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Methods of producing stable B-lymphocytes

a technology of b-lymphocytes and stable cells, applied in the field of improving the survival and stability of cells, can solve the problems of unstable hybridoma generation, inefficient method, and loss of a large number of cells

Inactive Publication Date: 2007-03-22
IQ THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] The invention is based on the discovery that repeated oligoclonal handpicking unexpectedly improves one or more characteristics of antibody-producing cell (e.g., cells within an immortalized population). For example, oliohandpicking results in B-lymphocytes that are more stable (i.e., more likely to survive (e.g. survive and or proliferate for a longer period in culture). Antibody producing cells that survive and or proliferate longer and more robustly produce more antibody as a result.
[0014] In some embodiments, the present methods will produce more stable populations of B-lymphocytes that produce antibodies that specifically bind a Staphylococcus or Staphylococcus antigen. The methods can also produce monoclonal antibodies that specifically bind a Staphylococcus or Staphylococcus antigen.

Problems solved by technology

However, this method is rather inefficient.
A great number of the cells are lost.
A major problem for the generation of human monoclonal antibodies is the fact that the hybridoma's generated are unstable in that they die, lose the ability to secrete antibody or stop proliferating shortly after the immortalization procedure.
Moreover, with traditional techniques only spleen cells can be used as the source of B-cells, because fusions with lymph node cells or peripheral blood cells only yield too limited a number of hybridomas or are not possible at all.

Method used

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  • Methods of producing stable B-lymphocytes

Examples

Experimental program
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Effect test

example 1

General Methods

[0073] Reagents

[0074] Culture medium DMEM / HAM's F12 (Cambrex Biosciences 12-719F) is prepared with 1300 mg / l sodium bicarbonate (Merck), 55 mg / l sodium pyruvate (Fluka), 2.3 mg / l 2-mercaptoethanol (Merck). 60 mg / l Gentamycin (Sigma), and 8% Fetal Bovine Serum (Wisent). In fusion experiments, the medium is further supplemented with 13.61 mg / l hypoxanthine (Fluka) and 3.83 mg / l thymidine (Fluka). This medium is referred to as DMEM / HAM's F12 / HT.

[0075] Selection of hybridomas is performed in DMEM / HAM's F12 / HT supplemented with 0.0004 mM aminopterin (Sigma) and optionally 1% of IL-6 containing supernatant of a human bladder carcinoma cell line T24 (T24CM). The selection medium is referred to as HAT-medium. Fusion medium: Ready to use hypo-osmotic buffer (Eppendorf AG)

[0076] Cell Cultures

[0077] Mutant EL-4 thymoma cells, EL-4 / B5 are routinely cultured in DMEM / HAM's F12 supplemented with 8% FCS) at cell concentrations between 1×104 to 1×106 c / ml. If the cells exceed a d...

example 2

EBV Transformation of Enriched Human B-Cells Isolated From an Anthrax-Vaccinated Donor Results in the Generation of a Stable B-Cell Culture Specific for Protective Antigen (PA)

[0105] Enriched B-cells from an Anthrax vaccinated donor were used to produce a stable B-cell culture producing monoclonal antibodies for Protective Antigen. In four independent experimental protocols as outlined below enriched B-cell isolated were seeded on 24 well plates with 3T6.CD40L cells. [0106] Protocol 1: Cells (5,000-10,000) were cultured on 100,000 3T6.CD40L cells / well in a 24 wells plate. Cells were EBV transformed immediately, by incubating with an adequate EBV stock. The EBV supernatant was replaced by standard growth medium plus IL-4 (10 ng / mL) after 2.5 hours. The B-cells were cultured in this medium for 2 to 3 weeks. [0107] Protocol 2: Cells (5,000-10,000) were cultured on 100,000 3T6.CD40L cells / well in a 24 wells plate and expanded for 3 days before EBV transformation. The EBV supernatant wa...

example 3

Handpicking of Fused Enriched Human B-cells Isolated from Anthrax Vaccinated Donor Results in of Production Antigen Specific Stable B-Cell Cultures

[0114] Enriched B-cells from an Anthrax vaccinated donor were panned as described above in a 6 wells plate coated with the desired antigen (Ag) Unbound cells were washed away and discarded, bound cells were removed from the plate by trypsin treatment (Protocol 1) or were left on the plate (Protocol 2). [0115] Protocol 1. Trypsin treated cells (1,000-1,500 cell per well) were added to 96-wells microtiter plates having 10,000 3T6.CD40L cells / well and IL4 (10 ng / mL). After 3 days of expansion, the wells were tested for anti-Ag antibody production. Cells from wells scoring positive for antibody were fused to K6H6 / B5 myeloma cells in a 1:1 ratio. Fusion either occurred by electrofusion or PEG fusion as described above). After the fusion, the fusion products are seeded into microtiter plates in a density of 10,000-20,000 cells / well and left in...

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Abstract

The invention provides a method of producing a stable lymphocyte culture and methods of producing monoclonal antibodies.

Description

RELATED APPLICATIONS [0001] This application claims priority to U.S. Ser. No. 60 / 710,626, filed Aug. 23, 2005 which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] The invention relates to improving the survival and stability of cells after an immortalization procedure. BACKGROUND OF THE INVENTION [0003] The standard procedure for the generation of monoclonal antibodies as originally described by Köhler and Milstein in 1975 (Nature 256, 495-497) involves the fusion of sensitized murine spleen cells with murine myeloma cells in the presence of polyethyleneglycol (PEG). However, this method is rather inefficient. Usually, at best only one B-cell at 2×105 spleen cells successfully fuses. A great number of the cells are lost. [0004] A major problem for the generation of human monoclonal antibodies is the fact that the hybridoma's generated are unstable in that they die, lose the ability to secrete antibody or stop proliferating shortly after the immorta...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/08C12N5/08C07K16/12
CPCC07K16/00C12N5/163C07K16/1271
Inventor GROEN, HERMANWESTRA, HANS H.
Owner IQ THERAPEUTICS
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