Monoclonal antibody isolation
AssigneeImperial College Innovations Limited
InventorsKonstantinos PASCHOS, Robin SHATTOCK
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Abstract
The invention relates to monoclonal antibody production and isolation, and particularly, although not exclusively, to a novel feeder cell line for culturing a monoclonal antibody-producing B cell. The invention also extends to the use of the feeder cell line in culturing a monoclonal antibody-producing B cell, and isolating the B cell from the cell culture media. The invention further extends to methods for culturing and isolating a monoclonal antibody-producing B cell, as well as a method for isolating a monoclonal antibody.
Claims30
A feeder cell line for culturing a monoclonal antibody-producing B cell, the feeder cell line expressing:
- mega CD40 ligand (mega CD40L), or a variant or fragment thereof;
- CD23, or a variant or fragment thereof;
- and/or
- a fluorescent protein that is not expressed by the B cell.
The feeder cell line according to any one of the preceding claims, wherein the feeder cell line is irradiated, preferably wherein the feeder cell line is irradiated using a 137Cs γ-ray irradiator or an X-ray irradiator, or wherein the feeder cell line is treated transiently with Mitomycin C.
The feeder cell line according to any one of the preceding claims, wherein the mega CD40L, or a variant or fragment thereof comprises an amino acid sequence substantially as set out in SEQ ID No:
- 1, or a fragment or variant thereof, and/or wherein the mega CD40L, or a variant or fragment thereof is encoded by a nucleotide sequence substantially as set out in SEQ ID No:
- 2, or a fragment or variant thereof.
The feeder cell line according to any one of the preceding claims, wherein the feeder cell line expresses
- (i) 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 a cell culture media;
- (ii) at least 10 ng/ml, at least 20 ng/ml, at least 30 ng/ml, at least 40 ng/ml, or at least 50ng/ml of mega CD40L, or a variant or fragment thereof, in a cell culture media;
- (iii) at least 51 ng/ml of mega CD40L, or a variant or fragment thereof, in a cell culture media;
- and/or
- (iv) at least 101 ng/ml of mega CD40L, or a variant or fragment thereof, in a cell culture media.
The feeder cell line according to any one of the preceding claims, wherein the CD23 or fragment or variant thereof, expressed by the feeder cell line, is CD23a or CD23b.
The feeder cell line according to any one of the preceding claims, wherein the feeder cell line expresses CD23b, preferably wherein CD23b comprises an amino acid sequence substantially as set out in SEQ ID No:
- 3, or a variant or fragment thereof, and/or wherein CD23b is encoded by a nucleotide sequence substantially as set out in SEQ ID No:
- 4, or a fragment or variant thereof.
The feeder cell line according to any one of the preceding claims, wherein the feeder cell line expresses a first fluorescent protein, and the B cell expresses a second fluorescent protein, wherein the first and second fluorescent proteins are different.
The feeder cell line according to any one of the preceding claims, wherein the B cell expresses enhanced green fluorescent protein (GFP).
The feeder cell line according to any one of the preceding claims, wherein the feeder cell line expresses mCherry.
The feeder cell line according to any one of the preceding claims, wherein the monoclonal antibody-producing B cell is cultured using Epstein-Barr virus (EBV), preferably wherein the EBV is recombinant EBV.
A lentiviral vector substantially as illustrated in FIG. 1A, 1B or 1C.
A kit for culturing a monoclonal antibody-producing B cell, the kit comprising a feeder cell line expressing:
- (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 that is not expressed by the B cell.
Dependencies are inferred from the claim text.