Modified antibody compositions, methods of making and using thereof

• US 8,563,269 B2
• Filed: 12/09/2011
• Issued: 10/22/2013
• Est. Priority Date: 01/12/2009
• Status: Active Grant

• Current Assignee: CytomX Therapeutics, Inc
• Original Assignee: CytomX Therapeutics, Inc
• Inventors: Stagliano, Nancy E., West, James W., Kamath, Kathryn, Bessette, Paul H., Gluck, Frederick W., Sagert, Jason, Daugherty, Patrick
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US13/315,623
• Publication Number:
  US 20120149061A1
• Time in Patent Office: 683 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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 method of manufacturing an activatable antibody, the method comprising:

(a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

1, 218-220, 236-265 and 266, a first peptide linker (LP1), a cleavable moiety (CM), a second peptide linker (LP2), and an antibody or an antigen binding fragment thereof (AB) that specifically binds Epidermal Growth Factor Receptor (EGFR),

(i) wherein;

the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to EGFR;

(ii) wherein the CM is a polypeptide that functions as a substrate for a protease selected from the group consisting of urokinase type plasminogen activator (uPA), legumain and matriptase (MT-SP1), and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease;

(iii) wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR;

(iv) wherein each of LP1 and LP2 is a peptide, and wherein the two LPs need not be identical to each other;

(v) wherein the activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows;

MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM;

(vi) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to EGFR and wherein the MM of the activatable antibody in a cleaved state does not interfere or compete with specific binding of the AB to EGFR; and

(vii) wherein the MM of the activatable antibody in an uncleaved state inhibits the binding of the AB to EGFR such that the dissociation constant (K_d) of the AB of the activatable antibody in an uncleaved state towards EGFR is at least 100 times greater than the K_d of the AB of the activatable antibody in a cleaved state towards EGFR;

and

(b) recovering the activatable antibody.

2. The method of claim 1, wherein the CM is a polypeptide of up to 15 amino acids in length.

3. The method of claim 1, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

4. The method of claim 1, wherein the AB is or is from cetuximab or panitumumab.

5. The method of claim 1, wherein the AB is or is from cetuximab.

6. The method of claim 1, wherein the CM is a substrate for a protease selected from the group consisting of uPA, legumain and MT-SP1 and is a substrate for at least one additional protease that is co-localized in a tissue with EGFR, wherein the additional protease is an enzyme selected from the group consisting of the enzymes in Table 3.

7. The method of claim 1, wherein the CM is a substrate for uPA and at least one additional protease that is co-localized in a tissue with EGFR, wherein the additional protease is legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, MT-SP1 or PSA.

8. The method of claim 1, wherein the antigen binding 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.

9. The method of claim 1, wherein the activatable antibody comprises at least a first CM that functions as a substrate for a protease-selected from the group consisting of uPA, legumain and matriptase MT-SP1 and a second CM.

10. The method of claim 9, wherein the first CM is cleavable by a first cleaving agent in a target tissue and wherein the second CM is cleavable by a second cleaving agent in a target tissue.

11. The method of claim 10, wherein the first cleaving agent and the second cleaving agent are the same enzyme, and where the first CM and the second CM are different substrates for the enzyme.

12. The method of claim 10, wherein the first cleaving agent and the second cleaving agent are different enzymes.

13. The method of claim 10, wherein the first cleaving agent and the second cleaving agent are co-localized in the tissue.

14. The method of claim 9, wherein the first CM and the second CM are cleavable by at least one cleaving agent in the tissue.

15. The method of claim 1, wherein the MM is a polypeptide of no more than 40 amino acids in length.

16. The method of claim 1, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_n, (GSGGS)_n (SEQ ID NO:

12) and (GGGS)_n (SEQ ID NO;

     13), where n is an integer of at least one.

17. The method of claim 1, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:

14), GGSGG (SEQ ID NO;

     15), GSGSG (SEQ ID NO;

     16), GSGGG (SEQ ID NO;

     17), GGGSG (SEQ ID NO;

     18), and GSSSG (SEQ ID NO;

     19).

18. The method of claim 1, wherein the CM is a substrate for uPA and at least one additional protease, wherein the additional protease is legumain, MT-SP1 or both legumain and MT-SP1.

19. The method of claim 1, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

267-279 and 280.

