Methods For The Generation Of Multispecific And Multivalent Antibodies

A method of preparing a bispecific antibody that specifically binds to a first antigen and a second antigen, wherein the first and second antigens are different, the method comprising:

• (a) providing a first nucleic acid molecule encoding a first polypeptide comprising a heavy variable chain region of an immunoglobulin polypeptide or fragment thereof that binds the first antigen, coupled to an immunoglobulin constant region;
• (b) providing a second nucleic acid molecule encoding a second polypeptide comprising a light chain variable region of the immunoglobulin polypeptide or fragment thereof that binds the first antigen coupled to a first Kappa-type or Lambda-type light chain constant region;
• (c) providing a third nucleic acid molecule encoding a third polypeptide comprising a light chain variable region of an immunoglobulin polypeptide or fragment thereof that shares the same heavy variable chain region of the immunoglobulin polypeptide or fragment thereof of step (a) and binds the second antigen, coupled to a second Kappa-type or Lambda-type light chain constant region, wherein the first and second light chain constant domains are different types;
• and
• (d) culturing a host cell comprising the first, second and third nucleic acid molecules under conditions that permit expression of the first, second and third polypeptides.

A method to generate an antibody mixture comprising two monospecific antibodies and one bispecific antibody, all having a common heavy chain, the method comprising:

• a. Isolating an antibody or antibody fragment region having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain;
• b. Isolating an antibody or antibody fragment region having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with a second light chain variable domain;
• c. Co-expressing in a cell:
• i. a heavy chain polypeptide comprising the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region;
• ii. a first light chain polypeptide comprising the first light chain variable domain fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type;
• and
• iii. a second light chain polypeptide comprising the second light chain variable domain fused to either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type;
• and
• d. recovering the heavy chain polypeptide, the first light chain polypeptide, and the second light chain polypeptide,
• thereby generating an antibody mixture comprising two monospecific antibodies and one bispecific antibody, all having a common heavy chain.

A method to generate an antibody mixture comprising three or more monospecific antibodies and three or more bispecific antibodies, all having a common heavy chain, the method comprising:

• a. Isolating an antibody or antibody fragment region having a specificity determined by a heavy chain variable domain combined with a first light chain variable domain;
• b. Isolating several antibodies or antibody fragments region having a different specificity determined by the same heavy chain variable domain as the antibody of step a) combined with different light chain variable domains;
• c. Co-expressing in a cell:
• i. a heavy chain polypeptide comprising the common heavy chain variable domain fused to an immunoglobulin heavy chain constant region;
• ii. light chain polypeptides comprising all the light chains of the antibodies isolated in step a) and b) fused either to a light chain constant domain of the Kappa type or fused to a light chain constant domain of the Lambda type;
• and
• d. Recovering the heavy chain polypeptide and the light chain polypeptides,
• thereby generating an antibody mixture comprising three or more monospecific antibodies and three or more bispecific antibodies, all having a common heavy chain.