Monoclonal antibodies against HER2 epitope

• US 9,714,294 B2
• Filed: 05/27/2011
• Issued: 07/25/2017
• Est. Priority Date: 05/27/2010
• Status: Active Grant

• Current Assignee: Genmab A/S
• Original Assignee: Genmab A/S
• Inventors: Satijn, David, Van De Winkel, Jan, De Goeij, Bart, Van Den Brink, Edward Norbert, De Haij, Simone, Riedl, Thilo, Hoet, Rene, Parren, Paul, Baadsgaard, Ole
• Primary Examiner(s): Wu, Julie
• Application Number:
  US13/700,246
• Publication Number:
  US 20130171148A1
• Time in Patent Office: 2,251 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

Isolated monoclonal antibodies which bind to human epidermal growth factor receptor 2 (HER2), and related antibody-based compositions and molecules, are disclosed. Pharmaceutical compositions comprising the antibodies and therapeutic and diagnostic methods for using the antibodies are also disclosed.

Claims

1. An isolated antibody which binds to human epidermal growth factor receptor 2 (HER2), comprising a VH region and a VL region selected from the group consisting of:

a) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

2, 3 and 4, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

6, GAS, and SEQ ID NO;

7, respectively;

b) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

9, 10 and 11, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

13, DAS, and SEQ ID NO;

14, respectively;

c) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

16, 17 and 18, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

20, GAS, and SEQ ID NO;

21, respectively;

d) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

23, 24 and 25, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

27, GAS, and SEQ ID NO;

28, respectively;

e) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

30, 31 and 32, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

34, GAS, and SEQ ID NO;

35, respectively; and

f) a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs;

37, 38 and 39, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

41, GAS, and SEQ ID NO;

42, respectively.

2. The antibody of claim 1, comprising a VH region and a VL region selected from the group consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5;

b) a VH region comprising the sequence of SEQ ID NO;

8 and a VL region comprising the sequence of SEQ ID NO;

12;

c) a VH region comprising the sequence of SEQ ID NO;

15 and a VL region comprising the sequence of SEQ ID NO;

19;

d) a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26;

e) a VH region comprising the sequence of SEQ ID NO;

29 and a VL region comprising the sequence of SEQ ID NO;

33;

f) a VH region comprising the sequence of SEQ ID NO;

36 and a VL region comprising the sequence of SEQ ID NO;

40; and

g) a variant of any of said antibodies, wherein said variant has at most 1, 2 or 3 amino acid substitutions.

3. The antibody of claim 1, which has an EC₅₀ value for binding to HER2-expressing cells lower than 0.80 μ

g/ml, and blocks the binding to soluble HER2 of a reference antibody comprising VH and VL regions selected from the group consisting of;

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5;

b) a VH region comprising the sequence of SEQ ID NO;

8 and a VL region comprising the sequence of SEQ ID NO;

12; and

c) a VH region comprising the sequence of SEQ ID NO;

15 and a VL region comprising the sequence of SEQ ID NO;

19.

4. The antibody of claim 1, which specifically binds HER2-positive Rhesus epithelial cells, and blocks the binding to soluble HER2 of a reference antibody comprising VH and VL regions selected from the group consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5;

b) a VH region comprising the sequence of SEQ ID NO;

8 and a VL region comprising the sequence of SEQ ID NO;

12;

c) a VH region comprising the sequence of SEQ ID NO;

15 and a VL region comprising the sequence of SEQ ID NO;

19;

d) a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26;

e) a VH region comprising the sequence of SEQ ID NO;

29 and a VL region comprising the sequence of SEQ ID NO;

33; and

f) a VH region comprising the sequence of SEQ ID NO;

36 and a VL region comprising the sequence of SEQ ID NO;

40.

5. The antibody of claim 1, which specifically binds HER2-expressing AU565 cells but promotes ligand-independent proliferation of the cells to a lesser extent than F5, and blocks the binding to soluble HER2 of a reference antibody comprising VH and VL regions selected from the group consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5; and

b) a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26.

6. The antibody of claim 1, wherein the antibody, when conjugated to a therapeutic moiety, which is a truncated form of the pseudomonas-exotoxin A, kills at least 60% of a HER2-expressing tumor cell-line, and blocks the binding to soluble HER2 of a reference antibody comprising the VH and VL regions selected from the groups consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5;

b) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26;

c) a VH region comprising the sequence of SEQ ID NO;

15 and a VL region comprising the sequence of SEQ ID NO;

19; and

d) a VH region comprising the sequence of SEQ ID NO;

36 and a VL region comprising the sequence of SEQ ID NO;

40.