20. The method of claim 9, wherein at least one of the first CM and the second CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

267-279 and 280.

37. The method of claim 20, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of (GS)_n, (GSGGS)_n (SEQ ID NO:

12) and (GGGS)_n (SEQ ID NO;

     13), where n is an integer of at least one.

38. The method of claim 20, wherein at least one of LP1 or LP2 comprises an amino acid sequence selected from the group consisting of GGSG (SEQ ID NO:

14), GGSGG (SEQ ID NO;

     15), GSGSG (SEQ ID NO;

     16), GSGGG (SEQ ID NO;

     17), GGGSG (SEQ ID NO;

     18), and GSSSG (SEQ ID NO;

     19).

21. A method of manufacturing an activatable antibody, the method comprising:

(a) culturing a cell comprising a nucleic acid construct that encodes the activatable antibody under conditions that lead to expression of the activatable antibody, wherein the activatable antibody comprises a masking moiety (MM) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

1, 218-220, 236-265 and 266, a first peptide linker (LP1), a cleavable moiety (CM) comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

267-279 and 280, a second peptide linker (LP2), and an antibody or an antigen binding fragment thereof (AB) that specifically binds Epidermal Growth Factor Receptor (EGFR),

(i) wherein;

the MM has an equilibrium dissociation constant for binding to the AB which is greater than the equilibrium dissociation constant of the AB for binding to EGFR;

(ii) wherein the CM is a polypeptide that functions as a substrate for urokinase type plasminogen activator (uPA), and wherein the protease cleaves the CM in the activatable antibody when the activatable antibody is exposed to the protease;

(iii) wherein the AB has an equilibrium dissociation constant of at most 100 nM for binding to EGFR;

(iv) wherein each of LP1 and LP2 is a peptide, and wherein the two LPs need not be identical to each other;

(v) wherein the activatable antibody in an uncleaved state has the structural arrangement from N-terminus to C-terminus as follows;

MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM;

(vi) wherein the MM of the activatable antibody in an uncleaved state interferes with specific binding of the AB to EGFR and wherein the MM of the activatable antibody in a cleaved state does not interfere or compete with specific binding of the AB to EGFR; and

(vii) wherein the MM of the activatable antibody in an uncleaved state inhibits the binding of the AB to EGFR such that the dissociation constant (K_d) of the AB of the activatable antibody in an uncleaved state towards EGFR is at least 100 times greater than the K_d of the AB of the activatable antibody in a cleaved state towards EGFR;

and

(b) recovering the activatable antibody.

22. The method of claim 21, wherein the CM is a polypeptide of up to 15 amino acids in length.

23. The method of claim 21, wherein each of LP1 and LP2 is a peptide of about 1 to 20 amino acids in length.

24. The method of claim 21, wherein the MM is a polypeptide of no more than 40 amino acids in length.

25. The method of claim 21, wherein the AB is or is derived from cetuximab or panitumumab.

26. The method of claim 21, wherein the AB is or is derived from cetuximab.

27. The method of claim 21, wherein the CM is a substrate for uPA and at least one additional protease that is co-localized in a tissue with EGFR, wherein the additional protease is an enzyme selected from the group consisting of the enzymes in Table 3.

28. The method of claim 21, wherein the CM is a substrate for uPA and at least one additional protease that is co-localized in a tissue with EGFR, wherein the additional protease is legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, MT-SP1 or PSA.

29. The method of claim 21, wherein the CM is a substrate for uPA and at least one additional protease, wherein the additional protease is legumain, MT-SP1 or both legumain and MT-SP1.

30. The method of claim 21, wherein the antigen binding 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.

31. The method of claim 21, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

267-279 and 280 and a second CM.

32. The method of claim 31, wherein the first CM is cleavable by uPA in a target tissue and wherein the second CM is cleavable by a second cleaving agent in a target tissue.

33. The method of claim 32, wherein the first cleaving agent and the second cleaving agent are uPA, and where the first CM and the second CM are different substrates for uPA.

34. The method of claim 32, wherein the first cleaving agent and the second cleaving agent are different enzymes.

35. The method of claim 32, wherein uPA and the second cleaving agent are co-localized in the tissue.

36. The method of claim 31, wherein the first CM and the second CM are cleavable by at least one cleaving agent in the tissue.