U.S. patent application number 12/676798 was filed with the patent office on 2011-10-20 for novel methods and antibodies for treating cancer.
This patent application is currently assigned to GENMAB A/S. Invention is credited to Sven Berger, Willem Karel Bleeker, Michael Dechant, Klaus Edvardsen, Paul Parren, Thomas Valerius, Jeroen Lammerts Van Bueren, Jan Van De Winkel, Wencke Weisner.
Application Number | 20110256142 12/676798 |
Document ID | / |
Family ID | 40224463 |
Filed Date | 2011-10-20 |
United States Patent
Application |
20110256142 |
Kind Code |
A1 |
Van De Winkel; Jan ; et
al. |
October 20, 2011 |
NOVEL METHODS AND ANTIBODIES FOR TREATING CANCER
Abstract
The invention relates to novel methods for the treatment of
tumors, comprising administration of a bispecific antibody or a
combination of two or more non-cross-blocking antibodies that
recognize the same target antigen or antigenic complex. In
particular, the invention relates to a method for inducing
complement-mediated cell killing in the treatment of a tumor, said
method comprising combined administration, to a human being in need
thereof, of a first antibody and a second antibody, wherein--said
first antibody binds EGFR, --said second antibody binds EGFR,
--said first and second antibody are non-cross-blocking, and--the
dosage regimen is such that CDC is obtained at the tumor site.
Inventors: |
Van De Winkel; Jan; (Zeist,
NL) ; Parren; Paul; (Utrecht, NL) ; Bleeker;
Willem Karel; (Amsterdam, NL) ; Edvardsen; Klaus;
(Klampenborg, DK) ; Van Bueren; Jeroen Lammerts;
(Harderwijk, NL) ; Valerius; Thomas;
(Neuwittenbek, DE) ; Dechant; Michael;
(Bordesholm, DE) ; Weisner; Wencke; (Bremen,
DE) ; Berger; Sven; (Haute-Savoie, FR) |
Assignee: |
GENMAB A/S
Copenhagen
DK
|
Family ID: |
40224463 |
Appl. No.: |
12/676798 |
Filed: |
September 5, 2008 |
PCT Filed: |
September 5, 2008 |
PCT NO: |
PCT/DK2008/050220 |
371 Date: |
December 21, 2010 |
Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/138.1; 530/387.3; 530/387.7 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 37/04 20180101; C07K 2317/24 20130101; A61P 35/00 20180101;
C07K 2317/732 20130101; A61K 2039/505 20130101; A61K 2039/507
20130101; C07K 16/2863 20130101; C07K 2317/21 20130101; A61P 17/06
20180101; C07K 2317/92 20130101; C07K 16/468 20130101; C07K
2317/734 20130101; G01N 33/573 20130101; C07K 16/40 20130101; C07K
2317/73 20130101; C07K 2317/54 20130101; C07K 16/44 20130101; A61P
37/06 20180101; C07K 2317/76 20130101; C07K 2317/31 20130101; C07K
2317/565 20130101 |
Class at
Publication: |
424/136.1 ;
424/138.1; 424/133.1; 530/387.7; 530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 37/04 20060101
A61P037/04; A61P 29/00 20060101 A61P029/00; A61P 17/06 20060101
A61P017/06; C07K 16/40 20060101 C07K016/40; A61P 37/06 20060101
A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 2007 |
DK |
PA 2007 01278 |
Jun 30, 2008 |
DK |
PA 2008 00912 |
Claims
1. A method for inducing complement-mediated cell killing (CDC) in
the treatment of a tumor, said method comprising combined
administration, to a human being in need thereof, of a first
antibody and a second antibody, wherein said first antibody binds
EGFR, said second antibody binds EGFR, said first and second
antibody are non-cross-blocking, and the dosage regimen is such
that CDC is obtained at the tumor site.
2. The method of claim 1, wherein said first antibody is an
antibody which is capable of binding an EGFR epitope which is found
on all wild-type-EGFR-expressing cells.
3. The method of claim 1, wherein said first antibody binds to
human EGFR with an equilibrium dissociation constant (KD) of at
most 10.sup.-8 M, preferably at most 10.sup.-10 M.
4. The method of claim 1, wherein said first antibody is an
antibody which is capable of inducing ADCC at the tumor site in the
absence of said second antibody.
5. The method of claim 1, wherein said first antibody is selected
from the group consisting of: an antibody which binds the same EGFR
epitope as zalutumumab, an antibody which binds the same EGFR
epitope as cetuximab, an antibody which binds the same EGFR epitope
as panitumumab, an antibody which binds the same EGFR epitope as
nimotuzumab, an antibody which binds the same EGFR epitope as
matuzumab, and an antibody which binds the same EGFR epitope as
528.
6. The method of claim 1, wherein said first antibody is selected
from the group consisting of: an antibody which comprises the same
heavy chain CDR3 sequence as zalutumumab and binds the same EGFR
epitope as zalutumumab, an antibody which comprises the same heavy
chain CDR3 sequence as cetuximab and binds the same EGFR epitope as
cetuximab, an antibody which comprises the same heavy chain CDR3
sequence as panitumumab and binds the same EGFR epitope as
panitumumab, an antibody which comprises the same heavy chain CDR3
sequence as nimotuzumab and binds the same EGFR epitope as
nimotuzumab, an antibody which comprises the same heavy chain CDR3
sequence as matuzumab and binds the same EGFR epitope as matuzumab,
and an antibody which comprises the same heavy chain CDR3 sequence
as 528 and binds the same EGFR epitope as 528.
7. The method of claim 1, wherein said first antibody is selected
from the group consisting of: an antibody which comprises the same
6 CDR sequences as zalutumumab, an antibody which comprises the
same 6 CDR sequences as cetuximab, an antibody which comprises the
same 6 CDR sequences as panitumumab, an antibody which comprises
the same 6 CDR sequences as nimotuzumab, an antibody which
comprises the same 6 CDR sequences as matuzumab, and an antibody
which comprises the same 6 CDR sequences as 528.
8. The method of claim 1, wherein said first antibody is selected
from the group consisting of: zalutumumab, cetuximab, panitumumab,
nimotuzumab, matuzumab and 528.
9. The method of claim 5, wherein said first antibody is an
antibody which binds the same EGFR epitope as zalutumumab and said
second antibody is selected from the group consisting of: an
antibody which binds the same EGFR epitope as nimotuzumab, and an
antibody which binds the same EGFR epitope as matuzumab.
10. The method of claim 5, wherein said first antibody is an
antibody which binds the same EGFR epitope as cetuximab and said
second antibody is an antibody which binds the same EGFR epitope as
matuzumab.
11. The method of claim 5, wherein said first antibody is an
antibody which binds the same EGFR epitope as panitumumab and said
second antibody is an antibody which binds the same EGFR epitope as
matuzumab.
12. The method of claim 5, wherein said first antibody is an
antibody which binds the same EGFR epitope as nimotuzumab and said
second antibody is an antibody which binds the same EGFR epitope as
matuzumab.
13. The method of claim 1, wherein said second antibody is capable
of binding an EGFR epitope which is found in tumor cells, but is
not detectable in normal cells.
14. The method of claim 13, wherein said EGFR epitope does not
demonstrate any amino acid sequence alterations or substitutions as
compared to wild-type EGFR.
15. The method of claim 13, wherein said second antibody binds an
EGFR epitope which is located within the region comprising residues
273-501 of EGFR.
16. The method of claim 1, wherein said second antibody binds an
EGFR epitope, which is located within the region comprising
residues 287-302 of EGFR.
17. The method of claim 1, wherein said second antibody is
cross-blocking with ch806.
18. The method of claim 1, wherein said second antibody binds the
same EGFR epitope as ch806.
19. The method of claim 18, wherein the second antibody comprises
SEQ ID NO:3 and one or more or all of SEQ ID NO: 1, 2, 4, 5 and
6.
20. The method of claim 18, wherein the second antibody is
ch806.
21. The method of claim 13, wherein the second antibody is
MR1-1.
23. The method of claim 1, wherein said second antibody binds to
EGFR-vIII with a KD which is at least 10 fold lower than the KD for
binding to wild-type EGFR.
24. The method of claim 1, wherein the first and/or the second
antibody is a human antibody.
25. The method of claim 1, wherein the dosage regimen of said first
antibody comprises administration, at least once per 14 days, of a
dosage of antibody of at least 0.1 mg/kg.
26. The method of claim 1, wherein the dosage regimen of said
second antibody comprises administration, at least once per 14
days, of a dosage of antibody of at least 0.1 mg/kg.
27. The method of claim 25, wherein the administration of said
first and second antibody is at least once per week.
28. The method of claim 1, wherein the dosage regimen for said
first antibody is lower than a standard dosage regimen for said
first antibody.
29. The method of claim 1, wherein the dosage regimen of said first
antibody comprises administration of a total dosage per 14 days of
between 0.01 mg/kg and 2 mg/kg.
30. The method of claim 1, wherein the dosage regimen for said
second antibody is lower than a standard dosage regimen for said
second antibody.
31. The method of claim 1, wherein the dosage regimen of said
second antibody comprises administration of a total dosage per 14
days of between 0.01 mg/kg and 2 mg/kg.
32. The method of claim 1, wherein the dosage regimen is such that
substantially no CDC is obtained at non-tumor sites.
33. The method of claim 1, wherein the dosage regimen ensures
efficient inhibition of ligand binding at tumor sites.
34. The method of claim 13, wherein the dosage regimen for said
first antibody is a dosage regimen which comprises an equal or a
higher dosage than a standard dosage regimen for said first
antibody.
35. The method of claim 13, wherein said first antibody is
administered at an at least 2 times higher dose than said second
antibody.
