embodiment, the EBV expresses a fluorescent protein.

[0095] Thus, in one embodiment, the feeder cell line expresses a first fluorescent protein and the B cells express a second fluorescent protein, wherein the first fluorescent protein is different from the second fluorescent protein. The first fluorescent protein and the second fluorescent protein can be selected from any fluorescent protein well known to those skilled in the art, such as green fluorescent protein (GFP) and variants thereof, 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).

[0096] Preferably, the first fluorescent protein and the second fluorescent protein emit fluorescent signals at different wavelengths. Thus, cell groups can be distinguished from each other.

[0097] Preferably, the EBV used to infect B cells expresses green fluorescent protein (GFP). More preferably, the EBV used to infect B cells expresses enhanced green fluorescent protein. Therefore, in a preferred embodiment, B cells express enhanced green fluorescent protein. This allows rapid detection of B cell populations infected by EBV.

[0098] One embodiment of the polypeptide sequence of enhanced GFP is represented herein as SEQ ID No: 8, as shown below:

[0099] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE DGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKSGLRSRAQASNSAVDGTAGPGSTGSR*

[0100] [SEQ ID No:8]

[0101] Therefore, preferably, the enhanced GFP comprises an amino acid sequence substantially as shown in SEQ ID No: 8 or a fragment or variant thereof.

[0102] In one embodiment, the nucleotide sequence encoding enhanced GFP is represented herein as SEQ ID No: 9, as shown below:

[0103] Atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtccggactcagatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccaccggatctagataa

[0104] [SEQ ID No:9]

[0105] Therefore, preferably, the enhanced GFP is encoded by a nucleotide sequence substantially as shown in SEQ ID No:9, or a fragment or variant thereof.

[0106] In another preferred embodiment, the feeder cell line expresses mCherry. An embodiment of the polypeptide sequence of mCherry is represented herein as SEQ ID No: 10, as shown below:

[0107] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0108] [SEQ ID No:10]

[0109] Therefore, preferably, mCherry comprises an amino acid sequence substantially as shown in SEQ ID No: 10 or a fragment or variant thereof.

[0110] In one embodiment, the nucleotide sequence encoding mCherry is represented herein as SEQ ID No: 11, as shown below:

[0111] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTG CCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGC AGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTA CGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAG

[0112] [SEQ ID No:11]

[0113] Therefore, preferably, mCherry is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 11 or a fragment or variant thereof.

[0114] 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.

[0115] This is because EBV-infected B cells express drug selection markers (e.g., hygromycin resistance genes) that allow selection against feeder cells after initial outgrowth of LCLs. Thus, advantageously, the use of drug selection markers allows for the elimination of the need for LCLs. 137 Selection for feeder cells is performed without irradiation with Cs gamma rays or X-rays (which require additional equipment) or treatment with cell cycle inhibitory drugs (which may affect the growth of monoclonal antibody-producing B cells). In other words, this allows actual killing of feeder cells without affecting overgrowing LCL cells.

[0116] In one embodiment, the method of the fifth to eighth aspects further comprises a step of isolating B cells from a sample obtained from a subject, preferably a step of isolating B cells from a sample obtained from a subject before the B cells are infected by EBV. Preferably, B cells are isolated from a sample before the B cells are contacted with a feeder cell line (i.e., before step (i) of the method of the fourth, fifth to sixth, and eighth aspects). Preferably, the method comprises isolating B cells with specificity for a target antigen. 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 demonstrate that the method of the present invention can be effectively used to isolate MABs with specificity for HIV-1 envelope immunogens (e.g., ConM and ConS) and SARS-CoV-2.

[0117] As used herein, the term "B cell" may refer to any type of B cell or its derivative that is capable of producing antibodies. For example, a B cell may be a B lymphocyte, a plasma B cell, an effector B cell, an activated B cell, or a memory B cell. B cells may be obtained from a person who has been immunized with an antigen or a person who has developed an immune response to an antigen due to a disease. Alternatively, B cells may be obtained from a person who has never been immunized. ) in persons who had not been previously exposed to the target antigen.

[0118] B cells can be isolated from any biological sample, such as 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 (PBMC). The blood can be venous blood or arterial blood. The blood sample can be measured immediately. Alternatively, the blood sample can be stored at low temperatures before the method is performed, such as in a refrigerator or even frozen. Alternatively, the blood sample can be stored at room temperature before the method is performed, such as between 18 and 22°C. PBMCs or B cells isolated from bone marrow, spleen or lymph nodes can be stored at -80°C.

[0119] A "subject" can be any human having B cells.

[0120] B cells can be isolated from biological samples 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 separating B cells by FACS. FACS can be used with any suitable marker panel to select B cells with specificity for the antigen. Preferably, the cells are kept at 4°C before EBV infection.

[0121] Then, EBV can be used to infect B cells to make them immortal (i.e., infinite division and proliferation). Therefore, in one embodiment, the method also includes the step of infecting B cells with EBV. Preferably, before B cells contact with feeder cell lines (i.e., before step (i) of the method of the fifth to eighth aspects), EBV is used to infect B cells. Still more preferably, after separating B cells from a sample obtained from a subject, EBV is used to infect B cells. Preferably, EBV is EBV as described above.

[0122] In one embodiment, infecting B cells with EBV includes contacting B cells with RPMI culture medium containing EBV. Such culture medium can be supplemented with fetal bovine serum (FBS, for example 10%), preferably pre-clarified by centrifugation (for example, at about 2000xg), preferably filtered (for example, by 0.45 μm filter). Preferably, B cells are contacted with a culture medium containing EBV 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 a culture medium containing EBV at a multiplicity of infection (MOI) of at least 50. Preferably, B cells are incubated with a culture medium containing EBV at about 37°C and 5% CO2. Still more preferably, B cells are incubated with a culture medium containing EBV for at least one hour, at least two hours, at least three hours, or at least four hours.