7. The antibody of claim 6, wherein the HER2-expressing tumor cell-line expresses less than an average of about 30000 HER2 molecules per cell, and wherein the antibody blocks the binding to soluble HER2 of a reference antibody comprising the VH and VL regions selected from the group consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5; and

b) an antibody comprising a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26.

8. The antibody of claim 1, wherein a higher amount of the antibody is internalized by a HER2-expressing tumor cell-line than the amount of trastuzumab internalized by the same cell line, and wherein the antibody cross-blocks a reference antibody comprising VH and VL regions selected from the group consisting of:

a) a VH region comprising the sequence of SEQ ID NO;

1 and a VL region comprising the sequence of SEQ ID NO;

5;

b) a VH region comprising the sequence of SEQ ID NO;

8 and a VL region comprising the sequence of SEQ ID NO;

12;

c) a VH region comprising the sequence of SEQ ID NO;

15 and a VL region comprising the sequence of SEQ ID NO;

19;

d) a VH region comprising the sequence of SEQ ID NO;

22 and a VL region comprising the sequence of SEQ ID NO;

26;

e) a VH region comprising the sequence of SEQ ID NO;

29 and a VL region comprising the sequence of SEQ ID NO;

33; and

f) a VH region comprising the sequence of SEQ ID NO;

36 and a VL region comprising the sequence of SEQ ID NO;

40.

9. The antibody of claim 3, which fully blocks the reference antibody from binding to soluble HER2.

10. The antibody of claim 1, wherein the antibody is a bivalent antibody.

11. The antibody of claim 1, wherein the antibody is an antigen-binding fragment.

12. The antibody of claim 1, wherein the antibody is a full-length antibody.

13. The antibody of claim 1, wherein the antibody is an effector-function-deficient antibody.

14. The antibody of claim 1, wherein the antibody is a monovalent antibody.

15. The antibody of claim 14, wherein the monovalent antibody further comprises a C_H region of an immunoglobulin or a fragment thereof comprising the C_H2 and C_H3 regions, wherein the C_H region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the C_H region do not comprise any amino acid residues which are capable of forming disulfide bonds with an identical C_H region or other covalent or stable non-covalent inter-heavy chain bonds with an identical C_H region in the presence of polyclonal human IgG.

16. A bispecific antibody comprising a first antigen-binding region of an antibody as defined in claim 1, and a second antigen-binding region having a different binding specificity than the first antigen-binding region.

17. The antibody of claim 1, wherein the antibody is conjugated to another moiety.

18. The antibody of claim 17, wherein the moiety is a cytotoxic moiety is selected from the group consisting of taxol;

cytochalasin B;

gramicidin D;

ethidium bromide;

emetine;

mitomycin;

etoposide;

tenoposide;

vincristine;

vinblastine;

colchicin;

doxorubicin;

daunorubicin;

dihydroxy anthracin dione;

a tubulin-inhibitor;

mitoxantrone;

mithramycin;

actinomycin D;

1-dehydrotestosterone;

a glucocorticoid;

procaine;

tetracaine;

lidocaine;

propranolol;

puromycin;

calicheamicin or an analog or derivative thereof;

an antimetabolite;

an alkylating agent;

an antibiotic;

an antimitotic agent;

diphtheria toxin;

ricin toxin;

cholera toxin;

a Shiga-like toxin;

LT toxin;

C3 toxin;

Shiga toxin;

pertussis toxin;

tetanus toxin;

soybean Bowman-Birk protease inhibitor;

Pseudomonas exotoxin;

alorin;

saporin;

modeccin;

gelanin;

abrin A chain;

modeccin A chain;

alpha-sarcin;

Aleurites fordii proteins;

dianthin proteins;

Phytolacca americana proteins;

momordica charantia inhibitor;

curcin;

crotin;

sapaonaria officinalis inhibitor;

gelonin;

mitogellin;

restrictocin;

phenomycin;

enomycin toxins;

ribonuclease (RNase);

DNase I;

Staphylococcal enterotoxin A;

pokeweed antiviral protein;

diphtherin toxin; and

Pseudomonas endotoxin.

19. The antibody of claim 17, wherein the moiety is a cytotoxic moiety selected from the group consisting of maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), monomethyl auristatin E, and an analog, derivative, or prodrug of any thereof.

20. The antibody of claim 17, which is conjugated to a cytokine selected from the group consisting of interleukin (IL)-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, keratinocyte growth factor, interferon (IFN)α

, IFNβ

, IFNγ

, granulocyte-macrophage colony-stimulating factor, CD40L, Flt3 ligand, stem cell factor, ancestim, and tumor necrosis factor (TNF)α

.