36. The method of claim 35, wherein said first antibody is
administered at a between 2 and 50 times higher dose than said
second antibody.
37. The method of claim 13, wherein: the dosage regimen of the
first antibody comprises administration, at least once per 14 days,
of a dose of antibody of at least 2 mg/kg, and the dosage regimen
of the second antibody comprises administration of a total dosage
per 14 days of between 0.1 mg/kg and 1 mg/kg.
38. The method of claim 13, wherein: the dosage regimen of the
first antibody comprises administration, at least once per 14 days,
of a dose of antibody of at least 4 mg/kg, and the dosage regimen
of the second antibody comprises administration of a total dosage
per 14 days of between 0.1 mg/kg and 2 mg/kg.
39. The method of claim 1, wherein said second antibody is
administered at least 15 minutes before the first antibody.
40. The method of claim 1, wherein the total duration of the
treatment is at least one month.
41. The method of claim 1, wherein said first and/or second
antibody is administered parenterally, preferably
intravenously.
42. The method of claim 1, further comprising administration of a
third antibody, wherein said third antibody is not cross-blocking
with either of said first and second antibody.
43. The method of claim 1, wherein said tumor is selected from the
group consisting of: breast cancer tumor, bladder cancer tumor,
uterine/cervical cancer tumor, esophageal cancer tumor, pancreatic
cancer tumor, colorectal cancer tumor, kidney cancer tumor, ovarian
cancer tumor, prostate cancer tumor, head and neck cancer tumor,
non-small cell lung cancer tumor, stomach tumor, glioblastoma and
other EGFR-expressing tumors.
44. The method of claim 1, wherein the EGFR levels in the tumor
cells to be treated are not below threshold for obtaining ADCC when
treated with the first antibody without co-administration of the
second antibody.
45. The method of claim 1, comprising administration of one or more
further therapies selected from chemotherapeutic agents,
immunosuppressive agents, anti-inflammatory agents, anti-psoriasis
agents, radiation therapy, hyperthermia, transplantation, surgery,
sunlight therapy, and phototherapy.
46. The method of claim 1, comprising administration of one or more
further therapies selected from the group consisting of nitrogen
mustards, aziridines, alkyl sulfonates, nitrosoureas, platinum
complexes, non-classical alkylating agents, folate analogs, purine
analogs, adenosine analogs, pyrimidine analogs, substituted ureas,
antitumor antibiotics, epipodophyllotoxins, microtubule agents,
camptothecin analogs, enzymes, cytokines, monoclonal antibodies,
recombinant toxins and immunotoxins, cancer gene therapies, and
cancer vaccines.
47. The method of claim 1, comprising administration of one or more
further therapies selected from the group consisting of
immunosuppressive antibodies against MHC, CD2, CD3, CD4, CD7, CD28,
B7, CD40, CD45, IFN-gamma, TNF-alpha, IL-4, IL-5, IL-6R, IL-7,
IL-10, CD11a, CD20, and CD58 or antibodies against their ligands,
soluble IL-15R, and IL-10.
48. The method of claim 1, comprising administration of one or more
further therapies selected from the group consisting of
cyclosporine, azathioprine, mycophenolic acid, mycophenolate
mofetil, corticosteroids, methotrexate, gold salts, sulfasalazine,
antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,
tacrolimus (FK-506), OKT3, anti-thymocyte globulin,
transplantation, and surgery.
49. The method of claim 1, comprising administration of one or more
further therapies selected from the group consisting of aspirin,
other salicylates, steroidal drugs, NSAIDs (nonsteroidal
anti-inflammatory drugs), Cox-2 inhibitors, and DMARDs (disease
modifying antirheumatic drugs).
50. The method of claim 1, comprising administration of one or more
further therapies selected from the group consisting of coal tar, A
vitamin, anthralin, calcipotrien, tarazotene, corticosteroids,
methotrexate, retinoids, cyclosporine, etanercept, alefacept,
efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus
(FK-506), hydroxyurea, sunlight therapy, and phototherapy.
51. The method of claim 1, comprising administration of one or more
tyrosine kinase inhibitors, such as gefitinib, erlotinib, XL-647,
JNJ-26483327, vandetanib, BMS-599626, AZD-9935, AEE-788, BIBW-2992,
ISU-101, HMPL-010, ON-012380, EKI-785, TX-2036, EHT-102, KI-6783,
KI-6896 and LFM-A12.
52. The method of claim 1, wherein said tumor is an
EGFRvIII-expressing tumor.
53. The method of claim 1, wherein the human being in need of the
treatment is a human being who has been diagnosed to have tumors
that exhibit EGFRvIII expression.
54. A first antibody for use in the treatment of a tumor in
combination with a second antibody, wherein said first antibody
binds EGFR, said second antibody binds EGFR, said first and second
antibody are non-cross-blocking, and the dosage regimen is such
that CDC is obtained at the tumor site.
55. A first antibody for use in the treatment of a tumor in
combination with a second antibody, wherein said first antibody
binds EGFR, said second antibody binds EGFR, said first and second
antibody are non-cross-blocking, and the dosage regimen is such
that CDC is obtained at the tumor site, wherein the first antibody,
the second antibody and/or the treatment comprises one or more of
the further features of claim 2.
56. A second antibody for use in the treatment of a tumor in
combination with a first antibody, wherein said first antibody
binds EGFR, said second antibody binds EGFR, and said first and
second antibody are non-cross-blocking, and the dosage regimen is
such that CDC is obtained at the tumor site.
57. A second antibody for use in the treatment of a tumor in
combination with a first antibody, wherein said first antibody
binds EGFR, said second antibody binds EGFR, and said first and
second antibody are non-cross-blocking, and the dosage regimen is
such that CDC is obtained at the tumor site, wherein the first
antibody, the second antibody and/or the treatment comprises one or
more of the further features of claim 2.
58. Use of a first antibody and a second antibody for the
preparation of a medicament for the treatment of a tumor, wherein
said first antibody binds EGFR, said second antibody binds EGFR,
and said first and second antibody are non-cross-blocking, and the
dosage regimen is such that CDC is obtained at the tumor site.
59. Use of a first antibody and a second antibody for the
preparation of a medicament for the treatment of a tumor, wherein
said first antibody binds EGFR, said second antibody binds EGFR,
and said first and second antibody are non-cross-blocking, and the
dosage regimen is such that CDC is obtained at the tumor site,
comprising one or more of the further features of claim 2.
60. A bispecific antibody comprising a first binding specificity
which binds an EGFR epitope which is found on all
wild-type-EGFR-expressing cells and a second binding specificity
which binds an EGFR epitope which is found in tumor cells, but is
not detectable in normal cells.
61. The bispecific antibody of claim 60, wherein the second binding
specificity binds an EGFR epitope is located within the region
comprising residues 273-501 of EGFR, preferably the same EGFR
epitope as bound by ch806, wherein said first and second binding
specificity are non-cross-blocking.
62. The bispecific antibody of claim 60, wherein the second binding
specificity is specific for EGFR-vIII.
63. The bispecific antibody of claim 60, wherein the antibody
comprises a first binding specificity which binds an epitope
selected from the group consisting of: the EGFR epitope bound by
zalutumumab, the EGFR epitope bound by cetuximab, the EGFR epitope
bound by panitumumab, the EGFR epitope bound by nimotuzumab, the
EGFR epitope bound by matuzumab, and--the EGFR epitope bound by
528.
64. A bispecific antibody as defined in claim 60 for use as a
medicament.
65. A bispecific antibody as defined in claim 60 for use as a
medicament for the treatment of cancer.
66. Use of a bispecific antibody as defined in claim 60 for the
preparation of a medicament for the treatment of cancer.
67. A method for the treatment of cancer comprising administration
of a bispecific antibody as defined in claim 60.
68. The bispecific antibody of claim 60, wherein said tumor is
selected from the group consisting of: breast cancer tumor, bladder
cancer tumor, uterine/cervical cancer tumor, esophageal cancer
tumor, pancreatic cancer tumor, colorectal cancer tumor, kidney
cancer tumor, ovarian cancer tumor, prostate cancer tumor, head and
neck cancer tumor, non-small cell lung cancer tumor, stomach cancer
tumor, glioblastoma and other EGFR-expressing tumors.
Description
[0001] All patents, patent applications and other publications
cited herein are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel methods for the
treatment of tumors, comprising administration of a bispecific
antibody or a combination of two or more non-cross-blocking
antibodies that recognize the same target antigen or antigenic
complex. Furthermore, the invention relates to novel anti-EGFR
antibodies.
BACKGROUND OF THE INVENTION
[0003] The erbB family of four receptor tyrosine kinases,
EGFR/HER1/erbB1, HER2/NEU/erbB2, HER3/erbB3 and HER4/erbB4 occupies
a central role in a wide variety of biological processes from
neuronal development to breast cancer. EGFR, the epidermal growth
factor receptor, is a tyrosine kinase receptor with critical
functions in the regulation of cell proliferation, differentiation
and survival (Ullrich and Schlessinger (1990) Cell 61:203-212).
Dysregulated function or expression of the EGFR is observed in
common cancers such as lung, colon, head and neck, and also in
non-epithelial malignancies such as glioblastomas--often
correlating with a poor prognosis for the patients. Due to its
documented involvement in tumorigenesis, EGFR constitutes a
promising molecule for targeted therapy (Mendelsohn (2002) J. Clin.
Oncol. 20:1S-13S).
[0004] So far, two EGFR-directed approaches have been successfully
introduced into clinical practice: small molecule tyrosine kinase
inhibitors, and EGFR-directed monoclonal antibodies (Baselga et al.
