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Method for the production of antibodies

a technology of antibody production and composition, applied in the field of antibodies, can solve the problems of limited diversity, specificity and specificity of transgenic mouse-derived human antibodies compared to natural human immune repertoires, and the failure of first-generation murine antibodies which were used in humans

Inactive Publication Date: 2007-11-15
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a method for producing human monoclonal antibodies from non-human animals. The method involves contacting a non-human animal with a human fetal liver stem cell to create an immune-transplanted animal. The animal is then exposed to an antigen, and a human cell that produces the antibody against the antigen is collected. The antibody is then isolated. The method can also involve creating an immortal antibody-producing cell that can be used for multiple passages. The non-human animal used in the method is typically a rodent, such as a mouse or rat. The FL cell used in the method is preferably a human hematopoietic fetal liver stem cell. The invention also includes a method for producing a plurality of human B cells that produce a complete plurality of antibodies against a specific antigen.

Problems solved by technology

However, the first generation of murine antibodies which were used in humans was rather unsuccessful due to human anti-mouse immune responses.
However such transgenic mice are only capable to provide antibodies with a restricted set of human Ig genes—those which result from the transfected immunoglobulin gene array.
However, transgenic mouse-derived human antibodies are limited in their diversity, affinities and specificities compared to natural human immune repertoires.
Similarly, a major drawback of phage or yeast display-based combinatorial library approaches is the random pairing of the antibody heavy and light chains.
In addition, such non-cognate pairs may display unwanted cross-reactivity to human self-antigens.
Finally, the genetic diversity of target-specific antibodies identified by selection and screening of combinatorial libraries is commonly limited due to inherent selection biases.
However, the method of Traggai et al utilized the non-human pseudomonas exotoxin antigen.

Method used

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Examples

Experimental program
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Effect test

example 1

Immunization Procedure of Reconstituted Mice

[0127] Three Rag2− / −γc− / − mice (3 females), were immunized with 100 μg plasmid DNA coding for human HER3 protein. In total up to six immunizations were given intramuscular (i.m.). When serum titers of anti-HER3 were found to be sufficient, mice were additionally boosted three times with 30 μg of purified HER3 proteine in 100 μl PBS intravenously (i.v.) 3,2 and 1 days before fusion.

example 2

Flowcytometric Analysis of Peripheral Blood of Reconstituted Mice on 15th Week after Engraftment

[0128] The mice were isofluran-anaesthized and then peripheral blood was collected from the orbital vene plexus for measuring the positive ratio of human hemocytes by use of flow cytomenty. The collected blood was instantly blended well with EDTA-2Na and kept at room temperature until analysis. An optimum amount of FITC conjugated anti-human CD45 antibodies was added to and reacted with 50 μl of the whole blood at room temperature for 20 minutes. Thereafter, hemolysis and immobilization were effected using FACS solution and the ratio was measured using FACS Calibur. FIG. 1 shows ratio (%) of human CD45 positive cells in peripheral blood of three tested mice.

example 3

Examination of the Production Ability of Human HER3 Antibodies in the Reconstituted Mice

[0129] The amount of total human IgG in pheripheral blood of reconstituted mice was measured by ELISA. Diluted mice serum was coated on MaxiSorb 96 well plate (NUNC) and incubated for lh at room temperature. After blocking with 2% CroteinC solution for 1 h at room temperature washing was performed and thereafter peroxidase conjugated anti-human IgG monoclonal antibodies were added. After incubation for 45 minutes at room temperature washing was performed and ABTS substrate solution was added for 10 minute reaction at room temperature. Then the absorbance was measured at 405 nm (FIG. 2). The IgG concentration was calculated in accordance with the standard curve.

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Abstract

The current invention is related to a method for the production of a human monoclonal antibody from a immundeficient non-human animal, said method comprising contacting a new borne immunodeficient non-human animal with a human fetal liver stem cell (FL cell) to generate an immune transplanted non-human animal (reconstituted animal), subsequently contacting said reconstituted animal with a antigen, collecting from said reconstituted animal a human cell producing human antibody against said antigen, and isolating said antibody from said antibody producing cell.

Description

PRIORITY OF RELATED APPLICATION(S) [0001] This application claims the benefit of European Patent Application No. 06009703.7, filed May 11, 2006, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION [0002] The present invention generally relates to methods for the production of antibodies, compositions of antibody producing cells, methods for their production and uses thereof. BACKGROUND OF THE INVENTION [0003] Monoclonal antibodies (MAB) have long been considered as magic bullets in immunotherapy of cancer, infection or autoimmune disease etc. However, the first generation of murine antibodies which were used in humans was rather unsuccessful due to human anti-mouse immune responses. [0004] Human antibodies have been mostly either derived from transgenic mice harboring a restricted set of human Ig genes or selected from large artificial antibody libraries using phage display, yeast display or similar recombinant technologies. These strategies have been d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C12P21/04C12N5/0781
CPCC07K16/00C07K16/2866C12P21/00C12N5/0635C07K16/32C07K16/18C12N15/63C12N5/0606
Inventor LIFKE, ALEXANDERLIFKE, VALERIAMUELLER-BECKMANN, BERNDSCHNITZER, TOBIAS
Owner F HOFFMANN LA ROCHE & CO AG
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