Antibodies and methods for generating genetically altered antibodies with enhanced effector function

Abstract

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within immunoglobulin genes directed against an antigen of interest to produce altered antibodies with enhanced biochemical activity. Moreover, these methods are useful for generating antibody-producing cells with increased level of antibody production. The invention also provides methods for increasing the effector function of monoclonal antibodies and monoclonal antibodies with increased effector function.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This claims the benefit of U.S. Provisional Application 60 / 491,310, filed Jul. 29, 2003, the entire contents of which are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The invention is related to the area of antibody effector function and cellular production. In particular, it is related to the field of mutagenesis. BACKGROUND OF THE INVENTION [0003] The use of antibodies to block the activity of foreign and / or endogenous polypeptides provides an effective and selective strategy for treating the underlying cause of disease. For example, monoclonal antibodies (MAb), such as the FDA-approved ReoPro (Glaser, V. (1996) “Can ReoPro repolish tarnished monoclonal therapeutics?”Nat. Biotechnol. 14:1216-1217), an anti-platelet MAb from Centocor; Herceptin (Weiner, L. M. (1999) “Monoclonal antibody therapy of cancer”Semin. Oncol. 26:43-51), an anti-Her2 / neu MAb from Genentech; and Synagis (Saez-Llorens, X. E., et al. (1998) “Safe...

AI Technical Summary

Benefits of technology

[0012] The invention provides methods for generating genetically altered antibodies (including single chain molecules) and antibody producing cell hosts in vitro and in vivo, whereby the antibody possesses a desired biochemical property(s), such as, but not limited to, increased antigen binding, increased gene expression, enhanced effector function and / or enhanced extracellular secretion by the cell host. One method for identifying antibodies with increased binding activity or cells with increased antibody production is through the screening of MMR defective antibody producing cell clones that produce molecules with enhanced binding properties, enhanced effector function such as (but not limited to) antibody-dependent cellular cytotoxicity (ADCC), or clones that have been genetically altered to produce enhanced amounts of antibody product.

[0020] Other aspects of the invention encompass methods of reversibly altering the hypermutability of an antibody producing cell, in which an inducible vector containing a dominant negative allele of a mismatch repair gene operably linked to an inducible promoter is introduced into an antibody-producing cell. The cell is treated with an inducing agent to express the dominant negative mismatch repair gene (which can be PMS2 (preferably human PMS2), MLH1, or PMS1). Alternatively, the cell may be induced to express a human mutL homolog or the first 133 amino acids of hPMS2. In another embodiment, the cells may be rendered capable of producing antibodies by co-transfecting a preselected immunoglobulin gene of interest. The immunoglobulin genes of the hypermutable cells, or the proteins produced by these methods may be analyzed for desired properties, and induction may be stopped such that the genetic stability of the host cell is restored.

[0027] In another embodiment of the invention, a method is provided for introducing a mutation into an endogenous gene encoding for an immunoglobulin polypeptide or a single chain antibody. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction and expression of the MMR gene allele. The cell further comprises an immunoglobulin gene of interest. The cell is grown and tested to determine whether the gene encoding for an immunoglobulin or a single chain antibody of interest harbors a mutation. In another aspect of the invention, the gene encoding the mutated immunoglobulin polypeptide or single chain antibody may be isolated and expressed in a genetically stable cell. In a preferred embodiment, the mutated antibody is screened for at least one desirable property such as, but not limited to, enhanced binding characteristics.

[0034] These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in cells and animals as well as providing cells and animals harboring potentially useful mutations for the large-scale production of high affinity antibodies with beneficial pharmacokinetic profiles.

Problems solved by technology

A common problem that exists during the “humanization” of rodent-derived MAbs (referred to hereon as HAb) is the loss of binding affinity due to conformational changes in the 3-dimensional structure of the CDR domain upon grafting onto the human Ig backbone (U.S. Pat. No. 5,530,101 to Queen et al.).

This process is a very time consuming procedure that involves the use of expensive computer modeling programs to predict changes that may lead to a high affinity HAb.

In some instances, the affinity of the HAb is never restored to that of the MAb, rendering them of little therapeutic use.

Another problem that exists in antibody engineering is the generation of stable, high yielding producer cell lines that are required for manufacturing of the molecule for clinical materials.

In all cases, the generation of a cell line that is capable of generating sufficient amounts of high affinity antibody poses a major limitation for producing sufficient materials for clinical studies.

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