(2005) J. Clin. Oncol. 23:2445-2459). Anti-EGFR antibodies that
have been tested in clinical trials include cetuximab (C225),
panitumumab (E7.6.3), nimotuzumab (hR3), matuzumab (425),
zalutumumab (2F8) and ch806. While most antibodies recognize both
wild-type and mutant forms of EGFR (e.g. the EGFR-vIII mutant),
antibody ch806 preferentially recognizes an epitope only exposed on
overexpressed, mutant or ligand-activated forms of EGFR (Scott et
al. (2007) PNAS 104:4071.4076; WO 02/092771). With the exception of
panitumumab, all these EGFR antibodies are of the human IgG1
isotype.
[0005] WO 2004/032960 describes that combination of two anti-EGFR
antibodies that bind to different epitopes on EGFR gives a minor
increase in inhibition of ligand binding and an increase in EGFR
down-modulation as compared to each of the antibodies alone.
Pharmaceutical compositions or kits comprising such antibody
combinations and their use in the treatment of tumors have been
proposed in WO 2004/032960.
[0006] WO 02/100348 discloses a composition comprising a
combination of two or more human anti-EGFR antibodies, wherein each
of said antibodies or antigen-binding portions thereof binds to a
distinct epitope of EGFR. WO 02/092771 discloses that a composition
comprising ch806 may be administered with, or may include
combinations along with other anti-EGFR antibodies.
[0007] Perera et al. (2005) Clin. Cancer Res. 11:6390-6399 describe
that treatment of human tumor xenografts with antibody ch806 in
combination with the non-tumor-specific monoclonal antibody 528
generates enhanced antitumor activity. A down-modulation of the
receptor was observed.
[0008] Antibodies can have activity on target cells via several
different mechanisms of action. Conceptually, these effector
mechanisms can be divided into direct mechanisms, mediated by the
antibodies' variable regions, and indirect mechanisms, which are
triggered by their constant regions. Direct mechanisms include
blockade of ligand binding and signalling, receptor
down-modulation, induction of apoptosis and inhibition of growth
and survival. Indirect mechanisms include complement-dependent
tumor cell lysis or complement-dependent cytotoxicity (CDC) and
effector-cell-mediated tumor killing or antibody-dependent
cell-mediated cytotoxicity (ADCC), tumor cell phagocytosis, and
potentially antibody-mediated antigen presentation.
[0009] The complement system is a phylogenetically old cascade of
proteases, which is tightly controlled by regulatory proteins in
the plasma and on cellular surfaces (Walport (2001) N. Engl. J.
Med. 344:1058-1066). Complement constitutes an integral link
between the innate and the adaptive immune systems (Carroll (1998)
Annu. Rev. Immunol. 16:545.568), and lack of critical components
predisposes to immunodeficiency and autoimmunity (Frank (1987) N.
Engl. J. Med. 316-1525-1530). Today, three pathways of complement
activation have been identified--with the "classical" pathway
triggered by C1q binding to complexed IgG. Complement activation
may lead to the formation of the "membrane attack complex" (MAC)
triggering lytic killing of bacteria and eukaryotic cells.
Furthermore, complement components like C5a and C3a are potent
chemoattractants for immune effector cells, and other complement
proteins like C3b and C3d effectively enhance antigen presentation
(Fearon et al. (1998) Semin. Immunol. 10:355-361).
[0010] The contribution of complement for the in vivo efficacy of
therapeutic antibodies has been investigated most extensively for
CD20 antibodies like e.g. rituximab (Taylor (2004) Blood 104:1592).
Here, data suggest that complement dependent killing mechanisms may
at least contribute to antibody efficacy under certain
conditions.
[0011] Complement-dependent tumor cell killing has so far not been
described for EGFR antibodies either in vivo or in vitro.
[0012] While the above-described antibody therapies have proved to
be of significant benefit for cancer therapy, a need for further
improvement of cancer therapy remains, in particular for aggressive
EGFR-associated cancers, e.g. lung and head and neck cancers, which
still have a poor prognosis.
SUMMARY OF THE INVENTION
[0013] It has now surprisingly been found that combinations of
non-cross-blocking anti-EGFR antibodies very potently deposit
complement components C1q and C4c on tumor cells, leading to highly
effective complement-mediated cell killing (CDC). This observation
has provided the basis for new and more efficient methods of
treatment of cancer.
[0014] Accordingly, in a first main aspect, the invention relates
to a method for inducing complement-mediated cell killing (CDC) in
the treatment of a tumor comprising combined administration, to a
human being in need thereof, of a first antibody and a second
antibody, wherein
[0015] said first antibody binds EGFR,
[0016] said second antibody binds EGFR,
[0017] said first and second antibody are non-cross-blocking,
and
[0018] the dosage regimen is such that CDC is obtained at the tumor
site.
[0019] In a particularly interesting embodiment of the method of
the invention, [0020] a) the first antibody is an antibody which
binds an EGFR epitope which is found in all
wild-type-EGFR-expressing cells, [0021] b) the second antibody is
an antibody, such as ch806, which binds an EGFR epitope which is
found in tumor cells, but is not detectable in normal cells, and
[0022] c) the dosage regimen is such that substantial CDC is
obtained at tumor sites, but substantially no CDC is obtained at
non-tumor sites.
[0023] For example, the dosage regimen defined in c) may be
obtained by dosing the first antibody in a dosage regimen which is
at least equal to what is usual for anti-EGFR antibody therapy and
dosing the second antibody in a dosage regimen which is
significantly lower than what is usual for anti-EGFR antibody
therapy. Without being bound by any specific theory, it is believed
that in such a dosage regimen: [0024] the full dosage of the first
antibody ensures efficient anti-tumor activity on cells that
overexpress wild-type EGFR, inter alia through efficient inhibition
of ligand binding and/or ADCC, and [0025] an additional therapeutic
effect is obtained from CDC due to the combination of two
non-cross-blocking antibodies, wherein the lower dosage of the
second antibody, together with the preferential binding to tumor
cells, ensures CDC activation at tumor sites, while avoiding
substantial CDC at non-tumor sites.
[0026] In a further main aspect, the invention relates to a
bispecific antibody comprising a first binding specificity which
binds an EGFR epitope which is found on all
wild-type-EGFR-expressing cells and a second binding specificity
which binds an EGFR epitope which is found in tumor cells, but is
not detectable in normal cells, preferably an EGFR epitope which is
located within the region comprising residues 273-501 of EGFR, more
preferably the same EGFR epitope as bound by ch806, wherein said
first and second binding specificity are non-cross-blocking.
[0027] In an even further aspect, the invention relates to an
isolated monoclonal antibody which binds to human EGFR, wherein the
antibody binds to the same epitope on EGFR as an antibody selected
from the group consisting of:
[0028] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 7 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
8;
[0029] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
10;
[0030] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
11;
[0031] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 12 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
13;
[0032] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
15;
[0033] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
16; and
[0034] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 17 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
18.
DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Inhibition of ligand binding to EGFR by
EGFR-antibodies. A431 cells were co-incubated with 2.5 .mu.g/ml
FITC-conjugated EGF and 200 .mu.g/ml antibodies. Ligand binding was
analyzed by flow cytometry. Blockade of ligand binding was
calculated by the formula: % inhibition of EGF-binding=(RFI
without-RFI with antibody)/(RFI without antibody).times.100. Data
are presented as mean.+-.SEM of three independent experiments.
[0036] FIG. 2: Inhibition of ligand-induced EGFR phosphorylation by
EGFR-antibodies. EGF-induced receptor phosphorylation of A431 cells
was measured in the absence or presence of EGFR-antibodies.
[0037] FIGS. 3 and 4: Inhibition of A431 cell proliferation by
EGFR-antibodies.
[0038] FIG. 5: Stimulation of PBMC-induced ADCC of A431 target
cells by EGFR-antibodies.
[0039] FIG. 6: FACS analyses of EGFR-antibody binding to A431
cells. Data are presented as mean.+-.SEM of two independent
observations. The concentrations of half-maximal binding (EC50) are
determined from a four-parameter logistic curve fit and expressed
in .mu.g/ml.
[0040] FIG. 7: Epitope analyses by competitive immunofluorescence.
Non-saturating concentrations of indicated FITC-conjugated EGFR
antibodies were incubated with 200-fold excess of unlabeled
antibodies. Immunofluoresence in the presence of KLH antibody
determined the maximum fluorescence. "% of maximal MFI" was
calculated. Data are presented as mean.+-.SEM of at least three
independent experiments, * indicates significant changes in binding
(p<0.05).
[0041] FIG. 8: Cartoon of EGFR epitopes recognized by different
antibodies. Significant inhibition in competitive
immunofluorescence experiments is indicated by overlapping circles,
while non-overlapping circles indicate that the respective
antibodies did not significantly cross-block each other.
[0042] FIG. 9: Binding of EGFR-antibodies to Ba/F3 cells expressing
EGFR-vIII deletion mutant, which lacks AA 6-273, the major portion
of domains I and II.
[0043] FIGS. 10 and 11: C1q deposition by EGFR antibody
combinations. C1q deposition on A431 cells was analyzed in the
presence of individual EGFR antibodies, or in the presence of
antibody combinations (final antibody concentration 10 .mu.g/mL).
(FIGS. 10a and 10b) While individual EGFR antibodies did not
trigger C1q deposition, all examined non cross-blocking
combinations led to C1q deposition (significance (p<0.05)
indicated by *). Data are presented as mean.+-.SEM of at least
three independent experiments. As shown in FIG. 11, the combination
of three non-blocking antibodies is superior to individual
combinations in C1q deposition (n=3; significant binding
(p<0.05) is indicated by *, significant difference between
triple and double combinations (p<0.05) by #).
