Title:  Multivalent and multispecific antigen-binding protein

United States Patent:  6,239,259

Assignee:  Unilever Patent Holdings B.V. (Vlaardingen, NL)

Filed:  December 28, 1998

PCT NO:  PCT/EP97/01609

102(e) Date:  December 28, 1998

PCT PUB. Date:  October 16, 1997

Foreign Application Priority Data:  Apr 04, 1996[EP] (96302412)

A multivalent antigen-binding protein comprises a first polypeptide comprising, in series, three or more variable domains of an antibody heavy chain and a second polypeptide comprising, in series, three of more variable domains of an antibody light chain, said first and second polypeptides being linked by association of the respective heavy chain and light chain variable domains, each associated variable domain pair forming an antigen binding site. Methods for their production and uses thereof, in particular for therapeutic and diagnostic applications, are disclosed.

SUMMARY OF THE INVENTION

According to the present invention there is provided a multivalent antigen binding protein comprising:

a first polypeptide comprising in series, three or more variable domains of an antibody heavy chain; and

a second polypeptide comprising, in series, three or more variable domains of an antibody light chain,

said first and second polypeptides being linked by association of the respective heavy chain and light chain variable domains, each associated variable domain pair forming an antigen binding site.

As used herein, the term multivalent means more than one antigen binding site.

Preferably the first polypeptide comprises three variable domains of an antibody heavy chain and the second polypeptide comprises three variable domains of an antibody light chain, providing a trivalent protein.

It will be appreciated that the polypeptides may comprise heavy or light chains, variable domains, as appropriate, or functional equivalents thereof.

The respective heavy or light-chain variable domains may suitably be linked without any intervening linker. According to a preferred embodiment, however, the variable domains contained in the individual polypeptides are linked by peptide linkers. Preferably the peptide linker is flexible, allowing the variable domains to flex in relation to each other such that they can bind to multiple antigenic determinants simultaneously. It will be appreciated that the binding of the linker to the individual heavy or light chain variable domains will be such that it does not affect the binding capacity of the binding site formed by the associated variable domain pair. Conveniently the peptide linker comprises from 16 to 19 amino acid residues. A preferred, peptide linker for heavy chain domains is (Gly₄ Ser)₃ AlaGlySerAla (residues numbered 121-139 of SEQ ID NO:27) and for the light chain domains is (Gly₄ Ser)₃ Val.

It will be appreciated that if two or more of the associated variable domain pairs (V_H/V_L pairs) have the same antigen specificity, for example if they are derived from the same parent antibody or fragment thereof or from different antibodies which bind the same epitope, then a binding protein which binds more than one molecule of the same type will be produced.

According to one embodiment, where the binding protein according to the invention comprises three antigen binding sites which are able to bind different epitopes from each other, a trivalent trispecific protein is produced.

In another embodiment, where the binding protein according to the invention comprises three associated variable domain pair binding sites, two of which sites bind the same epitopes, a trivalent, bispecific protein is provided. Where all three binding sites have the same antigen specificity, a trivalent, monospecific binding protein is provided.

The invention also provides nucleotide sequences coding for the polypeptides of the multivalent antigen binding protein according to the invention and cloning and expression vectors containing such nucleotide sequences.

The invention further provides host cells transformed with vectors containing such nucleotide sequences and methods of producing such polypeptides by expression of the nucleotide sequences in such hosts.

The invention further provides a process for preparing a multivalent antigen binding protein as set forth above comprising:

(i) transforming one or more hosts by incorporating genes encoding said first and second polypeptides;

(ii) expressing said genes in said host or hosts;

(iii) allowing said first and second polypeptides to combine to form the antigen binding protein.

Suitably the host or hosts may be selected from prokaryotic bacteria, such as Gram-negative bacteria, for example E. coli, and Gram-positive bacteria, for example B. subtilis or lactic acid bacteria, lower eukaryotes such as yeasts, for example belonging to the genera Saccharomyces Kluyveromyces or Trichoderma, moulds such as those belonging to the genera Aspergillus and Neurospora and higher eukaroytes, such as plants, for example tobacco, and animal cells, examples of which are myeloma cells and CHO, COS cells and insect cells. A particularly preferred host for use in connection with the present invention is COS (monkey kidney) cells.

Techniques for synthesising genes, incorporating them into hosts and expressing genes in hosts are well known in the art and the skilled person would readily be able to put the invention into effect using common general knowledge. Proteins according to the invention may be recovered and purified using conventional techniques such as affinity chromatography, ion exchange chromatography or gel filtration chromatography.

The activity of the multivalent binding proteins according to the invention may conveniently be measured by standard techniques known in the art such as enzyme-linked immunosorbant assay (ELISA), radioimmune assay (RIA) or by using biosensors.

The multivalent antigen binding proteins of the present invention may suitably be used in diagnostics or therapy for example in targeting a tumour cell with natural killer cells and cytotoxic agent. Other uses for which the multivalent binding proteins according to the invention are useful include those uses for which antibodies or fragments thereof are commonly used, including for immunoassays of a test sample and in purification. According to a particular preferred embodiment, multi-enzyme complexes may be assembled, at a target, for example a cell surface. As an illustration, multivalent binding proteins according to the invention may be used to target cell killing enzymes such as an oxidase (for example glucose oxidase) and peroxidase (for example horseradish peroxidase) to a target species which is an antigenic component of dental plaque, such as S. sanguis or S. mutans. Complexes comprising enzyme, coenzyme and target antigen may also conveniently be assembled.

Accordingly, the invention also provides compositions comprising the multivalent antigen binding proteins according to the invention, conveniently in combination with a cosmetically or pharmaceutically acceptable carrier, diluent or excipient. Methods of treatment using the multivalent antigen binding proteins according to the invention are also provided.

For use in diagnosis or therapy, the multivalent antigen binding proteins according to the invention may conveniently be attached to an appropriate diagnostically or therapeutically effective agent or carrier by methods conventional in the art.

An advantage of using multivalents antigen binding proteins according to the invention over multivalent binding proteins prepared by existing techniques known in the art is that the "self-assembling" association of the respective heavy and light chain variable domains to form the multivalent binding sites avoids the need for chemical coupling steps or the introduction of linking residues to stabilise the multivalent constructs, thereby minimising the risk of eliciting an immune response to such molecules when the resulting multivalent binding proteins are used in therapy.

A particular advantage of molecules according to the present invention is that they may conveniently be purified straight from the supernatant using conventional purification techniques. As they are self-assembling, there is no need to purify individual subunits prior to coupling as in existing techniques.

Claim 1 of 11 Claims

What is claimed is:

1. A multivalent antigen binding protein comprising:

a first polypeptide comprising, in series, three or more variable domains of an antibody heavy chain; and

a second polypeptide comprising, in series, three or more variable domains of an antibody light chain,

said first and second polypeptides being linked by association of the respective heavy chain and light chain variable domains, each associated variable domain pair forming an antigen binding site.

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