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Publication Number
US-11952586-B2
Publication Date
2024-04-09
Expiration Date
Abstract
Herein is reported a method for the co-cultivation of single deposited B-cells, which can be of any source, with EL-4 B5 feeder cells in a suitable co-cultivation medium. In the herein reported methods the EL-4 B5 cells have been irradiated with a dose of less than 40 Gy, preferably 9.5 Gy or less. Thereby the EL-4 B5 cells have a higher viability and maintain the ability to divide in cultivation at doses less than 6 Gy.
Core Innovation
The invention describes a method for cultivating one or more B-cells by co-cultivating the B-cells with EL-4 B5 cells. The EL-4 B5 cells are irradiated prior to co-cultivation with a dose of 9.5 Gy or less but greater than 0 Gy, and the method starts the co-cultivation using fewer than 5×10^4 EL-4 B5 cells per B-cell.
The disclosure further refines co-cultivation conditions by using fewer irradiated EL-4 B5 cells per B-cell and by adjusting irradiation dose and the feeder mix composition. A defined cytokine mix includes IL-1β, TNFα, IL-2, IL-10, and IL-6, and feeder mix options include thymocyte cultivation supernatant, SAC, and BAFF, with PMA as an optional additive. The co-cultivation is associated with dependence on the irradiation dose, feeder ratio, and concentration fraction of the cytokine mix in the feeder mix.
The disclosure also describes downstream processing after cultivation from single deposited, FACS-selected, B-cells, including cloning and sequencing of variable domains (VH/VL) and expression in mammalian cells. Antibody generation is supported by selection approaches using markers such as IgG/CD19, IgG/CD38, IgG−/CD138, and IgG+/IgM−, together with IgG-related readouts such as IgG production in terms of IgG-positive wells/clones and IgG concentration.
Claims Coverage
Independent claim clm-00001 covers cultivating one or more B-cells by co-cultivating them with EL-4 B5 feeder cells that are irradiated at >0 Gy and ≤9.5 Gy, while starting the co-cultivation with fewer than 5×10^4 feeder cells per B-cell. The dependent claims further refine the irradiation dose windows, feeder cell number ranges, and feeder mix composition constraints, and can narrow the cultivation to a single deposited B-cell.
Irradiated EL-4 B5 feeders with reduced cell number per B-cell
Co-cultivating one or more B-cells with EL-4 B5 cells that have been irradiated prior to the co-cultivation with a dose of 9.5 Gy or less but greater than 0 Gy, wherein the number of EL-4 B5 cells at the start of the co-cultivating is less than 5×10^4 per B-cell.
Co-cultivating with gamma-irradiated EL-4 B5 cells and feeder mix with IL-1β, TNFα, IL-2, IL-10, IL-6, and PMA
Co-cultivating one or more B-cells with gamma-irradiated EL-4 B5 cells using a feeder mix containing specified concentrations of IL-1β, TNFα, IL-2, IL-10, IL-6, and PMA.
Feeder mix comprising one or more cytokines, SAC, BAFF, and thymocyte cultivation supernatant
A feeder mix comprising one or more specified cytokines, SAC cells, BAFF, and thymocyte cultivation supernatant.
Feeder mix fraction constraint relative to IL-1β, TNFα, IL-2, IL-10, and IL-6
The fraction of the feeder mix is within 1.0 to 0.015 times each concentration of IL-1β, TNFα, IL-2, IL-10, and IL-6.
Cultivating a single deposited B-cell
Cultivating a single deposited B-cell.
Overall, the claims coverage emphasizes co-cultivation using irradiated EL-4 B5 feeder cells at >0 Gy and ≤9.5 Gy with a reduced starting feeder cell number per B-cell, together with dependent claim refinements on specific feeder cell number ranges, irradiation dose windows, and feeder mix composition and constraints involving named cytokines and additives, and further narrows cultivation to a single deposited B-cell.
Stated Advantages
Improved feeder viability.
Higher B-cell IgG productivity, including a higher frequency of IgG+ wells/clones.
Higher IgG concentration.
Documented Applications
Generation of antibodies from single deposited, FACS-selected B-cells, including cloning and sequencing of variable domains (VH/VL) and expression in mammalian cells.
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