[0044] FIG. 12: Complement dependent killing by individual EGFR
antibodies and by antibody combinations. Individual EGFR
antibodies, cross-blocking and non cross-blocking combinations were
analyzed for their capacity to trigger CDC of (a) A431 cells and
(b) A1207 cells. While none of the individual EGFR antibodies and
none of the cross-blocking combinations triggered CDC, most of the
non cross-blocking combinations led to significant CDC (p<0.05,
indicated by *). Data are presented as mean.+-.SEM of "% specific
lysis" from at least three independent experiments.
[0045] FIG. 13: CDC in correlation to antibody concentrations. CDC
of A431 (13a) and A1207 (13b) cells by individual EGFR antibodies
and by antibody combinations was analyzed at various antibody
concentrations. Indicated antibody concentrations refer to each
individual antibody. Results are presented as mean.+-.SEM of "%
specific lysis" of three independent experiments, significant CDC
(p<0.05) is indicated by *.
[0046] FIG. 14: CDC by EGFR antibody combinations is mediated by
the classical complement pathway. To identify the respective
contribution of the alternative and the classical complement
pathways in CDC by EGFR antibody combinations, CDC assays were
performed in the presence of Mg-EGTA (inactivation of the classical
pathway) or EDTA (inactivation of both pathways), and after
inactivation of the alternative (50.degree. C. for 15 min) or both
(56.degree. C. for 30 min) pathways. Data are presented as
mean.+-.SEM of two independent experiments.
[0047] FIGS. 15 and 16: C1q and C4c binding (FIG. 15, n=1) and CDC
(FIG. 16, n=2) by EGFR antibody combinations. Combinations of EGFR
antibody LC1006-003 with 2F8-IgG1, 2F8-IgG4 or 2F8-F(ab).sub.2 were
tested.
[0048] FIG. 17: Alignment of VH (17a) and VL (17b) sequences of
LC1006-003, LC1006-005, LC1006-008, LC1006-0011 and LC1006-018.
Dots indicate identity to the reference sequence.
[0049] FIG. 18: Comparison of ch806, MR1-1 and zalutumumab binding
properties to untransfected or EGFR-vIII transfected A431 cells,
and wild type or EGFR-vIII transfected Ba/F3 cells.
[0050] FIG. 19: CDC of untransfected and EGFR-vIII transfected A431
cells by combinations of EGFR antibodies with ch806 or MR1-1. Cells
were incubated with individual antibodies, or with antibody
combinations at additive antibody concentrations of 10
.mu.g/ml/well.
[0051] FIG. 20: CDC induction by double and triple combinations
with ch806 or MR1-1. Cells were incubated with individual
antibodies, or with antibody combinations at additive antibody
concentrations of 20 .mu.g/ml/well. * marks significant CDC, a
significant difference between double and triple combinations
(p<0.05) is indicated by #.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0052] The term "erbB protein", when used herein, refers to a
protein of the erbB protein family. This family is composed of four
members: EGFR/HER1/erbB1, HER2/NEU/erbB2, HER3/erbB3 and
HER4/erbB4. Unless specified otherwise, the term "EGFR" includes
both wild-type EGFR and mutant or variant forms of EGFR, such as
EGFRvIII.
[0053] When used herein, the terms "cross-blocking" or
"non-cross-blocking" in the context of two antibodies, refer to two
antibodies which, respectively, do and do not significantly compete
for binding to an antigen, such as EGFR, in the assay described in
Example 5, with a threshold for significance of 50%. Thus, two
cross-blocking antibodies cannot be bound to the target antigen at
the same time. Two non-cross-blocking antibodies on the other hand
can be bound to the target antigen at the same time.
[0054] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. Methods for epitope
mapping are well-known in the art.
[0055] When used herein, the term "same epitope", e.g. in the
context of two antibodies that bind the same epitope, refers to
antibodies that bind the same amino acid residues on the target
antigen. Thus, "same epitope" is a more narrow concept than
"cross-blocking", since two antibodies can e.g. bind different
epitopes, i.e. different amino acid residues on the target antigen,
but still be cross-blocking due to steric hindrance. Typically, two
antibodies that cross-block with each other, but differ with
respect to cross-blocking with a third antibody, do not bind the
same epitope.
[0056] When used herein, the term "normal" cells in the context of
EGFR-expressing cells refers to a cell, e.g. a keratinocyte, which
expresses endogenous EGFR, but not the truncated de2-7 form of EGFR
(EGFR-vIII). Furthermore, the term specifically excludes a cell
that overexpresses the EGFR gene (see also WO 02/092771,
incorporated herein by reference).
[0057] The term "all wild-type-EGFR-expressing cells", on the other
hand, refers to all cells that express non-truncated wild-type
EGFR, regardless of whether the cells is a tumor cell or not and
regardless of whether EGFR is overexpressed or not.
[0058] When used herein, the term "immunoglobulin" refers to a
class of structurally related glycoproteins consisting of two pairs
of polypeptide chains, one pair of light (L) low molecular weight
chains and one pair of heavy (H) chains, all four inter-connected
by disulfide bonds. The structure of immunoglobulins has been well
characterized. See for instance Fundamental Immunology Ch. 7 (Paul,
W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy
chain typically is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H) and a heavy chain constant region.
The heavy chain constant region typically is comprised of three
domains, C.sub.H1, C.sub.H2, and C.sub.H3. Each light chain
typically is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region typically is comprised of one
domain, C.sub.L. The V.sub.H and V.sub.L regions may be further
subdivided into regions of hyper-variability (or hypervariable
regions which may be hypervariable in sequence and/or form of
structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each V.sub.H and V.sub.L
is typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.
Biol. 196, 901-917 (1987)). Typically, the numbering of amino acid
residues in this region is performed by the method described in
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md. (1991) (phrases such as variable domain residue numbering as in
Kabat or according to Kabat herein refer to this numbering system
for heavy chain variable domains or light chain variable domains).
Using this numbering system, the actual linear amino acid sequence
of a peptide may contain fewer or additional amino acids
corresponding to a shortening of, or insertion into, a FR or CDR of
the variable domain. For example, a heavy chain variable domain may
include a single amino acid insert (residue 52a according to Kabat)
after residue 52 of V.sub.H CDR2 and inserted residues (for
instance residues 82a, 82b, and 82c, etc. according to Kabat) after
heavy chain FR residue 82. The Kabat numbering of residues may be
determined for a given antibody by alignment at regions of homology
of the sequence of the antibody with a "standard" Kabat numbered
sequence.
[0059] The term "antibody" (Ab) in the context of the present
invention refers to an immunoglobulin molecule, a fragment of an
immunoglobulin molecule, or a derivative of either thereof, which
has the ability to specifically bind to an antigen under typical
physiological conditions for significant periods of time such as at
least about 30 minutes, at least about 45 minutes, at least about
one hour, at least about two hours, at least about four hours, at
least about 8 hours, at least about 12 hours, about 24 hours or
more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days,
etc., or any other relevant functionally-defined period (such as a
time sufficient to modulate a physiological response associated
with antibody binding to the antigen and/or time sufficient for the
antibody to recruit an Fc-mediated effector activity). The variable
regions of the heavy and light chains of the immunoglobulin
molecule contain a binding domain that interacts with an antigen.
The constant regions of the antibodies (Abs) may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (such as effector cells) and
components of the complement system such as C1q, the first
component in the classical pathway of complement activation.
[0060] As indicated above, the term antibody herein, unless
otherwise stated or clearly contradicted by context, includes
fragments of an antibody that retain the ability to specifically
bind to an antigen. It has been shown that the antigen-binding
function of an antibody may be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antibody" include, but are not limited to: (i) a
Fab fragment, a monovalent fragment consisting of the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; (ii) F(ab).sub.2 and
F(ab').sub.2 fragments, bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting essentially of the V.sub.H and C.sub.H1
domains; (iv) a Fv fragment consisting essentially of the V.sub.L
and V.sub.H domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., Nature 341, 544-546 (1989)), which consists
essentially of a V.sub.H domain and also called domain antibodies
(Holt et al; Trends Biotechnol 2003 November; 21(11):484-90); (vi)
camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005
January; 5(1):111-24), and (vii) an isolated complementarity
determining region (CDR). Furthermore, although the two domains of
the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they may be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain antibodies or single
chain Fv (scFv), see for instance Bird et al., Science 242, 423-426
(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such
single chain antibodies are encompassed within the term antibody
unless otherwise noted or clearly indicated by context.
[0061] Antibodies interact with target antigens primarily through
amino acid residues that are located in the six heavy and light
chain CDRs. For this reason, the amino acid sequences within CDRs
are more diverse between individual antibodies than sequences
outside of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted into framework sequences from a different antibody
with different properties (see for instance Riechmann, L. et al.,
Nature 332, 323-327 (1998), Jones, P. et al., Nature 321, 522-525
(1986) and Queen, C. et al., PNAS USA 10029-10033 (1989)).
[0062] It also should be understood that the term antibody also
generally includes polyclonal antibodies, monoclonal antibodies
(mAbs), antibody-like polypeptides, such as chimeric antibodies and
humanized antibodies, anti-idiotypic (anti-Id) antibodies to
antibodies, and antibody fragments retaining the ability to
specifically bind to the antigen (antigen-binding fragments)
provided by any known technique, such as enzymatic cleavage,
peptide synthesis, and recombinant techniques. An antibody as
generated can possess any isotype.
[0063] The term "bispecific molecule" is intended to include any
agent, such as a protein, peptide, or protein or peptide complex,
which has two different binding specificities. For example, the
molecule may bind to, or interact with, (a) a cell surface antigen
and (b) an Fc receptor on the surface of an effector cell.
[0064] The term "bispecific antibodies" is intended to include any
EGFR antibody, which is a bispecific molecule. The term "bispecific
antibodies" also includes diabodies and SMIP.TM.s (Trubion).
