Engineered heterodimeric protein domains

• US 8,871,912 B2
• Filed: 03/23/2007
• Issued: 10/28/2014
• Est. Priority Date: 03/24/2006
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Davis, Jonathan H., Huston, James S.
• Primary Examiner(s): DUFFY, BRADLEY
• Application Number:
  US11/728,048
• Publication Number:
  US 20070287170A1
• Time in Patent Office: 2,776 Days
• Field of Search: 530/350, 530/387.1, 530/387.3
• US Class Current: 530/387.3
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/246 IL-2
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention provides an engineered multidomain protein including at least two nonidentical engineered domains, each of which contains a protein-protein interaction interface containing amino acid sequence segments derived from two or more existing homologous parent domains, thereby conferring on the engineered domains assembly specificities distinct from assembly specificities of the parent domains. In particular, the engineered domains form heterodimers with one another preferentially over forming homodimers. Methods of designing and using the engineered proteins are also included.

Claims

1. A heterodimeric fusion protein comprising first and second polypeptides, each comprising a CH3 domain mediating the heterodimerization of the first and second polypeptides, wherein each CH3 domain is a modified IgG CH3 domain substituted with corresponding amino acid residues from an IgA CH3 domain,

wherein each modified IgG CH3 domain comprises substitutions selected from the group consisting of:

Glu at Kabat position 347, His at Kabat position 349, Pro at Kabat position 354, Leu at Kabat position 359, Asn at Kabat position 360, Leu at Kabat position 362, Thr at Kabat position 364, Arg at Kabat position 370, Lys at Kabat position 390, Leu at Kabat position 392, Trp at Kabat position 394, Ala at Kabat position 395, Arg at Kabat position 397, Glu at Kabat position 399, Pro at Kabat position 400, Ala at Kabat position 405, Thr at Kabat position 407, Ile at Kabat position 409, and Arg at Kabat position 411;

wherein the substitutions in the first and second polypeptides occur at different positions; and

wherein heterodimerization of the modified IgG CH3 domains is enhanced compared to IgG CH3 domains without the substitutions.

2. The heterodimeric fusion protein of claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:

10 and the second polypeptide comprises the amino acid sequence of SEQ ID NO;

11.

3. The heterodimeric fusion protein of claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:

3 and the second polypeptide comprises the amino acid sequence of SEQ ID NO;

6.

4. The heterodimeric fusion protein of claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:

5 and the second polypeptide comprises the amino acid sequence of SEQ ID NO;

8.

5. A heterodimeric fusion protein comprising first and second polypeptides, each comprising a CH3 domain mediating the heterodimerization of the first and second polypeptides, wherein each CH3 domain is a modified IgG CH3 domain substituted with corresponding amino acid residues from an IgA CH3 domain,

wherein each modified IgG CH3 domain comprises substitutions selected from the group consisting of:

Glu at Kabat position 347, His at Kabat position 349, Pro at Kabat position 354, Leu at Kabat position 359, Asn at Kabat position 360, Leu at Kabat position 362, Thr at Kabat position 364, Arg at Kabat position 370, Lys at Kabat position 390, Leu at Kabat position 392, Trp at Kabat position 394, Ala at Kabat position 395, Arg at Kabat position 397, Glu at Kabat position 399, Pro at Kabat position 400, Thr at Kabat position 407, Ile at Kabat position 409, and Arg at Kabat position 411;

wherein the substitutions in the first and second polypeptides occur at different positions; and

wherein heterodimerization of the modified IgG CH3 domains is enhanced compared to IgG CH3 domains without the substitutions.