[0123] The method may further comprise the step of washing and resuspending the B cell and EBV mixture with RPMI (preferably containing FBS, most preferably containing 20% ​​FBS). Preferably, the B cell and EBV mixture is washed with RPMI before the B cell is contacted with the feeder cell line (i.e. before step (i) of the method of the fifth to eighth aspects). Preferably, the RPMI medium is RPMI 1640 medium, GlutaMAX TM Supplement (ThermoFisher, 61870143).

[0124] Then, the method includes the step of contacting the B cells with the feeder cell line of the present invention (i.e., step (i) of the fifth to eighth aspects). Preferably, the feeder cell line is a feeder cell line as described above. Then, the B cells and the feeder cell line are cultured under conditions that support the growth of B cells producing monoclonal antibodies (i.e., step (ii) of the fifth and eighth aspects). Therefore, it should be understood that the method produces a culture of B cells that produce monoclonal antibodies. For example, the method may include contacting the B cells with RPMI culture medium (preferably containing FBS, more preferably containing 20% ​​FBS).

[0125] The method may also include contacting the B cell with a CpG oligonucleotide, preferably after the B cell is contacted with a feeder cell line (i.e., after step (i) of the fifth to eighth aspects). CpG is an unmethylated DNA oligonucleotide that serves as a polyclonal activator for the B cell. Preferably, the CpG oligonucleotide is a nuclease-resistant phosphorothioate oligonucleotide. Preferably, the CpG oligonucleotide is CpG ODN 2006. An embodiment of the nucleotide sequence of CpG ODN 2006 is provided herein as SEQ ID No: 12, as shown below:

[0126] 5'-tcgtcgttttgtcgttttgtcgtt-3'

[0127] [SEQ ID No:12]

[0128] Therefore, preferably, the CpG oligonucleotide comprises a nucleotide sequence substantially as shown in SEQ ID No: 12 or a fragment or variant thereof.

[0129] In one embodiment, the method includes 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 oligonucleotides. In another embodiment, the method includes 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 oligonucleotides. In a preferred embodiment, the method includes 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 oligonucleotides. Most preferably, the method includes contacting B cells with at least 2.5 μg / ml of CpG oligonucleotides.

[0130] In one embodiment, the method comprises culturing the B cells for at least two days, at least three days, at least four days, at least five days or at least six days, most preferably at least seven days, preferably culturing the B cells for at least two days, at least three days, at least four days, at least five days or at least six days, most preferably at least seven 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).

[0131] Then, the method may include separating the B cells that produce the monoclonal antibody (i.e., step (ii) of the sixth and seventh aspects). Preferably, the method of the fifth and eighth aspects also includes separating the B cells that produce the monoclonal antibody after step (ii). As described herein, the feeder cell line expresses a fluorescent protein that the B cells do not express. Therefore, the method may include identifying the feeder cell line that expresses the fluorescent protein to allow the B cells that produce the monoclonal antibody to be separated. The method may include using FACS to separate the B cells that produce the monoclonal antibody.

[0132] The method may also include cultivating the B cells producing monoclonal antibodies with RPMI (preferably containing FBS, most preferably containing 20% ​​FBS). Preferably, after separating the B cells producing monoclonal antibodies, the B cells producing monoclonal antibodies are cultivated with RPMI, i.e., step (ii) of the fifth aspect. The B cells producing monoclonal antibodies can also be cultivated with CpG oligonucleotides (preferably ODN 2006) 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 B cells producing monoclonal antibodies are cultivated with CpG oligonucleotides (preferably ODN 2006) at a concentration of at least 5.0 μg / ml. Preferably, the culture is fed twice weekly with fresh culture medium. Still more preferably, the culture medium of the first feeding only comprises CpG oligonucleotides (preferably ODN 2006). This step obtains the monoclonal B cell culture of amplification.

[0133] The monoclonal B cell culture of amplification can then be assessed by the presence of cells infected by fluorescently labeled EBV. Can be assessed after one or two weeks, for example, the presence of cells infected by fluorescently labeled EBV is detected under a fluorescence microscope. The B cell culture of amplification produces monoclonal antibodies, which are preferably capable of binding to specific antigens. As used herein, the term "monoclonal antibody" refers to an antibody from a substantially homogeneous antibody group. Substantially homogeneous antibody groups comprise antibodies that are substantially similar and combined with the same epitope, except for variants that may normally occur during the monoclonal antibody production process. Such variants are usually only present in small quantities.

[0134] Therefore, the method may include separating the monoclonal antibody from the B cell culture that produces the monoclonal antibody, i.e., step (iii) of the eighth aspect. Separating the monoclonal antibody from the B cell culture that produces the monoclonal antibody may include harvesting, centrifuging and / or filtering the cell culture medium to obtain a cell culture supernatant containing the monoclonal antibody. The method may also include separating and purifying the monoclonal antibody from the cell culture supernatant.

[0135] Advantageously, the feeder cell lines of the present invention can culture monoclonal antibody-producing B cells indefinitely, preferably to the point where there is sufficient antibody in the culture supernatant for use in a variety of assays, without the need for molecular cloning and expression in a secondary specialized protein-expressing cell line.