Diabodies are bivalent, bispecific antibodies in which the V.sub.H
and V.sub.L domains are expressed on a single polypeptide chain,
but using a linker that is too short to allow for pairing between
the two domains on the same chain, thereby forcing the domains to
pair with complementary domains of another chain and creating two
antigen binding sites (see for instance Holliger, P. et al., PNAS
USA 90, 6444-6448 (1993), Poljak, R. J. et al., Structure 2,
1121-1123 (1994)). Methods for construction of bispecific
antibodies have e.g. been discussed in Marcin and Zhu (2005) Acta
Pharmacol Sin. 26:649.
[0065] In one embodiment, the two different binding specificities
of the bispecific antibody are each contained within a
half-molecule. A half-molecule typically consists of one heavy
chain molecule and one light chain molecule.
[0066] As used herein, the terms "inhibits binding" and "blocks
binding" (for instance when referring to inhibition/blocking of
binding of a ligand to EGFR) are used interchangeably herein and
encompass both partial and complete inhibition/blocking. The
inhibition/blocking of binding of a ligand to a receptor normally
reduces or alters the normal level or type of cell signaling that
occurs when a ligand binds to the receptor. Inhibition and blocking
are also intended to include any measurable decrease in the binding
affinity of a ligand to its receptor due to a binding protein, e.g.
an antibody. Binding of a ligand to a receptor may e.g. be
inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 99%, or 100%.
[0067] As used herein, the term "effector cell" refers to an immune
cell which is involved in the effector phase of an immune response,
as opposed to the cognitive and activation phases of an immune
response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, for instance lymphocytes (such as B cells and T
cells including cytolytic T cells (CTLs)), killer cells, natural
killer cells, macrophages, monocytes, eosinophils, neutrophils,
polymorphonuclear cells, granulocytes, mast cells, and basophiles.
Some effector cells express specific Fc receptors and carry out
specific immune functions. In some embodiments, an effector cell is
capable of inducing antibody-dependent cellular cytotoxicity
(ADCC), such as a neutrophil capable of inducing ADCC. For example,
monocytes, macrophages, which express FcR are involved in specific
killing of target cells and presenting antigens to other components
of the immune system, or binding to cells that present antigens. In
some embodiments, an effector cell may phagocytose a target
antigen, target cell, or microorganism. The expression of a
particular FcR on an effector cell may be regulated by humoral
factors such as cytokines. For example, expression of Fc.gamma.RI
has been found to be up-regulated by interferon .gamma.
(IFN-.gamma.) and/or G-CSF. This enhanced expression increases the
cytotoxic activity of Fc.gamma.RI-bearing cells against targets. An
effector cell can phagocytose or lyse a target antigen or a target
cell.
[0068] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the present invention may include amino acid residues not
encoded by human germ line immunoglobulin sequences (for instance
mutations introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germ line of another mammalian
species, such as a mouse, have been grafted into human framework
sequences. As used herein, a human antibody is "derived from" a
particular germ line sequence if the antibody is obtained from a
system using human immunoglobulin sequences, for instance by
immunizing a transgenic mouse carrying human immunoglobulin genes
or by screening a human immunoglobulin gene library, and wherein
the selected human antibody is at least 90%, such as at least 95%,
for instance at least 96%, such as at least 97%, for instance at
least 98%, or such as at least 99% identical in amino acid sequence
to the amino acid sequence encoded by the germ line immunoglobulin
gene. Typically, outside the heavy chain CDR3, a human antibody
derived from a particular human germ line sequence will display no
more than 10 amino acid differences, such as no more than 5, for
instance no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germ line immunoglobulin
gene.
[0069] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies. The term "chimeric
antibody" includes monovalent, divalent, or polyvalent antibodies.
Chimeric antibodies are produced by recombinant processes well
known in the art (see for instance Cabilly et al., PNAS USA 81,
3273-3277 (1984), Morrison et al., PNAS USA 81, 6851-6855 (1984),
Boulianne et al., Nature 312, 643-646 (1984), EP125023, Neuberger
et al., Nature 314, 268-270 (1985), EP171496, EP173494, WO86/01533,
EP184187, Sahagan et al., J. Immunol. 137, 1066-1074 (1986),
WO87/02671, Liu et al., PNAS USA 84, 3439-3443 (1987), Sun et al.,
PNAS USA 84, 214-218 (1987), Better et al., Science 240, 1041-1043
(1988) and Harlow et al., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1988)).
[0070] A "humanized antibody" is an antibody that is derived from a
non-human species, in which certain amino acids in the framework
and constant domains of the heavy and light chains have been
mutated so as to avoid or abrogate an immune response in humans.
Humanized forms of non-human (for instance murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, a humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. A humanized
antibody typically also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature 321,
522-525 (1986), Riechmann et al., Nature 332, 323-329 (1988) and
Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992).
[0071] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions derived from
human germ line immunoglobulin sequences. The human monoclonal
antibodies may be generated by a hybridoma which includes a B cell
obtained from a transgenic or transchromosomal nonhuman animal,
such as a transgenic mouse, having a genome comprising a human
heavy chain transgene and a light chain transgene, fused to an
immortalized cell.
[0072] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (such as a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared there from (described further elsewhere herein), (b)
antibodies isolated from a host cell transformed to express the
antibody, such as from a transfectoma, (c) antibodies isolated from
a recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germ line
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies may be subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences
is used, in vivo somatic mutagenesis) and thus the amino acid
sequences of the V.sub.H and V.sub.L regions of the recombinant
antibodies are sequences that, while derived from and related to
human germ line V.sub.H and V.sub.L sequences, may not, naturally
exist within the human antibody germ line repertoire in vivo.
[0073] As used herein, "antibody which binds X" refers to the
binding of an antibody to a predetermined antigen X. Typically, the
antibody binds with an affinity corresponding to a K.sub.D of about
10.sup.-7 M or less, such as about 10.sup.-8 M or less, such as
about 10.sup.-9 M or less, about 10.sup.-10 M or less, or about
10.sup.-11 M or even less, when determined by for instance surface
plasmon resonance (SPR) technology in a BIAcore 3000 instrument
using the antigen as the ligand and the antibody as the analyte,
and binds to the predetermined antigen with an affinity
corresponding to a K.sub.D that is at least ten-fold lower, such as
at least 100 fold lower, for instance at least 1000 fold lower,
such as at least 10,000 fold lower, for instance at least 100,000
fold lower than its affinity for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen. The amount with which the affinity is
lower is dependent on the K.sub.D of the antibody, so that when the
K.sub.D of the antibody is very low, then the amount with which the
affinity for the antigen is lower than the affinity for a
non-specific antigen may be at least 10,000 fold. Binding affinity
also may be determined by equilibrium methods (for instance
enzyme-linked immunoabsorbent assay (ELISA) or radioimmuno-assay
(RIA)).
[0074] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular antibody-antigen
interaction.
[0075] Antigen binding is preferably specific. The term "specific"
herein refers to the ability of an antibody, e.g. an anti-EGFR
antibody, to recognize an epitope within an antigen, e.g. EGFR,
while only having little or no detectable reactivity with other
portions of the antigen or with another, unrelated, antigen.
Specificity may be relatively determined by competition assays as
described herein. Specificity can more particularly be determined
by any of the epitope identification/characterization techniques
described herein or their equivalents known in the art. An antibody
specific for a particular antigenic determinant may nonetheless
cross-react with other biomolecules. For instance, an anti-EGFR
antibody that binds human EGFR may cross-react with EGFR homologues
from other species.
[0076] As used herein, "isotype" refers to the immunoglobulin class
(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgA2, IgE, or IgM)
that is encoded by heavy chain constant region genes.
[0077] The terms "transgenic, non-human animal" refers to a
non-human animal having a genome comprising one or more human heavy
and/or light chain transgenes or transchromosomes (either
integrated or non-integrated into the animal's natural genomic DNA)
and which is capable of expressing fully human antibodies. For
example, a transgenic mouse can have a human light chain transgene
and either a human heavy chain transgene or human heavy chain
transchromosome, such that the mouse produces human anti-EGFR
antibodies when immunized with human EGFR antigen and/or cells
expressing EGFR. The human heavy chain transgene may be integrated
into the chromosomal DNA of the mouse, as is the case for
transgenic mice, for instance HuMAb mice, such as HCo7 or HCo12
mice, or the human heavy chain transgene may be maintained
extrachromosomally, as is the case for transchromosomal KM mice as
described in WO02/43478. Such transgenic and transchromosomal mice
(collectively referred to herein as "transgenic mice") are capable
of producing multiple isotypes of human monoclonal antibodies to a
given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing
V-D-J recombination and isotype switching. Transgenic, nonhuman
animal can also be used for production of antibodies against a
specific antigen by introducing genes encoding such specific
antibody, for example by operatively linking the genes to a gene
which is expressed in the milk of the animal.
[0078] The antibodies used in the present invention are typically
used in and provided in an at least substantially isolated form. An
"isolated" molecule refers to a molecule that is not associated
with significant levels (such as more than about 1%, more than
about 2%, more than about 3%, or more than about 5%) of any
extraneous and undesirable physiological factors, such as non-EGFR
biomolecules contained within a cell or animal in which the
antibody is produced. An isolated molecule also refers to any
molecule that has passed through such a stage of purity due to
human intervention (whether automatic, manual, or both).
[0079] "Treatment" means the administration of an effective amount
of a therapeutically active compound of the present invention with
the purpose of easing, ameliorating or eradicating (curing)
symptoms or disease states.
Further Aspects and Embodiments of the Invention
[0080] As explained above, in a first main aspect, the invention
relates to a method for the treatment of a tumor comprising
combined administration, to a human being in need thereof, of a
first antibody and a second antibody, wherein
[0081] said first antibody binds EGFR,
[0082] said second antibody binds EGFR,
[0083] said first and second antibody are non-cross-blocking,
and
[0084] the dosage regimen is such that CDC is obtained at the tumor
site.
