Antibodies and methods for making and using them

a technology of antigen binding fragments and cell culture, which is applied in the field of cell culture production of can solve the problems of not providing an antibody that can be produced in high yield in cell culture, and reducing the time to prepare humanized antibodies or antigen binding fragments, so as to and improve the yield of antibodies

Inactive Publication Date: 2008-08-07
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]The present invention concerns methods for improving the process of humanizing an antibody or antigen binding fragment and for improving the yield of antibodies or antigen binding fragments in cell culture, especially bacterial cell culture. The invention is based on the discovery that the primary sequence of antibody variable domains can be designed or modified to contribute to correct folding, assembly, and yield of antibodies or antigen binding fragments. The invention involves identifying not only which residues should be substituted but also identifying which substitutions to make at those residues in a more predictable manner to result in improved yield of the antibodies.
[0011]One method for humanizing antibodies involves combining HVRs from a non-human antibody with human consensus framework regions that were derived from the most commonly occurring heavy and light chain subgroups in the sequence compilation of Kabat et al, Sequences of Proteins of Immunological Interest, NIH 1991. It has been discovered that selecting the most commonly occurring heavy and light chain consensus sequences may not provide an antibody that can be produced in high yield in cell culture. In one embodiment, the invention provides a method for selecting a human subgroup consensus sequence for at least one of the framework region or regions based on identifying the subgroup consensus sequence that has the most sequence identity with the HVR1 and / or HVR2 sequence of the non-human antibody. The method may decrease the time to prepare a humanized antibody or antigen binding fragment that can be produced in high yield in cell culture.

Problems solved by technology

It has been discovered that selecting the most commonly occurring heavy and light chain consensus sequences may not provide an antibody that can be produced in high yield in cell culture.
The method may decrease the time to prepare a humanized antibody or antigen binding fragment that can be produced in high yield in cell culture.

Method used

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  • Antibodies and methods for making and using them
  • Antibodies and methods for making and using them
  • Antibodies and methods for making and using them

Examples

Experimental program
Comparison scheme
Effect test

example 1

Heavy Chain Aggregation During Expression and Folding of Antibodies

[0210]Antibodies produced in cell culture can accumulate intracellularly, in the periplasm or in the extracellular medium. Antibody production typically involves expression of the light and heavy chains in the cytoplasm, secretion into the periplasmic space, folding of the light and heavy chains, and assembly of the folded light and heavy chains to form an antibody molecule. Multiple covalent and non-covalent interactions occur between and within the heavy and light chains during these folding and assembly processes. Antibody yield can be greatly affected by the efficiency and fidelity of these processes. Following synthesis of the heavy and light chains, protein aggregation or proteolysis can occur thereby reducing the yield of the antibody. Solubility experiments were carried out in order to assess the efficiency of steps in the antibody production pathway in bacterial cells for the purpose of improving antibody yi...

example 2

Preparation of Anti-VEGF Antibodies with Improved Yield

[0226]The yield of antibodies or antigen binding fragments from cells can be influenced by the in vivo folding and / or assembly of the antibody. In order to increase antibody or antigen binding fragment yield, the sequence of the antibody or fragment was modified and the effect on folding and yield on the antibody was examined. As described in Example 1, aggregation of heavy chains may contribute to a decrease in antibody folding, assembly and yield. Targeted modification of the heavy chain variable region FR1 of anti-VEGF antibodies was performed. Antibody variants with different human variable domain subgroup consensus sequences in the FR1 were prepared and the yield of completely assembled antibody products was examined.

Materials and Methods

A. Preparation of Expression Vectors Encoding Modified Anti-VEGF Antibodies

[0227]To evaluate the effect of different heavy chain FR1 human subgroup consensus sequences on the expression, fo...

example 3

Selection of a Heavy Chain FR1 Sequence for Antibodies Based on Comparison of HVR1 Antibody Sequences to HVR1 Consensus Subgroup I-III Sequences

[0278]Substitution of heavy chain FR1 consensus sequence subgroup III with consensus sequence subgroup I in anti-VEGF antibodies increased assembled antibody yield, presumably due to improved folding efficiency. Framework region sequences that provide for improved yield for other antibodies or antigen binding fragments can be identified based upon identifying the consensus sequence subgroup that has the most sequence identity in the HVR1 region of the variable domain when the HVR1 region of the antibody or fragment is compared to the corresponding HVR1 sequences of each of the subgroup consensus sequences. The method of identifying a heavy chain FR1 sequence for increasing the yield of antibodies based on HVR1 consensus subgroup I-III comparisons was tested for an anti-IgE antibody, E25.

A. Identification of a Heavy Chain FR1 Sequence for Hum...

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Abstract

The present invention provides methods for producing humanized antibodies and increasing the yield of antibodies and / or antigen binding fragments when produced in cell culture. In one aspect of the invention, at least one framework region amino acid residue of the variable domain is substituted by a corresponding amino acid from a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and / or HVR2 amino acid sequence of the variable domain. In another aspect, an amino acid is placed at a position proximal to a cys residue that participates in an intrachain variable domain disulfide bond that corresponds to an amino acid found at that position in a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and / or HVR2 amino acid sequence of the variable domain.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Ser. No. 60 / 442,484, filed Jan. 23, 2003, under 35 U.S.C. 119(e), which application is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The present invention generally concerns the production of antibodies or antigen binding fragments in cell culture. More specifically, the invention provides methods for improving the yield of recombinant antibodies or antigen binding fragments in cell culture.BACKGROUND[0003]Antibodies, particularly humanized antibodies, have become very useful for diagnostic and therapeutic purposes. Humanized antibodies are antibodies in which CDRs or hypervariable regions (HVRs) from a non-human antibody are combined with human framework regions to form an antigen binding molecule. This exchange is sometimes known as a “CDR swap”. There are different ways of selecting human framework sequences for humanized antibodies. One method involves selecting a human variable ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C07K16/00C07K16/22C07K16/42
CPCC07K16/00C07K16/22C07K2317/567C07K16/4291C07K16/42
Inventor SIMMONS, LAURA
Owner GENENTECH INC
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