MODIFIED ANTIBODY COMPOSITIONS, METHODS OF MAKING AND USING THEREOF

• US 20100189651A1
• Filed: 01/12/2010
• Published: 07/29/2010
• Est. Priority Date: 01/12/2009
• Status: Abandoned Application

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, LLC (CytomX Therapeutics, Inc)
• Inventors: Daugherty, Patrick, Gluck, Frederick, West, James W., Sagert, Jason, Bessette, Paul H., Kamath, Kathryn, Stagliano, Nancy E.
• Application Number:
  US12/686,344
• Publication Number:
  US 20100189651A1
• Time in Patent Office: Days
• US Class Current: 424/9.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 39/3955 against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• 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: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/001 by chemical synthesis
• 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 : against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/241 : Tumor Necrosis Factors
• CPC Class Codes: C07K 16/28 : against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2818 : against CD28 or CD152
• CPC Class Codes: C07K 16/2845 : against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: C07K 16/2863 : against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 : against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2875 : against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 16/2896 : against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 2317/20 : characterized by taxonomic origin
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• 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 2317/40 : characterized by post-translational modification
• CPC Class Codes: C07K 2317/51 : Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 : Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• 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 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• 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
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/50 : containing protease site
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 : Immunoglobulins

Abstract

The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolysed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolysing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.

Claims

1. A modified antibody comprising:

an antibody or antibody fragment (AB), capable of specifically binding its target, coupled to a masking moiety (MM), wherein the coupling of the MM reduces the ability of the AB to bind its target such that that the dissociation constant (K_d) of the AB when coupled to the MM towards the target is at least 100 times greater than the K_d of the AB when not coupled to the MM towards the target.

4. The modified antibody of claim 1 wherein in the presence of a target, the coupling of the MM to the AB reduces the ability of the AB to bind its target by at least 90%, as compared to the ability of the AB to bind its target when not coupled to the MM, when assayed in vitro using a target displacement assay.

5. The modified antibody of claim 4 wherein the coupling of the MM to the AB reduces the ability of the AB to bind its target for at least 12 hours.

8. The modified antibody of claim 1 wherein the dissociation constant (K_d) of the MM towards the AB is at least 100 times greater than the K_d of the AB towards the target.

9. The modified antibody of claim 8 wherein the K_d of the MM towards the AB is lower than 10 nM.

12. The modified antibody of claim 1 further coupled to a cleavable moiety (CM).

13. The modified antibody of claim 12 wherein the CM is capable of being cleaved by an enzyme, reduced by a reducing agent, or photolysed.

16. The modified antibody of claim 1 wherein the MM is capable of specifically binding to the antigen-binding domain of the AB.

17. The modified antibody of claim 16 wherein the binding of the MM to the antigen-binding domain is non-covalent.

18. The modified antibody of claim 1 wherein the MM reduces the AB'"'"'s ability to bind its target allosterically.

19. The modified antibody of claim 1 wherein the MM reduces the AB'"'"'s ability to bind its target sterically.

23. The modified antibody of claim 1 wherein the antibody fragment is selected from the group consisting of a Fab′

fragment, a F(ab′

) 2 fragment, a scFv, a scAB a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

24. The modified antibody of claim 1 wherein the AB is selected from the group consisting of the antibodies in Table 2.

26. The modified antibody of claim 24 wherein the AB is cetuximab, panitumumab, infliximab, adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab, or figitumumab.

27. The modified antibody of claim 1 wherein the target is selected from the group consisting of the targets in Table 1.

29. The modified antibody of claim 27 wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4.

30. The modified antibody of claim 1 further comprising a second AB wherein the target for the second AB is selected from the group consisting of the targets in Table 1.

31. The modified antibody of claim 12 wherein the CM is located within the MM.

32. The modified antibody of claim 12 wherein the CM is a substrate for an enzyme selected from the group consisting of the enzymes in Table 3.

33. The modified antibody of claim 32 wherein the CM is a substrate for legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA.

34. The modified antibody of claim 32 wherein the AB is selected from the group consisting of the antibodies in Table 2.

36. The modified antibody of claim 34 wherein the AB is cetuximab, panitumumab, infliximab, adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab, or figitumumab.

37. The modified antibody of claim 32 wherein the target is selected from the group consisting of the targets in Table 1.