[0085] In the present context, the term "comprising" means
"consisting at least of". Thus, the treatment may include further
steps, including the administration of a third, fourth, fifth, etc,
anti-EGFR antibody.
[0086] Similarly, the invention relates to a first antibody for use
in the treatment of a tumor in combination with a second antibody,
wherein
[0087] said first antibody binds EGFR,
[0088] said second antibody binds EGFR, and
[0089] said first and second antibody are non-cross-blocking,
and
[0090] the dosage regimen is such that CDC is obtained at the tumor
site.
[0091] Furthermore, the invention relates to a second antibody for
use in the treatment of a tumor in combination with a first
antibody, wherein
[0092] said first antibody binds EGFR,
[0093] said second antibody binds EGFR,
[0094] said first and second antibody are non-cross-blocking,
and
[0095] the dosage regimen is such that CDC is obtained at the tumor
site.
[0096] Moreover, the invention relates to the use of a first
antibody and a second antibody for the preparation of a medicament
for the treatment of a tumor, wherein
[0097] said first antibody binds EGFR,
[0098] said second antibody binds EGFR,
[0099] said first and second antibody are non-cross-blocking,
and
[0100] the dosage regimen is such that CDC is obtained at the tumor
site.
Antibodies Suitable for Use in the Invention
[0101] In one embodiment of the method of the invention, the first
and/or second antibody used is a monoclonal antibody. In a further
embodiment, the first and/or second antibody used is a human
antibody. In another embodiment, the first and/or second antibody
used is a chimeric or humanized antibody. In a further preferred
embodiment, the first and/or second antibody used is an intact
antibody, i.e. a full-length antibody rather than a fragment.
[0102] In one embodiment of the method of the invention, the first
and/or second antibody used binds to human EGFR with an equilibrium
dissociation constant (K.sub.D) of 10.sup.-8 M or less, more
preferably 10.sup.-10 M or less.
[0103] Further preferred anti-EGFR antibodies for use in the
invention comprise antibodies that have one or more of the
following properties:
[0104] a) the ability to opsonize a cell expressing EGFR;
[0105] b) the ability to inhibit growth and/or mediate phagocytosis
and killing of cells expressing EGFR (e.g., a tumor cell) in the
presence of human effector cells at a concentration of about 10
.mu.g/ml or less (e.g., in vitro).
[0106] Antibodies used in the present invention may be in any
suitable form with respect to multimerization. Also, if desired,
the class of antibody used in the present invention may be switched
by known methods. Thus, the effector function of the antibodies of
the present invention may be changed by isotype switching to, e.g.,
an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for
various therapeutic uses.
[0107] In one embodiment, the anti-EGFR antibody used in the
present invention is an IgG1 antibody, for instance an IgG1,.kappa.
or IgG1,.lamda. isotype. In another embodiment, the anti-EGFR
antibody used in the present invention is an IgG3 antibody, for
instance an IgG3,.kappa. or IgG3,.lamda. isotype. In yet another
embodiment, the antibody used is an IgG4 antibody, for instance an
IgG4,.kappa. or IgG4,.lamda. isotype. In a further embodiment, the
anti-EGFR antibody used in the present invention is an IgA1 or IgA2
antibody. In an even further embodiment, the anti-EGFR antibody
used in the present invention is an IgM antibody.
[0108] In a further embodiment, the first or second anti-EGFR
antibody used is an antibody as described in WO02/100348,
WO04/056847, WO200556606, WO05/012479, WO05/10151, U.S. Pat. No.
6,794,494, EP1454917, WO0314159, WO02092771, WO0312072, WO02066058,
WO0188138, WO98/50433, WO98/36074, WO96/40210, WO 96/27010,
US2002065398, WO95/20045, EP586002, U.S. Pat. No. 5,459,061 or U.S.
Pat. No. 4,943,533.
[0109] In one embodiment of the method of the invention, said first
antibody is an antibody which is capable of binding an EGFR epitope
which is found on all wild-type-EGFR-expressing cells. Typically,
such an epitope is also detectable on all wild-type-EGFR-expressing
cells, either tumor cells or non-tumor cells expressing EGFR, such
as cells in the skin, e.g. keratinocytes. Furthermore, such
antibodies typically bind to wild-type EGFR regardless of whether
or not it is in a ligand-activated form.
[0110] Preferably, said first antibody binds to human EGFR with an
equilibrium dissociation constant (K.sub.D) of at most 10.sup.-8 M,
preferably at most 10.sup.-10 M and/or said first antibody is an
antibody which is capable of inducing ADCC at the tumor site in the
absence of said second antibody.
[0111] In one embodiment of the method of the invention, said first
antibody is an antibody selected from the group consisting of:
zalutumumab (2F8, described in WO02/100348 and WO04/056847),
cetuximab (see e.g. Wong 2005 Clin Ther 27:684 and references
therein), panitumumab (see e.g. Cohernuram and Saif (2007)
Anticancer Drugs 18:7 and references therein), nimotuzumab (h-R3,
see e.g. Spicer (2005) Curr Opin Mol Ther 7:182 and references
therein), matuzumab (EMD72000, see e.g. Kim (2004) Curr Opin Mol
Ther 6:96 and references therein), 528 (see e.g. Reilly 3 (2000)
Nucl Med 41:903), LC1006-003 (described herein), LC1006-005
(described herein), LC1006-008 (described herein), LC1006-011
(described herein), LC1006-018 (described herein), and a variant
antibody of any of these, such as a variant as described herein
below.
[0112] Antibodies that bind to the same epitope of EGFR as the
above mentioned antibodies are also suitable for use in the method
of the invention.
[0113] Protein electron tomography analyses of zalutumumab in
complex with EGFR showed that zalutumumab binds an epitope located
in EGFR domain III (amino acid 313-482), which is one of the two
ligand binding domains of EGFR. Cetuximab also binds an epitope
located in EGFR domain III.
[0114] In one embodiment, the first antibody used in the method of
the invention is an antibody which binds to an epitope within
domain III of EGFR, i.e. within the region of amino acid
313-482.
[0115] In a further embodiment, first antibody is selected from the
group consisting of:
[0116] an antibody which binds the same EGFR epitope as
zalutumumab,
[0117] an antibody which binds the same EGFR epitope as
cetuximab,
[0118] an antibody which binds the same EGFR epitope as
panitumumab,
[0119] an antibody which binds the same EGFR epitope as
nimotuzumab,
[0120] an antibody which binds the same EGFR epitope as
matuzumab,
[0121] an antibody which binds the same EGFR epitope as 528,
[0122] an antibody which binds the same EGFR epitope as
LC1006-003,
[0123] an antibody which binds the same EGFR epitope as
LC1006-005,
[0124] an antibody which binds the same EGFR epitope as
LC1006-008,
[0125] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0126] an antibody which binds the same EGFR epitope as
LC1006-018.
[0127] Li et al, Cancer Cell, April 2005 vol. 7:301-311 showed in
the crystal structure of sEGFR in complex with the cetuximab Fab
fragment that cetuximab specifically interacts with EGFR amino
acids R353, Q384, Q408, H409, F412, S418, S440, K443, K465, I467,
S468, and N473.
[0128] Cross blocking studies using EGFR expressing cells showed
that cetuximab cross blocks with zalutumumab and mAb 528.
Zalutumumab, however, does not cross block with mAb 528, which
shows that cetuximab and zalutumumab bind different epitopes.
Fine-epitope mapping performed in binding studies using
transiently-transfected, human-to-murine EGFR point-mutants showed
that zalutumumab specifically interacts with amino acids K465,
I467, K443 and S468.
[0129] Accordingly, in one embodiment, the first antibody is an
antibody which interacts with one, more or all of amino acids R353,
Q384, Q408, H409, F412, S418, S440, K443, K465, I467, S468 and N473
of EGFR, such as one, more or all of amino acids K465, I467, K443
and S468 of EGFR. Amino acids N473 and G471 are involved in binding
of cetuximab, but are not important for binding of zalutumumab. In
a further embodiment, the first antibody is an antibody which
interacts with one, more or all of amino acids K465, I467, K443 and
S468, but not with N473 and G471.
[0130] Similarly, in another embodiment, said first antibody is
selected from the group consisting of:
[0131] an antibody which comprises the same heavy chain CDR3
sequence as zalutumumab and binds the same EGFR epitope as
zalutumumab,
[0132] an antibody which comprises the same heavy chain CDR3
sequence as cetuximab and binds the same EGFR epitope as
cetuximab,
[0133] an antibody which comprises the same heavy chain CDR3
sequence as panitumumab and binds the same EGFR epitope as
panitumumab,
[0134] an antibody which comprises the same heavy chain CDR3
sequence as nimotuzumab and binds the same EGFR epitope as
nimotuzumab,
[0135] an antibody which comprises the same heavy chain CDR3
sequence as matuzumab and binds the same EGFR epitope as
matuzumab,
[0136] an antibody which comprises the same heavy chain CDR3
sequence as 528 and binds the same EGFR epitope as 528,
[0137] an antibody which comprises the same heavy chain CDR3
sequence as LC1006-003 and binds the same EGFR epitope as
LC1006-003,
[0138] an antibody which comprises the same heavy chain CDR3
sequence as LC1006-005 and binds the same EGFR epitope as
LC1006-005,
[0139] an antibody which comprises the same heavy chain CDR3
sequence as LC1006-008 and binds the same EGFR epitope as
LC1006-008,
[0140] an antibody which comprises the same heavy chain CDR3
sequence as LC1006-011 and binds the same EGFR epitope as
LC1006-011, and
[0141] an antibody which comprises the same heavy chain CDR3
sequence as LC1006-018 and binds the same EGFR epitope as
LC1006-018.