[0136] Furthermore, the feeder cell lines of the present invention support monoclonal cultures until they are self-sustaining indefinitely, and ideally, until there are thousands or even millions of identical antibody-producing cells in a single culture, which can be harvested for efficient determination of antibody sequences by molecular biology methods, far exceeding the efficiency of molecular cloning from single cells according to current methods. Thus, in a preferred embodiment, the method comprises obtaining the gene sequence of a monoclonal antibody from mRNA produced by B cells in a monoclonal culture (as described in Example 8).

[0137] The method may be an in vitro or ex vivo method. Preferably, the method is an in vitro method.

[0138] As described herein, the feeder cell lines of the present invention are obtained by transducing a starting cell line (preferably U2OS cells) with one or more expression vectors encoding mega CD40L, CD23b and a fluorescent marker (e.g., mCherry). Therefore, the inventors have developed a novel expression vector that can be used to transduce the feeder cell lines of the present invention.

[0139] Therefore, the ninth aspect provides a lentiviral vector, which is essentially as follows Figure 1A , 1B Or as shown in 1C.

[0140] The tenth aspect provides a kit for culturing B cells that produce monoclonal antibodies, the kit comprising:

[0141] (i) a feeder cell line expressing:

[0142] (a) mega CD40 ligand (mega CD40L) or a variant or fragment thereof;

[0143] (b) CD23 or a variant or fragment thereof; and / or

[0144] (c) Fluorescent proteins not expressed by B cells.

[0145] Preferably, the kit is used to perform the method of any one of the fifth to eighth aspects.

[0146] In one embodiment, the kit further comprises EBV. In another embodiment, the kit further comprises a reagent for isolating B cells from a biological sample. EBV and reagents are as defined in the previous aspects of the invention.

[0147] The kit may also include instructions for use and / or a container for obtaining a biological sample from a subject.

[0148] It should be understood that the present invention extends to any nucleic acid or peptide comprising an amino acid or nucleic acid sequence (including variants or fragments thereof) of any sequence mentioned herein, or a variant, derivative or analog thereof. The terms "substantially ... an amino acid / nucleotide / peptide sequence", "variant" and "fragment" may be a sequence having at least 40% sequence identity with an amino acid / nucleotide / peptide sequence of any sequence mentioned herein, for example a sequence having 40% identity with any sequence identified herein.

[0149] Also contemplated are amino acid / polynucleotide / polypeptide sequences having greater than 65%, more preferably greater than 70%, still more preferably greater than 75%, and still more preferably greater than 80% sequence identity with any of the sequences mentioned. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity, more preferably at least 90% identity, still more preferably at least 92% identity, still more preferably at least 95% identity, still more preferably at least 97% identity, still more preferably at least 98% identity, and most preferably at least 99% identity with any of the sequences mentioned herein.

[0150] Those skilled in the art will understand how to calculate the percent identity between two amino acid / polynucleotide / peptide sequences. In order to calculate the percent identity between two amino acid / polynucleotide / peptide sequences, the comparison of the two sequences must first be prepared, and then the value of sequence identity is calculated. The percent identity of two sequences may take different values, depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (Smith-Waterman) (performed in different programs) or the structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, such as local relative global alignment, the pairing scoring matrix used (such as BLOSUM62, PAM250, Gonnet, etc.) and gap penalties, such as function forms and constants.

[0151] Once the alignment is complete, there are many different ways to calculate the percent identity between two sequences. For example, the number of identities can 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. In addition, it will be appreciated that the percent identity also depends greatly on length. Thus, the shorter a pair of sequences is, the higher the sequence identity that can be expected to occur by chance.

[0152] Therefore, it should be understood that accurate alignment of protein or DNA sequences is a complex process. According to the present invention, the popular 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. Suitable ClustalW parameters may be as follows: for DNA alignments: gap open penalty = 15.0, gap extension penalty = 6.66, matrix = identity. For protein alignments: gap open penalty = 10.0, gap extension penalty = 0.2, matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will recognize that these and other parameters may need to be changed to achieve optimal sequence alignment.

[0153] Preferably, the percent identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from such an alignment as (N / T)*100, where N is the number of positions of identical residues shared by the sequences and T is the total number of positions compared, including gaps and with or without overhangs. Preferably, the calculation includes overhangs. Thus, the most preferred method for calculating the percent identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable parameter set (e.g., as described above); and (ii) substituting the values ​​for N and T into the following formula: sequence identity=(N / T)*100.

[0154] Those skilled in the art will be aware of alternative methods for identifying similar sequences. For example, substantially similar nucleotide sequences will be encoded by sequences that hybridize to the DNA sequence or its complement under stringent conditions. By stringent conditions, the inventors mean hybridization of nucleotides to DNA or RNA bound to a filter in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at least once in 0.2x SSC / 0.1% SDS at about 20-65°C. Alternatively, substantially similar polypeptides may differ from any sequence described herein by at least 1 amino acid, but by less than 5, 10, 20, 50, or 100 amino acids.

[0155] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be modified or changed without significantly affecting the protein sequence encoded by it, thereby providing its functional variant. Suitable nucleotide variants are those variants having sequences that are changed by replacing different codons encoding the same amino acid in the sequence to produce silent (synonymous) changes. Other suitable variants are those variants having homologous nucleotide sequences but containing sequences that are changed in whole or in part by replacing different codons to produce conservative changes, and the amino acids encoded by the different codons have side chains with biophysical properties similar to the amino acids they replace. For example, small non-polar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline and methionine; large non-polar 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. Therefore, it is understood which amino acids can be substituted by amino acids having similar biophysical properties, and those skilled in the art are aware of the nucleotide sequences encoding these amino acids.