21. The antibody of claim 17, which is conjugated to a radioisotope.

22. A pharmaceutical composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

23. A pharmaceutical comprising an antibody as defined in claim 17, and a pharmaceutically acceptable carrier.

24. A method for inhibiting growth and/or proliferation of one or more tumor cells expressing HER2, comprising administration, to an individual in need thereof, of an antibody according to claim 1.

25. The method of claim 24, wherein the one or more tumor cell co-expresses HER2 and EGFR and/or HER3.

26. A method for treating cancer, comprising

a) selecting a subject suffering from a cancer comprising tumor cells co-expressing HER2 and EGFR and/or HER3, and

b) administering to the subject the antibody according to claim 1.

27. The method of claim 26, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, endometrial/cervical cancer, lung cancer, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, testis cancer, a soft-tissue tumor, and bladder cancer.

28. A method for detecting the presence of HER2 in a sample, comprising:

contacting the sample with an antibody of claim 1 under conditions that allow for formation of a complex between the antibody and HER2; and

analyzing whether a complex has been formed.

29. A kit for detecting the presence of HER2 in a sample comprising an antibody of claim 1;

and

instructions for use of the kit.

30. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

2, 3 and 4, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

6, GAS, and SEQ ID NO;

7, respectively.

31. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

9, 10 and 11, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

13, DAS, and SEQ ID NO;

14, respectively.

32. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

16, 17 and 18, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

20, GAS, and SEQ ID NO;

21, respectively.

33. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

23, 24 and 25, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

27, GAS, and SEQ ID NO;

28, respectively.

34. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

30, 31 and 32, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

34, GAS, and SEQ ID NO;

35, respectively.

35. The antibody of claim 1, wherein the antibody comprises a VH region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs:

37, 38 and 39, respectively; and

a VL region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO;

41, GAS, and SEQ ID NO;

42, respectively.

36. The bispecific antibody of claim 16, wherein the second antigen-binding site binds to a molecule selected from the group consisting of:

a cancer- or tumor-associated antigen;

a cancer-associated integrin;

a T cell and/or NK cell antigen;

an angiogenic factor, receptor for an angiogenic factor, or other cancer-associated growth factor; and

a receptor associated with cancer progression.

37. The bispecific antibody of claim 36, wherein the cancer- or tumor-associated antigen is selected from the group consisting of:

a carcinoembryonic antigen, prostate specific antigen, renal antigen, α

-fetoprotein, CTL-recognized antigen on melanoma, a CT antigen, a mucin antigen, a ganglioside antigen, tyrosinase, gp75, c-Met, c-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, and Ep-CAM.

38. The bispecific antibody of claim 37, wherein:

a) the CT antigen is selected from the group consisting of;

MAGE-B5, MAGE-B6, MAGE-C2, MAGE-C3, MAGE-D, Mage-12, CT10, NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE;

b) the mucin antigen is MUC1 or mucin-CA125;

c) the cancer-associated integrin is α

5β

3 integrin;

d) the T cell and/or NK cell antigen is CD3 or CD16;

e) the angiogenic factor or other cancer-associated growth factor is selected from the group consisting of vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, angiogenin, vascular endothelial growth factor receptor, fibroblast growth factor receptor, epidermal growth factor receptor, and angiogenin receptor;

or

f) the receptor associated with cancer progression is selected from the group consisting of HER1, HER3, and HER4.

39. The bispecific antibody of claim 16, wherein the second antigen-binding region binds to a different site on HER2 than the first antigen-binding region.

40. The bispecific antibody of claim 39, wherein the second antigen-binding region cross-blocks the binding of trastuzumab, pertuzumab, F5, or C1 to HER2.

41. The antibody of claim 12, which is an IgG1 antibody.

42. The antibody of claim 41, which is an IgG1, κ

antibody.

43. The antibody of claim 18, wherein

a) the tubulin-inhibitor is a maytansine or an analog or derivative thereof;

b) the antimetabolite is selected from the group consisting of methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, and cladribine;

c) the alkylating agent is selected from the group consisting of mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, carboplatin, duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), and an analog or derivative thereof;

d) the antibiotic is selected from the group consisting of;

dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, and anthramycin (AMC));

e) the antimitotic agent is monomethyl auristatin E or F or an analog or derivative thereof;

f) the diphtheria toxin is diphtheria A chain or an active fragment thereof;

g) the ricin toxin is ricin A or a deglycosylated ricin A chain toxin;

h) the Shiga-like toxin is selected from the group consisting of;

SLT I, SLT II, and SLT IIV;

or

i) the Phytolacca americana protein is selected from the group consisting of;

PAPI, PAPII, and PAP S.