[0142] In an even further embodiment, said first antibody is
selected from the group consisting of:
[0143] an antibody which comprises the same 6 CDR sequences as
zalutumumab,
[0144] an antibody which comprises the same 6 CDR sequences as
cetuximab,
[0145] an antibody which comprises the same 6 CDR sequences as
panitumumab,
[0146] an antibody which comprises the same 6 CDR sequences as
nimotuzumab,
[0147] an antibody which comprises the same 6 CDR sequences as
matuzumab,
[0148] an antibody which comprises the same 6 CDR sequences as
528,
[0149] an antibody which comprises the same 6 CDR sequences as
LC1006-003,
[0150] an antibody which comprises the same 6 CDR sequences as
LC1006-005,
[0151] an antibody which comprises the same 6 CDR sequences as
LC1006-008,
[0152] an antibody which comprises the same 6 CDR sequences as
LC1006-011, and
[0153] an antibody which comprises the same 6 CDR sequences as
LC1006-018.
[0154] In a preferred embodiment of the method of the invention,
said first antibody is an antibody which binds the same EGFR
epitope as zalutumumab and said second antibody is selected from
the group consisting of:
[0155] an antibody which binds the same EGFR epitope as
nimotuzumab,
[0156] an antibody which binds the same EGFR epitope as
matuzumab,
[0157] an antibody which binds the same EGFR epitope as
LC1006-003,
[0158] an antibody which binds the same EGFR epitope as
LC1006-005,
[0159] an antibody which binds the same EGFR epitope as
LC1006-008,
[0160] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0161] an antibody which binds the same EGFR epitope as
LC1006-018.
[0162] In another preferred embodiment of the method of the
invention, said first antibody is an antibody which binds the same
EGFR epitope as cetuximab and said second antibody is selected from
the group consisting of:
[0163] an antibody which binds the same EGFR epitope as
matuzumab,
[0164] an antibody which binds the same EGFR epitope as
LC1006-003,
[0165] an antibody which binds the same EGFR epitope as
LC1006-005,
[0166] an antibody which binds the same EGFR epitope as
LC1006-008,
[0167] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0168] an antibody which binds the same EGFR epitope as
LC1006-018.
[0169] In a further preferred embodiment of the method of the
invention, said first antibody is an antibody which binds the same
EGFR epitope as panitumumab and said second antibody is selected
from the group consisting of:
[0170] an antibody which binds the same EGFR epitope as
matuzumab,
[0171] an antibody which binds the same EGFR epitope as
LC1006-003,
[0172] an antibody which binds the same EGFR epitope as
LC1006-005,
[0173] an antibody which binds the same EGFR epitope as
LC1006-008,
[0174] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0175] an antibody which binds the same EGFR epitope as
LC1006-018.
[0176] In an even further preferred embodiment of the method of the
invention, said first antibody is an antibody which binds the same
EGFR epitope as nimotuzumab and said second antibody is selected
from the group consisting of:
[0177] an antibody which binds the same EGFR epitope as
LC1006-003,
[0178] an antibody which binds the same EGFR epitope as
LC1006-005,
[0179] an antibody which binds the same EGFR epitope as
LC1006-008,
[0180] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0181] an antibody which binds the same EGFR epitope as
LC1006-018.
[0182] In another preferred embodiment of the method of the
invention, said first antibody is an antibody which binds the same
EGFR epitope as matuzumab and said second antibody is selected from
the group consisting of:
[0183] an antibody which binds the same EGFR epitope as
LC1006-003,
[0184] an antibody which binds the same EGFR epitope as
LC1006-005,
[0185] an antibody which binds the same EGFR epitope as LC1006-008,
and
[0186] an antibody which binds the same EGFR epitope as
LC1006-011.
[0187] In a further preferred embodiment of the method of the
invention, said first antibody is an antibody which binds the same
EGFR epitope as 528 and said second antibody is selected from the
group consisting of:
[0188] an antibody which binds the same EGFR epitope as
LC1006-003,
[0189] an antibody which binds the same EGFR epitope as
LC1006-005,
[0190] an antibody which binds the same EGFR epitope as
LC1006-008,
[0191] an antibody which binds the same EGFR epitope as LC1006-011,
and
[0192] an antibody which binds the same EGFR epitope as
LC1006-018.
[0193] In a yet even further preferred embodiment of the method of
the invention, said first antibody is an antibody which binds the
same EGFR epitope as LC1006-018 and said second antibody is
selected from the group consisting of:
[0194] an antibody which binds the same EGFR epitope as
nimotuzumab,
[0195] an antibody which binds the same EGFR epitope as
LC1006-003,
[0196] an antibody which binds the same EGFR epitope as
LC1006-005,
[0197] an antibody which binds the same EGFR epitope as LC1006-008,
and
[0198] an antibody which binds the same EGFR epitope as
LC1006-011.
[0199] Further embodiments of the antibodies used in the present
invention are given below in the section "Production of
antibodies".
Combinations of Antibodies Wherein One Antibody Preferentially
Binds Tumor Cells
[0200] In a particularly interesting embodiment of the method of
the invention, treatment with one of the antibodies mentioned above
is combined with an anti-EGFR antibody which preferentially binds
tumor cells. In this embodiment of the method of the invention, the
CDC induced by the combination therapy is more specifically
directed to tumor cells, and strong CDC at healthy tissues, which
may be undesirable for safety reasons, is avoided or at least
reduced.
[0201] Thus, in one embodiment, the first antibody is one of the
antibodies mentioned herein above, preferably one of the anti-EGFR
antibodies mentioned herein above, and the second antibody is an
antibody which is capable of binding to an EGFR epitope which is
found in tumor cells, but is not detectable in normal cells.
[0202] In one embodiment, such a tumor-cell-specific anti-EGFR
antibody is an antibody which is specific for an EGFR variant which
is only expressed on tumor cells, e.g. EGFRvIII. Such an
anti-EGFRvIII antibody may either recognize the neo-epitope in
EGFRvIII, formed by the junction of sequences due to the deletion
of exons 2-7 (see e.g. WO2005012479), or the anti-EGFRvIII antibody
may recognize an epitope which does not demonstrate any amino acid
sequence alterations or substitutions as compared to wild-type
EGFR, but is only exposed on EGFRvIII.
[0203] In another embodiment, such a tumor-cell-specific anti-EGFR
antibody binds preferentially to EGFRvIII, but also exhibits
residual binding to wild-type EGFR, wherein the binding is
detectable when EGFR is overexpressed, e.g. on tumor cells. Such
antibodies typically recognize an EGFR epitope which does not
demonstrate any amino acid sequence alterations or substitutions as
compared to wild-type EGFR. In a preferred embodiment, such a
tumor-cell-specific anti-EGFR antibody binds an EGFR epitope which
is located within the region comprising residues 273-501 of EGFR,
more preferably an EGFR epitope which is located within the region
comprising residues 287-302 of EGFR. In a further embodiment, said
second antibody is cross-blocking with ch806, such as a second
antibody which binds the same EGFR epitope as ch806, e.g. a second
antibody which comprises SEQ ID NO:3 and optionally one or more or
all of SEQ ID NO:1, 2, 4, 5 and 6.
[0204] In an even further embodiment, said second antibody is
ch806.
[0205] In another embodiment, said second, tumor-cell-specific,
anti-EGFR antibody is MR1-1.
[0206] In another embodiment, the second antibody is an antibody
which is specific for EGFR-vIII (i.e. does not bind wild-type
EGFR). Such antibodies have e.g. been described by Hills et al.
(1995) Int. J. Cancer 63:537-543, Humphrey et al. 1990 PNAS
87:4207-4211, and Wikstrand et al. 1995 Cancer Res.
55:3140-3148).
[0207] In a further embodiment, the second antibody binds to
EGFR-vIII with a K.sub.D which is at least 10 fold lower, such as
at least 50 fold lower, e.g. at least 100 fold lower than the
K.sub.D for binding to wild-type EGFR.
Production of Antibodies
[0208] A monoclonal antibody refers to a composition comprising a
homogeneous antibody population having a uniform structure and
specificity. That an antibody is monoclonal is not to be construed
as requiring production of the antibody by any particular method.
For example, the monoclonal antibodies used in the present
invention may be produced by the hybridoma method first described
by Kohler et al., Nature 256, 495 (1975), or may be produced by
recombinant DNA methods. Monoclonal antibodies may also be isolated
from phage antibody libraries using the techniques described in,
for example, Clackson et al., Nature 352, 624-628 (1991) and Marks
et al., J. Mol. Biol. 222, 581-597 (1991).
[0209] Monoclonal antibodies may be obtained from any suitable
source. Thus, for example, monoclonal antibodies may be obtained
from hybridomas prepared from murine splenic B cells obtained from
mice immunized with an antigen of interest, for instance in form of
cells expressing the antigen on the surface, or a nucleic acid
encoding an antigen of interest. Monoclonal antibodies may also be
obtained from hybridomas derived from antibody-expressing cells of
immunized humans or non-human mammals such as rats, dogs, primates,
etc. In one embodiment, human monoclonal antibodies may be
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. Such
transgenic and transchromosomic mice include mice referred to
herein as HuMAb mice and KM mice, respectively, and are
collectively referred to herein as "transgenic mice".
[0210] The HuMAb mouse contains a human immunoglobulin gene
miniloci that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa. and in response to immunization, the introduced human heavy
and light chain transgenes, undergo class switching and somatic
mutation to generate high affinity human IgG,.kappa. monoclonal
antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg,
N. Handbook of Experimental Pharmacology 113, 49-101 (1994),
Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93
(1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci. 764
536-546 (1995)). The preparation of HuMAb mice is described in
detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295
(1992), Chen, J. et al., International Immunology 5, 647-656
(1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor,
L. et al., International Immunology 6, 579-591 (1994), Fishwild, D.
et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.