[0156] All features described herein (including any accompanying claims, abstract and drawings) and / or all steps of any method or process disclosed may be combined with any of the above aspects in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] For a better understanding of the invention and to show how embodiments of the invention may be carried into effect, reference will now be made by way of example to the accompanying drawings, in which:

[0158] FIG. 1 shows a map of three embodiments of various lentiviral vectors used for cloning transgenes for the “Amalthea” feeder cell line (ie, the feeder cell line of the present invention). Figure 1A The lentiviral expression vector used to clone mega CD40L is shown. Figure 1BThe lentiviral expression vector used to clone CD23B is shown. Figure 1C Lentiviral expression vectors used to clone the red fluorescent protein mCherry by Gateway cloning are shown. Custom vectors from Vector Builder contain puromycin (PURO- for mega CD40L), blasticidin (BLAST- for CD23b), and neomycin (NEO- for mCherry) selection markers. DNA fragments encoding transgenes and necessary Gateway cloning regions were manufactured by GeneArt services of ThermoFisherScientific.

[0159] Figure 2 Shown is the expression of mega CD40L in cell culture medium by feeder cells (i.e., MRC5, U2OS, and LCL). 10,000 cells of each cell line were seeded into wells of a 96-well culture plate and expressed mega CD40L after lentiviral transduction and puromycin selection. Two days later, supernatants were collected and analyzed by ELISA using Bio-Techne's Quantikine Human CD40 Ligand Immunoassay (Cat. No. DCDL40) according to the manufacturer's instructions.

[0160] Figure 3 The ability of Amalthea feeder cells (i.e., feeder cell lines of the present invention) to improve the efficiency of B cell transformation was demonstrated. B cells were isolated and infected with recombinant EBV expressing GFP. The infected cells were co-cultured with allogeneic PBMC or Amalthea cells as feeder cells. Flow cytometry was performed two days later. 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 (Biolegend338522) (13).

[0161] Figure 4 The presence of CD23b in feeder cells was shown to increase LCL overgrowth. B cells newly infected with recombinant EBV were placed in wells of a 96-well culture plate containing irradiated feeder cells (with or without CD23b expression). An average of two B cells were placed in each well, and after two weeks of cell culture, the number of wells with LCL overgrowth was assessed by microscopy. The percentage of wells with LCL overgrowth in each feeder cell was determined. The average percentage and standard deviation of two experiments are shown.

[0162] Figure 5It is shown that Amalthea feeder cells (i.e., feeder cell lines of the present invention) stably express mCherry. After G418 selection, Amalthea feeder cells were confirmed to be mCherry positive and, therefore, easily distinguished by flow cytometry. The GFP channel is clear and can be used for GFP-positive recombinant EBV-infected cells if necessary.

[0163] Figure 6 Indexed FACS of EBV infected cells for a single B cell clone is shown. Co-cultures of EBV infected LCLs and Amalthea feeder cells were centrifuged once at 400 g for two minutes and resuspended in RPMI containing 5% FBS. Live mCherry negative cells were single cell sorted into 96 well plate wells. The upper panel shows the FACS gating strategy for the entire population, and the lower panel shows the same gating strategy applied to indexed single cell sorting in one 96 well plate. Cells were sorted only in the inner 60 wells of the plate.

[0164] Figure 7 Fluorescence microscopy of an outgrowing monoclonal LCL culture is shown. GFP-expressing LCLs are detectable early after single-cell sorting (here, four days after sorting), and GFP expression is retained for a long time (here, 21 days), ensuring detection of growth. Amalthea mCherry-positive feeder cells can also be clearly visualized and their status assessed during culture.

[0165] Figure 8 Shown is the FACS gating strategy for sorting HIV envelope-specific B cells. PBMCs from the blood of volunteers in an HIV vaccine trial were used to isolate live (Draq7 negative) B cells (CD19 positive) that were class-switched (IgM and IgD negative) and bound to the HIV envelope probes ConM GFP and / or ConS Scarlet.

[0166] Fig. 9ELISA data of supernatants from monoclonal LCLs specific for HIV-1 envelope immunogens are shown. A) Human IgG production in supernatants from monoclonal cultures of 326 LCLs derived from cells sorted for binding to HIV envelope immunogens ConM and ConS was verified. Points represent the concentrations obtained by binding of antibodies to anti-human antibodies and comparison with known standard curves. Error bars are standard deviations of three replicates. B) After performing the ELISA, in order to determine the binding of supernatant antibodies to ConM SOSIP HIV envelope immunogens, the ratio of ConM concentrations bound to IgG was calculated to assess the affinity of the MAB to ConM. A value of 1 indicates that the affinity for the immunogen is as high as the affinity of the anti-human antibodies in the supernatant to IgG. C) Same as B), but involving ConS UFO HIV envelope immunogens.

[0167] Fig.10 The FACS gating strategy for sorting SARS-CoV-2 spike-specific B cells is shown. PBMCs from the blood of COVID-19 convalescent patients were used to isolate live (Draq7 negative) B cells (CD19 positive) that were class-switched (IgM negative, IgA negative) and could bind to the probe, as shown by staining with anti-MYC-AF488 antibody.

[0168] Fig.11 Neutralization of pseudovirus by monoclonal LCL culture supernatants is shown. Antibodies in the supernatants were evaluated for their ability to neutralize SARS-CoV-2 pseudovirus expressing luciferase and prevent infection of susceptible cells. Results for one 96-well plate are shown. Only the inner 60 wells of the plate were used for incubation. Each line represents the level of neutralization of the supernatant from the well at the corresponding position on the plate relative to the pseudovirus-only control. Neutralization levels were determined by luciferase expression assay.

