Bispecific EGFR/IGFIR binding molecules

• US 8,343,501 B2
• Filed: 11/24/2009
• Issued: 01/01/2013
• Est. Priority Date: 11/24/2008
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Emanuel, Stuart, Engle, Linda, Camphausen, Ray, Wright, Martin C., Chao, Ginger, Gottardis, Marco, Carboni, Joan
• Primary Examiner(s): Bunner, Bridget E
• Application Number:
  US12/625,217
• Publication Number:
  US 20100179094A1
• Time in Patent Office: 1,134 Days
• Field of Search: None
• US Class Current: 424/185.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• 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 relates to bispecific molecules comprising an EGFR binding domain and a distinct IGFIR binding domain for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and vectors comprising the polynucleotides encoding the innovative proteins. Exemplary bispecific molecules include antibody-like protein dimers based on the tenth fibronectin type III domain.

Claims

1. An antibody-like protein dimer comprising (a) a tenth fibronectin type III domain (¹⁰Fn3) that binds insulin-like growth factor 1 receptor (IGF-IR) with a K_D of less than 500 nM covalently or non-covalently linked to (b) a ¹⁰Fn3 that binds epidermal growth factor receptor (EGFR) with a K_D of less than 500 nM;

wherein the IGF-IR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequence set forth in amino acids 21-30 of SEQ ID NO;

3, a DE loop having the amino acid sequence set forth in amino acids 51-56 of SEQ ID NO;

3, and an FG loop having the amino acid sequence set forth in amino acids 76-83 of SEQ ID NO;

3, and

wherein the EGFR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequence set forth in amino acids 21-30 of SEQ ID NO;

112, a DE loop having the amino acid sequence set forth in amino acids 51-56 of SEQ ID NO;

112, and an FG loop having the amino acid sequence set forth in amino acids 76-92 of SEQ ID NO;

112.

2. The antibody-like protein dimer of claim 1, wherein the IGF-IR binding ¹⁰Fn3 is covalently linked to the EGFR binding ¹⁰Fn3 via a polypeptide linker or a polyethylene glycol moiety.

3. The antibody-like protein dimer of claim 2 comprising an amino acid sequence at least 90% identical to any one of SEQ ID NOs:

126-132.

4. The antibody-like protein dimer of claim 3, comprising an amino acid sequence at least 90% identical to SEQ ID NO:

130.

5. The antibody-like protein dimer of claim 4, comprising an amino acid sequence at least 95% identical to SEQ ID NO:

130.

6. The antibody-like protein dimer of claim 5, comprising an amino acid sequence at least 98% identical to SEQ ID NO:

130.

7. The antibody-like protein dimer of claim 6, further comprising a C-terminal tail comprising the amino acid sequence of SEQ ID NO:

217.

8. The antibody-like protein dimer of claim 7, further comprising one or more pharmacokinetic (PK) moieties.

9. The antibody-like protein dimer of claim 8, wherein the PK moiety is a polyoxyalkylene moiety and said polyoxyalkylene moiety is polyethylene glycol.

10. The antibody-like protein dimer of claim 9, wherein the polyethylene glycol is between 0.1 kDa and 150 kDa.

11. The antibody-like protein dimer of claim 10, wherein the polyethylene glycol is linked to the antibody-like protein dimer via a Cys amino acid residue.

12. The antibody-like protein dimer of claim 6, comprising the amino acid sequence set forth in SEQ ID NO:

130.

15. The antibody-like protein dimer of any one of claims 1-14, wherein the IGF-IR binding ¹⁰Fn3 has an amino acid sequence at least 90% identical to SEQ ID NO:

3 and the EGFR binding ¹⁰Fn3 has an amino acid sequence at least 90% identical to SEQ ID NO;

112.

16. The antibody-like protein dimer of claim 15, wherein the IGF-IR binding ¹⁰Fn3 has an amino acid sequence at least 95% identical to SEQ ID NO:

3 and the EGFR binding ¹⁰Fn3 has an amino acid sequence at least 95% identical to SEQ ID NO;

112.

17. The antibody-like protein dimer of claim 16, wherein the IGF-IR binding ¹⁰Fn3 has an amino acid sequence at least 98% identical to SEQ ID NO:

3 and the EGFR binding ¹⁰Fn3 has an amino acid sequence at least 98% identical to SEQ ID NO;

112.

18. The antibody-like protein dimer of claim 17, wherein the IGF-IR binding ¹⁰Fn3 has the amino acid sequence of SEQ ID NO:

3 and the EGFR binding ¹⁰Fn3 has the amino acid sequence of SEQ ID NO;

112.

19. A pharmaceutically acceptable composition comprising the antibody-like protein dimer of any one of claim 1-14, 2-3, or 4-10, wherein the composition is essentially endotoxin free.

13. An antibody-like protein dimer comprising a tenth fibronectin type III domain (¹⁰Fn3 ) that binds insulin-like growth factor 1 receptor (IGF-IR) with a K_D of less than 500 nM covalently or non-covalently linked to a ¹⁰Fn3 that binds epidermal growth factor receptor (EGFR) with a K_D of less than 500 nM;

wherein the IGF-IR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequence X_aSARLKVAX_b (SEQ ID NO;

     46), a DE loop having the amino acid sequence X_CKNVYX_d (SEQ ID NO;

     48), and an FG loop having the amino acid sequence X_eRFRDYQX_f (SEQ ID NO;

     50);

wherein the EGFR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequence X_gWAPVDRYQX_h (SEQ ID NO;

     137), a DE loop having the amino acid sequence X_iRDVYX_j (SEQ ID NO;

     138), and an FG loop having the amino acid sequence X_kDYKPHADGPHTYHESX_l (SEQ ID NO;

     139); and

wherein X is any amino acid and a, b, c, d, e, f, g, h, i, j, k, and l are integers independently selected from 0 to 5.

14. The antibody-like protein dimer of claim 13,

wherein the IGF-IR binding ¹⁰Fn3 comprises a BC loop having the amino acid sequence SWSARLKVAR (SEQ ID NO:

45), a DE loop having the amino acid sequence PKNVYT (SEQ ID NO;

     47), and an FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO;

     49); and

an EGFR binding ¹⁰Fn3 comprising a BC loop having the amino acid sequence SWWAPVDRYQ (SEQ ID NO;

     115), a DE loop having the amino acid sequence PRDVYT (SEQ ID NO;

     116), and an FG loop having the amino acid sequence TDYKPHADGPHTYHESP (SEQ ID NO;

     117).