39. The modified antibody of claim 37 wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4.

42. The modified antibody of claim 1 wherein the MM does not comprise more than 50% amino acid sequence similarity to a natural binding partner of the AB.

46. The modified antibody of claim 12 further comprising two linker peptides, wherein the first linker peptide is between the AB and the CM and the second linker peptide is positioned between the MM and the CM.

50. The modified antibody of claim 46 wherein the linkers are selected from the group consisting of a cleavable linker, a non-cleavable linker, and a branched linker.

54. The modified antibody of claim 1 further comprising an agent conjugated to the AB.

55. The modified antibody of claim 54 wherein the agent is a therapeutic agent.

56. The modified antibody of claim 54 further comprising a cleavable moiety (CM) coupled to the AB, capable of being specifically cleaved.

57. The modified antibody of claim 56 wherein the agent is an antineoplastic agent.

70. The modified antibody of claim 12 wherein the serum half-life of the composition is at least 5 days when administered to an organism.

71. The modified antibody of claim 1 wherein the consensus sequence of the MM is CISPRGC, C(N/P)H(HVF)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG, xCxxYQCLxxxxxx, XXQPxPPRVXX, PxPGFPYCxxxx, xxxxQxxPWPP, GxGxCYTILExxCxxxR, GxxxCYxlxExxCxxxx, GxxxCYxIxExWCxxxx, xxxCCxxYxIxxCCxxx, or xxxxxYxILExxxxx.

72. The modified antibody of claim 57 wherein the consensus sequence is specific for binding to an anti-VEGF antibody, an anti-EFGR antibody, or an anti-CTLA-4 antibody.

165. A method of inhibiting angiogenesis in a mammalian subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of claim 12.

183. The antibody of claim 1 wherein the affinity of binding of the antibody to its target is lower in a first tissue when compared to the binding of the antibody to its target in a second tissue.

184. The antibody therapeutic of claim 183 wherein the target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4.

185. The antibody of claim 183 wherein the first tissue is a healthy tissue and the second tissue is a diseased tissue;

the first tissue is an early stage tumor and the second tissue is a late stage tumor;

the first tissue is a benign tumor and the second tissue is a malignant tumor;

the first tissue and second tissue are spatially separated;

or the first tissue is epithelial tissue and the second tissue is breast, head, neck, lung, pancreatic, nervous system, liver, prostate, urogenital, or cervical tissue.

2-3. -3. (canceled)

6-7. -7. (canceled)

10-11. -11. (canceled)

14-15. -15. (canceled)

20-22. -22. (canceled)

25. (canceled)

28. (canceled)

35. (canceled)

38. (canceled)

40-41. -41. (canceled)

43-45. -45. (canceled)

47-49. -49. (canceled)

52. (canceled)

58-69. -69. (canceled)

73-133. -133. (canceled)

134. An activatable antibody complex (AAC) comprising:

(a) two antibodies or antibody fragments (AB1 and AB2), each capable of specifically binding its target;

(b) at least one masking moiety (MM) coupled to either AB1 or AB2, capable of inhibiting the specific binding of AB1 and AB2 to their targets; and

(c) at least one cleavable moiety (CM) coupled to either AB1 or AB2, capable of being specifically cleaved by an enzyme whereby activating the AAC composition;

wherein when the AAC is in an uncleaved state, the MM inhibits the specific binding of AB1 and AB2 to their targets and when the AAC is in a cleaved state, the MM does not inhibit the specific binding of AB1 and AB2 to their targets.

135-152. -152. (canceled)

153. A method of treating or diagnosing a condition in a subject including administering to the subject a composition comprising:

(a) an antibody or antibody fragment (AB), capable of specifically binding its target;

(b) a masking moiety (MM) coupled to the AB, capable of inhibiting the specific binding of the AB to its target; and

(c) a cleavable moiety (CM) coupled to the AB, capable of being specifically cleaved by an enzyme;

wherein upon administration to the subject, when the AA is not in the presence of sufficient enzyme activity to cleave the CM, the MM reduces the specific binding of the AB to its target by at least 90% when compared to when the AA is in the presence of sufficient enzyme activity to cleave the CM and the MM does not inhibit the specific binding of the AB to its target.

154-164. -164. (canceled)

166-182. -182. (canceled)

186-234. -234. (canceled)