[0169] Fig.12 Neutralization of pseudovirus by purified anti-SARS-CoV2 antibodies is shown. Assay-validated MABs were purified and their neutralization potency was investigated by detailed neutralization assays with serial dilutions as shown.

[0170] Table 1 summarizes the outgrowth of LCLs after index single cell sorting. Fluorescence microscopy was used to assess the outgrowth of green LCLs in wells after two weeks of culture after index cell sorting. The number and mean percentage of outgrowth + / - standard deviation of different cell culture plates from two independent experiments are shown.

[0171] Table 2 summarizes the antibody information for 12 antibodies that were found to have high and broad affinity for HIV envelope immunogens. DETAILED DESCRIPTION

[0172] Example

[0173] As mentioned above, current methods for isolating antigen-specific human monoclonal antibodies from B cells have several disadvantages. Therefore, the inventors set out to test whether their modified feeder cell lines could be used to increase the efficiency and yield of culturing and isolating monoclonal antibodies from B cells.

[0174] The inventors used three lentiviral vectors (see Figure 1) to modify feeder cell lines to express mega CD40L, CD23b and mCherry fluorescent proteins. Then, the inventors tested whether the mega CD40L and CD23b expressed by the feeder cell lines can improve the conversion efficiency and overgrowth of B cells producing monoclonal antibodies (Examples 2 and 3). The inventors further set out to test whether fluorescent proteins different from those expressed by B cells can be used to distinguish feeder cell lines from B cells producing MABs (Example 4). Finally, the inventors set out to demonstrate whether their feeder cell lines can be used to separate MABs with specificity for HIV and SARS-CoV-2 antigens (Examples 8 and 9).

[0175] Materials and methods

[0176] Production of recombinant EBV for infection of B cells

[0177] As described in reference (17). In short:

[0178] a) EBV producer cells HEK293 were grown in RPMI medium containing 10% FBS until confluence.

[0179] b) To produce recombinant EBV, producer cells are transfected with a plasmid expressing a transgene that induces EBV lytic cycle and viral shedding.

[0180] c) Harvesting the supernatant containing the recombinant EBV.

[0181] d) Virus titer was determined by an assay involving infection of susceptible lymphoma cells using serial dilutions of harvested supernatant and counting GFP positive (green) cells. EBV stocks can be stored at 4°C for >1 year.

[0182] Lentiviral transduction of feeder cells

[0183] Exogenous expression was achieved by lentiviral transduction of U2OS cells. A lentiviral expression vector with a puromycin selection cassette ( Figure 1A) clone mega CD40L and generate lentiviral particles. Two days after transduction, 1 μg / ml puromycin was used for selection. Full-length CD23b expression was achieved by lentiviral transduction of U2OS cells expressing mega CD40L / puromycin. A lentiviral expression vector with a blasticidin selection cassette ( Figure 1B ) to clone CD23b and produce viral particles. Two days after lentiviral transduction, blasticidin was added to the culture medium at a concentration of 10 μg / ml. The feeder cell line also expressed the fluorescent protein mCherry for easy identification, which was achieved by a custom lentiviral vector with a neomycin resistance gene ( Figure 1C Two days after transduction, selection was performed using 250 μg / ml of G418.

[0184] Cultivation and application of Amalthea feeder cells

[0185] a) Amalthea cells were adherent cells grown in RPMI medium containing 10% FBS.

[0186] b) To support the growth of LCLs, they were irradiated the day before to retard their growth and prevent them from becoming dominant in the culture. 137 Cs gamma ray irradiator, irradiation dose is 30Gy. Alternatively, X-ray irradiator can be used. Alternatively, transient mitomycin C treatment can be used.

[0187] c) After irradiation, the cells were counted and seeded into the cell culture vessel of choice. To form a confluent monolayer, they were plated at 8.3*10 4 cells / cm 2 The density of inoculation.

[0188] Methods for MAB separation

[0189] a) Select live, class-switched B cells with specificity for the antigen using FACS and an appropriate panel of markers. The cells are always kept at 4°C. This is a well-established procedure in the art and is adapted for each specific antigen.

[0190] b) The sorted cells were infected with recombinant EBV by mixing the sorted cells with medium containing EBV at an MOI of 50 and incubating at 37°C for 3 hours.

[0191] c) Wash and resuspend the cells with 0.5 ml RPMI medium containing 20% ​​fetal bovine serum (FBS).

[0192] d) The resuspended cells were added to the wells of a 48-well culture plate containing feeder cells that had been irradiated the day before and added to the wells in 0.5 ml of RPMI medium containing 20% ​​FBS (enough time for the cells to settle, attach and produce the appropriate recombinant protein for their feeding function). CpG ODN2006 (Invivogentlrl-2006) was added to a final concentration of 2.5 μg / ml.

[0193] e) The infected cells were grown in bulk for 7 days.

[0194] f) After the infected cells have grown for 7 days (now LCL), single live mCherry negative (non-feeder) cells are placed into wells of a 96-well culture plate using FACS. The cell culture wells contain 2.5*10 50 μl RPMI (containing 20% ​​FBS) prepared the day before. 4 Each culture is now a single clone.

[0195] g) Add 50 μl of fresh RPMI (containing 20% ​​FBS and CpG ODN 2006 at a concentration of 5 μg / ml) to each well. The final concentration of CpG ODN 2006 is 2.5 μg / ml.

