Antibody fragment-polymer conjugates and uses of same

• US 7,122,636 B1
• Filed: 01/21/2000
• Issued: 10/17/2006
• Est. Priority Date: 02/21/1997
• Status: Expired due to Fees

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Shahrokh, Zahra, Koumenis, Iphigenia, Leong, Steven, Hsei, Vanessa, Zapata, Gerardo
• Primary Examiner(s): Nickol, Gary
• Assistant Examiner(s): Aeder, Sean E
• Application Number:
  US09/489,394
• Time in Patent Office: 2,461 Days
• Field of Search: 530/387.1, 530/387.3, 530/388.85, 530/391.5, 530/388.23, 530/388.24, 530/391.1, 424/123.1, 424/156.1, 424/193.1, 424/178.1
• US Class Current: 530/387.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/6845 the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/244 Interleukins [IL]
• CPC Class Codes: C07K 17/02 Peptides being immobilised on, or in, an organic carrier
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 : Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

Described are conjugates formed by an antibody fragment covalently attached to a non-proteinaceous polymer, wherein the apparent size of the conjugate is at least about 500 kD. The conjugates exhibit substantially improved half-life, mean residence time, and/or clearance rate in circulation as compared to the underivatized parental antibody fragment. Also described are conjugates directed against human vascular endothelial growth factor (VEGF), human p185 receptor-like tyrosine kinase (HER2), human CD20, human CD18, human CD11a, human IgE, human apoptosis receptor-2 (Apo-2), human tumor necrosis factor-α (TNF-α), human tissue factor (TF), human α4β7 integrin, human GPIIb-IIIa integrin, human epidermal growth factor receptor (EGFR), human CD3, and human interleukin-2 receptor α-chain (TAC) for diagnostic and therapeutic applications.

Claims

1. A conjugate consisting essentially of an antibody fragment covalently modified by one or two nonproteinaceous polymer molecules at a free sulfhydryl group of a cysteine residue within the hinge region of the antibody fragment, wherein a disulfide bridge within the hinge region is avoided by substituting another amino acid for the corresponding cysteine residue within the hinge region in the opposite chain of said antibody fragment, and wherein (a) the apparent molecular weight of the conjugate, as determined by size exclusion chromatography, is at least about 500 kD, (b) the average actual molecular weight of each nonproteinaceous polymer molecule is at least 20 kD, (c) the conjugate has an apparent size that is at least about 8-fold greater than the apparent size of the parental antibody fragment, and (d) the conjugate binds the same antigen as the parental molecule that is not covalently modified by one or two nonproteinaceous polymer molecules.

2. The conjugate of claim 1 wherein the nonproteinaceous polymer molecule is polyethylene glycol (PEG).

3. The conjugate of claim 2 wherein the antibody fragment is selected from the group consisting of Fab′

, Fab′

-SH, and F(ab′

)₂.

4. The conjugate of claim 2 wherein the antibody fragment is selected from the group consisting of Fab′

and Fab′

-SH modified by one PEG molecule.

5. The conjugate of claim 4 wherein the PEG molecule has an average molecular weight of at least 30 kD.

6. The conjugate of claim 1 wherein the apparent molecular weight of the conjugate is at least about 800 kD.

7. The conjugate of claim 1 wherein the apparent molecular weight of the conjugate is at least about 1,800 kD.

8. The conjugate of claim 1 comprising the antigen binding site of HER2.

9. The conjugate of claim 1 comprising the antigen binding site of CD20.

11. The conjugate of claim 2 comprising the antigen binding site of HER2.

12. The conjugate of claim 2 comprising the antigen binding site of CD20.

13. The conjugate of claim 3 comprising the antigen binding site of HER2.

14. The conjugate of claim 3 comprising the antigen binding site of CD20.

15. The conjugate of claim 4 comprising the antigen binding site of HER2.

16. The conjugate of claim 4 comprising the antigen binding site of CD20.

17. The conjugate of claim 5 comprising the antigen binding site of HER2.

18. The conjugate of claim 5 comprising the antigen binding site of CD20.

19. The conjugate of claim 6 comprising the antigen binding site of HER2.

20. The conjugate of claim 6 comprising the antigen binding site of CD20.

21. The conjugate of claim 7 comprising the antigen binding site of HER2.

22. The conjugate of claim 7 comprising the antigen binding site of CD20.

10. A conjugate consisting essentially of an antibody fragment covalently modified by two nonproteinaceous polymer molecules at a free sulfhydryl group of a cysteine residue within the hinge region of the antibody fragment, wherein a disulfide bridge within the hinge region is avoided by substituting another amino acid for the corresponding cysteine residue within the hinge region in the opposite chain of said antibody fragment, and wherein (a) the apparent molecular weight of the conjugate, as determined by size exclusion chromatorgraphy, is at least about 500 kD, (b) the average actual molecular weight of each nonproteinaceous polymer molecule is at least 20 kD, (c) the conjugate has an apparent size that is at least about 8-fold greater than the apparent size of the parental antibody fragment, and (d) the conjugate binds the same antigen as the parental molecule that is not covalently modified by two nonproteinaceous polymer molecules.