Multivalent and multispecific binding proteins and their use

A dimer of

• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker having a length selected from the group consisting of 9, 8, 7 and 6 amino acids;
• and
• (ii) a second polypeptide which has a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site.

A dimer of

• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker;
• and
• (ii) a second polypeptide which has a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker having a length selected from the group consisting of 9, 8, 7 and 6 amino acids;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site;
• the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site.

A dimer of

• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 6 amino acids;
• and
• (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 6 amino acids;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site.

A dimer of

• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 7 amino acids;
• and
• (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 7 amino acids;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site.

A population of dimers wherein each dimer is of

• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker;
• and
• (ii) a second polypeptide which has a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site;
• the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site, wherein said population has a diverse repertoire of antigen binding activities.

An assay for an antigen using a dimer comprising the steps:

• (a) incubating the dimer and an antigen, wherein said antigen binds to either or both of the first and second antigen binding sites of the dimer, under conditions which permit binding of the dimer to the antigen, and
• (b) detecting the antigen-dimer binding using surface plasmon resonance;
• said dimer being of
• (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker;
• and
• (ii) a second polypeptide which has a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker;
• the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site;
• and
• the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
• said first antigen binding site being different from said second antigen binding site;
• the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site.