[0196] h) The culture was fed twice a week by changing 50 μl of medium. Only at the first feeding the fresh medium contained CpG ODN 2006. The feeding was performed using a pipetting robot (Integra Viaflo96).

[0197] i) After two weeks, the growth of monoclonal LCL cultures was assessed under a fluorescent microscope by the presence of GFP-positive EBV-infected cells. Alternatively, a fluorescent plate reader can be used.

[0198] j) Harvest the supernatant containing the MAB to evaluate the MAB and harvest cells from the overgrown culture to determine the gene sequence of the MAB produced. If a larger amount of the MAB is required for a molecular assay, the monoclonal LCL culture can be cultured continuously and the antibody-containing supernatant harvested as needed.

[0199] Results and Discussion

[0200] Example 1 - Lentiviral transduction of feeder cell lines

[0201] To clone the transgenes of the "Amalthea" feeder cell line (i.e., the feeder cell line of the present invention), the inventors used three different lentiviral vectors, such as Figure 1A , 1Band 1C. The first and second lentiviral expression vectors ( Figure 1A and 1B ) are used to clone mega CD40L and CD23b into feeder cell lines, respectively, to support the growth of B cells producing monoclonal antibodies. A third lentiviral vector is used to clone the fluorescent protein mCherry to facilitate identification of feeder cell lines.

[0202] Example 2 - Amalthea feeder cells improve transformation efficiency

[0203] To test the B cell transformation efficiency of Amalthea feeder cells (i.e., the feeder cell line of the present invention), B cells were isolated from human blood samples using the Miltenyi B cell isolation kit (130-091-151). These cells were infected with recombinant EBV by mixing the cells with the virus stock solution (infection multiplicity (MOI) of 50), incubating for three hours at 37°C and 5% CO2, then washing with RPMI medium and resuspending in RPMI supplemented with 20% fetal bovine serum (FBS). Half of the infected cells were placed in 24-well plates with 2.5*10 5 The other half was placed in a well with 1.75*10 5 irradiated (30 Gy) Amalthea cells as feeder cells. CpG ODN2006 (Invivogen tlrl-2006) was added to all wells at a final concentration of 2.5 μg / ml. Cyclosporin A was added to the wells with allogeneic PBMCs at 1 μg / ml. Feeder cells were irradiated and inoculated one day before infection.

[0204] Two days after infection, flow cytometry was performed to determine the percentage of infected (GFP positive) and activated (CD23 positive) B cells. Two days after infection, cells are activated (13) but proliferation has not yet begun (18). Figure 3 As shown, in the case of Amalthea feeder cells, the number of B cells infected and activated in the process of becoming LCL increased by more than 4 times. Specifically, when supported by PBMC feeder cells, only 15% of the live cells were infected and activated, while when supported by Amalthea feeder cells, 62.8% of the live cells were infected and activated.

[0205] Example 3 - Amalthea feeder cells increase LCL outgrowth by expressing CD23b

[0206] Prepare two 96-well culture plates and place 2.5*10 4irradiated feeder cells were seeded into each well. The feeder cells of one culture plate were U2OS cells expressing exogenous mega CD40L and mCherry (Amalthea precursor cells before the introduction of lentivirus for expressing CD23b). The feeder cells of the other culture plate were Amalthea cells additionally transduced with CD23b lentivirus. The next day, B cells were isolated and infected with recombinant EBV as described above. Viable cells were counted and serially diluted in RPMI medium containing 20% ​​FBS, so that an average of two viable cells were placed in each well of a 96-well culture plate containing feeder cells expressing or not expressing CD23b. RPMI medium was supplemented with CpG ODN2006 as described above. The cultures were grown for two weeks and fed on days 2, 6 and 12 by replacing 50 μl of medium, and CpGODN2006 was supplemented again at the first feeding. On day 14, the growth of LCLs was assessed by fluorescence microscopy and the percentage of wells with growth was plotted ( Figure 4 ). In both experiments, the number of outgrowing LCL cultures increased by approximately 20% when Amalthea feeder cells expressing CD23b were used.

[0207] Example 4 - Amalthea feeder cells are easily distinguished from MAB-producing B cells

[0208] Amalthea feeder cells were stably transduced with lentivirus to express mCherry and neomycin resistance genes. Selection was performed using 2 mg / ml of G418, and flow cytometry was used to confirm that almost all cells were mCherry positive ( Figure 5 ). mCherry expression in Amalthea cells allows them to be easily distinguished from mCherry-negative LCLs, so even when using non-recombinant, non-GFP-expressing EBV, it is not necessary to stain new LCLs prior to FACS for single B-cell cloning. This is a significant advantage, as additional staining steps can result in loss of target cells.

[0209] Example 5 - Amalthea feeder cells can be used without irradiation

[0210] Amalthea feeder cells do not express the hygromycin resistance gene, whereas LCL infected with recombinant EBV do. This means that when using recombinant EBV, when feeder cells are no longer needed, hygromycin can be used to kill feeder cells in the co-culture without affecting the hygromycin-resistant LCL cells producing antibodies. This makes the platform flexible enough to be used in situations where no irradiator is used to inhibit feeder cells and drug treatment is not appropriate.

[0211] Example 6 - Amalthea feeder cells promote confirmed single B cell clones

[0212] B cells were first sorted into wells of a 96-well plate using indexed single-cell FACS for B cell (LCL) culture cloning. Antigen-specific, class-switched B cells were infected with recombinant EBV and cultured in bulk for the first seven days on irradiated (30 Gy) Amalthea feeder cells that had been cultured the day before at 1.75*10 5 Cells were seeded into wells of a 24-well plate. Seven days later, cells from early bulk cultures (new LCLs and Amalthea feeder cells) 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 ). The outermost wells were filled with sterile PBS to prevent evaporation of the culture wells. As described above, the sorted cells were cultured in 96-well plates for two weeks and then the LCL growth in the wells was evaluated. In two independent experiments, 540 and 1500 cells were sorted in separate wells, respectively, of which growth was observed in 198 and 273 wells (Table 1).

[0213] Example 7 - LCLs expressing GFP can be easily visualized for overgrowth assessment

[0214] Single-cell sorted cultures can be tracked and evaluated throughout their outgrowth period ( Figure 7 ), thereby easily and accurately identifying overgrowing cultures and allowing for forward planning without culture loss. The platform has the potential to automate overgrowth detection with a fluorescent plate reader, greatly increasing yield potential.

[0215] Example 8 - MAB against HIV envelope protein

[0216] The following example demonstrates the effectiveness of the present invention in isolating useful MABs. The yield of the method is very high and after three weeks the supernatant containing antigen specific MABs can be used in a variety of assays, here shown for ELISA as well as neutralization assays. This means that only assay validated MABs are selected for sequence analysis and downstream applications, reducing cost and workload. The present invention is unique in that this is done directly in B cell cultures that have been validated as monoclonal from the outset.

[0217] 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 variants of the immunogen were used, called ConM SOSIP and ConSUFO, which represent two different stable soluble immunogen strategies of the HIV Env-like native configuration.

[0218] PBMCs were isolated by gradient centrifugation and live, class-switched B cells specific for the immunogen were sorted by FACS. To sort immunogen-specific B cells, two probes were made containing the protein sequence of ConM SOSIP or ConS UFO fused to superfolded GFP (sfGFP) or mScarlet-I fluorescent protein, respectively (ConM-GFP and ConS-Scarlet). Figure 8 B cells with specificity for ConM-GFP and / or ConS-Scarlet were sorted as indicated.

[0219] As described above, the sorted cells were infected with recombinant EBV and co-cultured with Amalthea feeder cells for seven days to generate proliferating LCLs from antigen-specific B cells. Figure 6 Gating strategy shown, LCL cells were single-cell sorted into 96-well plates and co-cultured with Amalthea feeder cells.

[0220] 326 monoclonal LCL cultures were grown and all supernatants and cells were harvested. The supernatants were tested for the presence of human IgG antibodies by ELISA and all supernatants were found to be antibody positive with concentrations ranging from 100-10000 ng / ml, with most antibody concentrations being 1-2 μg / ml ( Fig. 9 A). Separate ELISAs were performed on the same supernatants to assess specificity for ConMSOSIP and ConS UFO, with the vast majority showing binding to at least one immunogen. The ratio of the concentration of ConMSOSIP or ConS UFO in the supernatant that bound to IgG provided a readout of antibody affinity for each immunogen ( Fig. 9 B and 9C). The affinity information of each monoclonal antibody generated helps to select 12 monoclonal antibodies with the best affinity level and breadth, and determine their sequences for further study (Table 2). Antibody sequences are determined using techniques widely used in the art. In brief, harvested LCL cells are frozen at -80°C in a lysis buffer containing 0.01M Tris pH 8.0 and Ribolock RNase inhibitor (ThermoFisher EO0381). cDNA is produced from the mRNA of the cell by reverse transcription using SuperScript IV reverse transcriptase (ThermoFisher 18090200) and random hexamer (ThermFisher N8080127). PCR amplification (4) is performed using a primer pool with specificity for human antibodies and PCR products are sequenced by Sanger sequencing, showing the MAB sequences.

[0221] Example 9 - Neutralizing MAB against SARS CoV2

[0222] Blood samples were obtained from recovered COVID-19 patients and PBMCs were isolated by gradient centrifugation. Soluble SARS-CoV-2 MYC marker protein (19) was produced and used as a probe to sort antigen-specific B cells ( Fig.10 ). As described above, recombinant EBV was used to infect sorted SARS-CoV-2-specific B cells and generate monoclonal LCL cultures. Two weeks after single cells were sorted into wells, supernatants of monoclonal cultures were harvested from 75 96-well plates. Supernatants were used directly in standard neutralization assays for SARS-CoV-2 pseudoviruses as described in (19) ( Fig.11 ). Several neutralization supernatants with different neutralization potencies were identified. For the best neutralizer, the antibody sequence was determined as described in Example 8, and more antibodies were generated and purified for more detailed studies. For example, Fig.12 A comprehensive neutralization study of two antibodies isolated and purified by the inventors and a control group of antibodies isolated by others is shown. The inventors' D2S15 MAB, tested at higher dilutions, was superior to the other antibodies in neutralization.

[0223] in conclusion

[0224] As shown in all the examples, the inventors unexpectedly discovered that the inventors' Amalthea feeder cell line increased the efficiency of EBV-mediated B cell transformation / immortality by more than 4-fold compared to the classic prior art EBV method. Specifically, the inventors have determined that modifying the feeder cell line to express mega CD40L and / or CD23 can produce a feeder cell line that is capable of significantly increasing the number of outgrowing monoclonal antibody-producing B cells.

[0225] Furthermore, using the inventors' novel feeder cell lines, it is now possible for the first time to support monoclonal overgrowth of EBV-infected cells without the need for a subcloning step, saving weeks and greatly improving efficiency compared to the classical EBV method for monoclonal antibody isolation. In addition, the supernatant of the resulting monoclonal culture contains antibodies that can be evaluated without the need for molecular cloning of the antibody sequence, which is required by the most popular methods currently. This reduces the cost of evaluating each antibody by 50 to 100 times compared to the currently preferred single-cell molecular cloning method.

[0226] Finally, by modifying feeder cell lines to express a fluorescent protein that is not expressed by B cells, the inventors demonstrated that feeder cells and B cells can be distinguished without any additional staining steps that would result in loss of target cells.

[0227] References

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Claims

1. A feeder cell line, wherein the feeder cell line is used to culture B cells that produce monoclonal antibodies, and the feeder cell line expresses: - 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.

2. The feeder cell line according to claim 1, wherein The feeder cell line is 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 an endothelial cell line, and the feeder cell line is preferably an osteosarcoma cell line.

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

4. A feeder cell line according to any one of the preceding claims, wherein The feeder cell line is irradiated. Preferably, the feeder cell line is irradiated. 137 Cs gamma ray irradiator or X-ray irradiator; or, the feeder cell line is transiently treated with mitomycin C.

5. A feeder cell line according to any one of the preceding claims, wherein The mega CD40L or variant or fragment thereof comprises an amino acid sequence substantially as shown in SEQ ID No: 1 or a fragment or variant thereof, and / or the mega CD40L or variant or fragment thereof is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 2 or a fragment or variant thereof.

6. A feeder cell line according to any one of the preceding claims, wherein The feeder cell line (i) expressing 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 cell culture medium; (ii) expressing 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; (iii) expressing at least 51 ng / ml of mega CD40L or a variant or fragment thereof in cell culture medium; and / or (iv) expressing at least 101 ng / ml of mega CD40L or a variant or fragment thereof in cell culture medium.

7. A feeder cell line according to any one of the preceding claims, wherein The CD23 or the fragment or variant thereof expressed by the feeder cell line is CD23a or CD23b.

8. A feeder cell line according to any one of the preceding claims, wherein The feeder cell line expresses CD23b, preferably, CD23b comprises an amino acid sequence substantially as shown in SEQ ID No: 3 or a variant or fragment thereof, and / or CD23b is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 4 or a fragment or variant thereof.

9. The feeder cell line according to any one of claims 1 to 7, wherein The feeder cell line expresses CD23a, preferably, CD23a comprises an amino acid sequence substantially as shown in SEQ ID No: 5 or a variant or fragment thereof, and / or CD23a is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 6 or a fragment or variant thereof.

10. A feeder cell line according to any one of the preceding claims, wherein The feeder cell line expresses a first fluorescent protein and the B cells express a second fluorescent protein, wherein the first fluorescent protein is different from the second fluorescent protein.

11. A feeder cell line according to any one of the preceding claims, wherein The B cells expressed enhanced green fluorescent protein (GFP).

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

13. A feeder cell line according to any one of the preceding claims, wherein The feeder cell line expresses mCherry.

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

15. A feeder cell line according to any one of the preceding claims, wherein The monoclonal antibody-producing B cells are cultured using Epstein-Barr virus (EBV), and the EBV is preferably a recombinant EBV.

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

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

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

19. Use of the feeder cell line according to any one of claims 1 to 17 for isolating B cells producing monoclonal antibodies.

20. A method for culturing B cells that produce monoclonal antibodies, the method comprising: (i) contacting B cells with a feeder cell line according to any one of claims 1 to 17; as well as (ii) culturing the B cells and the feeder cell line under conditions that support the growth of the monoclonal antibody-producing B cells.

21. A method for isolating B cells producing monoclonal antibodies from a cell culture medium, the method comprising: (i) contacting B cells with a feeder cell line according to any one of claims 1 to 17; as well as (ii) Identifying a feeder cell line expressing a fluorescent protein that is not expressed by B cells to isolate the monoclonal antibody-producing B cells.

22. A method for isolating B cells producing monoclonal antibodies from a cell culture medium, the method comprising: (i) contacting B cells with the feeder cell line according to claim 16 or 17; as well as (ii) culturing the B cells and the feeder cell line in the presence of a drug selection marker to isolate the monoclonal antibody-producing B cells.

23. The method according to any one of claims 20 to 22, wherein: The method further comprises isolating the monoclonal antibody from the monoclonal antibody-producing B cells.

24. A method for isolating a monoclonal antibody from a B cell producing the monoclonal antibody, the method comprising: (i) contacting B cells with a feeder cell line according to any one of claims 1 to 17; (ii) culturing the B cells and the feeder cell line under conditions that support the growth of the monoclonal antibody-producing B cells; and (iii) isolating monoclonal antibodies from the monoclonal antibody-producing B cells.

25. The method according to any one of claims 20 to 24, wherein: The method further comprises isolating B cells from a sample obtained from the subject, the B cells preferably being specific for the antigen of interest.

26. The method according to any one of claims 20 to 25, wherein: The method further comprises contacting the B cell with a CpG oligonucleotide, preferably a nuclease resistant phosphorothioate oligonucleotide.

27. The method according to claim 26, wherein: The CpG oligonucleotide comprises a nucleotide sequence substantially as shown in SEQ ID No: 12 or a fragment or variant thereof.

28. A lentiviral vector substantially as shown in Figure 1A, 1B or 1C.

29. A kit for culturing B cells that produce monoclonal antibodies, the kit comprising a feeder cell line that expresses: (a) mega CD40 ligand (mega CD40L) or a variant or fragment thereof; (b) CD23 or a variant or fragment thereof; and / or (c) Fluorescent proteins not expressed by B cells.

30. The kit according to claim 29, for carrying out the method according to any one of claims 20 to 27, and / or for using the feeder cell line according to any one of claims 1 to 17.

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