No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126,
U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No.
5,877,397, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S.
Pat. No. 5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No.
5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO
92/03918 and WO 01/09187.
[0211] The HCo7 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen et al., EMBO J. 12,
821-830 (1993)), a CMD disruption in their endogenous heavy chain
genes (as described in Example 1 of WO 01/14424), a KCo5 human
kappa light chain transgene (as described in Fishwild et al.,
Nature Biotechnology 14, 845-851 (1996)), and a HCo7 human heavy
chain transgene (as described in U.S. Pat. No. 5,770,429).
[0212] The HCo12 mice have a JKD disruption in their endogenous
light chain (kappa) genes (as described in Chen et al., EMBO J. 12,
821-830 (1993)), a CMD disruption in their endogenous heavy chain
genes (as described in Example 1 of WO 01/14424), a KCo5 human
kappa light chain transgene (as described in Fishwild et al.,
Nature Biotechnology 14, 845-851 (1996)), and a HCo12 human heavy
chain transgene (as described in Example 2 of WO 01/14424). In the
KM mouse strain, the endogenous mouse kappa light chain gene has
been homozygously disrupted as described in Chen et al., EMBO J.
12, 811-820 (1993) and the endogenous mouse heavy chain gene has
been homozygously disrupted as described in Example 1 of WO
01/09187. This mouse strain carries a human kappa light chain
transgene, KCo5, as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996). This mouse strain also carries a
human heavy chain transchromosome composed of chromosome 14
fragment hCF (SC20) as described in WO 02/43478.
[0213] The KM mouse contains a human heavy chain transchromosome
and a human kappa light chain transgene. The endogenous mouse heavy
and light chain genes also have been disrupted in the KM mice such
that immunization of the mice leads to production of human
immunoglobulins rather than mouse immunoglobulins. Construction of
KM mice and their use to raise human immunoglobulins is described
in detail in WO 02/43478. Splenocytes from these transgenic mice
may be used to generate hybridomas that secrete human monoclonal
antibodies according to well known techniques.
[0214] Human monoclonal or polyclonal antibodies for use in the
present invention, or antibodies for use in the present invention
originating from other species may also be generated transgenically
through the generation of another non-human mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form
therefrom. In connection with the transgenic production in mammals,
antibodies may be produced in, and recovered from, the milk of
goats, cows, or other mammals. See for instance U.S. Pat. No.
5,827,690, U.S. Pat. No. 5,756,687, U.S. Pat. No. 5,750,172 and
U.S. Pat. No. 5,741,957.
[0215] Further, human antibodies for use in the present invention
or antibodies for use in the present invention from other species
may be generated through display-type technologies, including,
without limitation, phage display, retroviral display, ribosomal
display, and other techniques, using techniques well known in the
art and the resulting molecules may be subjected to additional
maturation, such as affinity maturation, as such techniques are
well known in the art (see for instance Hoogenboom et al., J. Mol.
Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature
Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS
USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith,
Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245
(1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et
al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al.,
Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH
80-84 (1992), and U.S. Pat. No. 5,733,743). If display technologies
are utilized to produce antibodies that are not human, such
antibodies may be humanized, for instance as described elsewhere
herein.
[0216] Antibodies may also be recovered from recombinant
combinatorial antibody libraries, such as a scFv phage display
library, which may be made with human V.sub.L and V.sub.H cDNAs
prepared from mRNA derived from human lymphocytes. Methods for
preparing and screening such libraries are known in the art.
[0217] A "variant" antibody is an antibody that differs from a
parent antibody (typically generated by immunization) by one or
more suitable amino acid residue alterations, that is
substitutions, deletions, insertions, or terminal sequence
additions, in the CDRs or other V.sub.H and/or V.sub.L sequences
(provided that at least a substantial amount of the epitope binding
characteristics of the parent antibody are retained, if not
improved upon, by such changes). Variations in an antibody variant
may be made in each of the framework regions, the constant domain,
and/or the variable regions (or any one or more CDRs thereof) in a
single variant antibody. Alternatively, variations may be made in
only one of the framework regions, the variable regions (or single
CDR thereof), or the constant domain in an antibody.
[0218] A suitable amino acid residue substitution in the context of
a CDR variant is any amino acid residue that permits the CDR to
interact with the epitope to which the parent CDR is
selective/specific and to cooperatively associate with other parent
CDRs and/or variant CDRs similarly specific/selective for that
epitope. Factors influencing the selection of a suitable amino acid
sequence substitution may include the impact of the residue on the
conformation of the CDR (e.g., retention of CDR loop structure and
flexibility) and the ability to engage in noncovalent interactions
(e.g., Van der Waals interactions, hydrogen bonding interactions,
ionic interactions, and/or other interactions characteristic of
epitope-variable region binding) with the epitope and/or other
similar CDRs in a manner similar to or advantageous over the
replaced residue in the parent CDR.
[0219] The percent identity between two sequences, e.g. variable
domain sequences or CDR3 sequences, is a function of the number of
identical positions shared by the sequences (i.e., % homology=# of
identical positions/total # of positions.times.100), taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences may be accomplished using a mathematical
algorithm, as described in the non-limiting examples below.
[0220] The percent identity between two nucleotide sequences may be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences may also be determined using the algorithm of E.
Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4. In addition, the percent identity between two amino
acid sequences may be determined using the Needleman and Wunsch, J.
Mol. Biol. 48, 444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at http://www.gcg.com), using either a Blossum 62 matrix
or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4
and a length weight of 1, 2, 3, 4, 5, or 6.
[0221] The sequence of CDR variants may differ from the sequence of
the CDR of the parent antibody sequences through mostly
conservative substitutions; for instance at least about 35%, about
50% or more, about 60% or more, about 70% or more, about 75% or
more, about 80% or more, about 85% or more, about 90% or more,
about 95% or more (e.g., about 65-99%) of the substitutions in the
variant are conservative amino acid residue replacements. In the
context of the present invention, conservative substitutions may be
defined by substitutions within the classes of amino acids
reflected in one or more of the following three tables:
TABLE-US-00001 Amino acid residue classes for conservative
substitutions Acidic Residues Asp and Glu Basic Residues Lys, Arg,
and His Hydrophilic Uncharged Residues Ser, Thr, Asn, and Gln
Aliphatic Uncharged Residues Gly, Ala, Val, Leu, and Ile Non-polar
Uncharged Residues Cys, Met, and Pro Aromatic Residues Phe, Tyr,
and Trp
[0222] Variant antibodies used in the present invention may
comprise framework (FR) alterations, that is outside the
hypervariable region, for instance in the Fc region, which
alterations may be associated with advantageous properties, such as
changing the functional or pharmacokinetic properties of the
antibodies. For example, a substitution or other modification
(insertion, deletion, terminal sequence additions or combination of
any thereof) in a framework region or constant domain may be
associated with an increase in the half-life of the variant
antibody with respect to the parent antibody, or may be made to
alter the immunogenicity of the variant antibody with respect to
the parent antibody, to provide a site for covalent or non-covalent
binding to another molecule, or to alter such properties as
complement fixation, for instance resulting in a decrease or
increase of C1q binding and CDC or of Fc.gamma.R binding and
antibody-dependent cellular cytotoxicity (ADCC). Substitutions may
for example be made in one or more of the amino acid residues 234,
235, 236, 237, 297, 318, 320, and 322 of the heavy chain constant
region, thereby causing an alteration in an effector function while
retaining binding to antigen as compared with the unmodified
antibody, cf. U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260.
Further reference may be had to WO 00/42072 disclosing antibodies
with altered Fc regions that increase ADCC, and WO 94/29351
disclosing antibodies having mutations in the N-terminal region of
the C.sub.H2 domain that alter the ability of the antibodies to
bind to FcRI and thereby decreases the ability of the antibodies to
bind to C1q which in turn decreases the ability of the antibodies
to fix complement. Furthermore, Shields et al., J. Biol. Chem. 276,
6591-6604 (2001) teaches combination variants, which improve
Fc.gamma.RIII binding, for instance T256A/S298A, S298A/E333A, and
S298A/E333A/K334A.
[0223] The in vivo half-life of the antibodies may also be improved
by modifying the salvage receptor epitope of the Ig constant domain
or an Ig-like constant domain such that the molecule does not
comprise an intact C.sub.H2 domain or an intact Ig Fc region, cf.
U.S. Pat. No. 6,121,022 and U.S. Pat. No. 6,194,551. The in vivo
half-life may furthermore be increased by making mutations in the
Fc region, e.g. by substituting threonine for leucine at position
252, threonine for serine at position 254, or threonine for
phenylalanine at position 256, cf. U.S. Pat. No. 6,277,375.
[0224] The present invention may also use fragments of antibodies
(including variant antibodies). Examples of such antibody fragments
include Fab, Fab', F(ab').sub.2, and Fv fragments. Thus, although
the discussion herein may focus on antibodies, it should be
understood that the embodiments and features of the antibodies may
equally be applied to antibody fragments, such as Fab fragments,
Fab' fragments, and scFv peptides, antibody-like peptides (peptides
comprising a CDR), and bi- and multi-specific antibodies as
appropriate, provided that the molecule retains at least a
substantial proportion of the antigen-binding properties of the
corresponding complete antibody. In some instances, antibody
fragments may be associated with lower antigen-binding affinity,
but may offer other advantageous features that may offset for any
such loss in affinity.
[0225] Antibodies used in the present invention also include
antibody derivatives. Such derivatives may be produced by
chemically conjugating a radioisotope, protein, or other
agent/moiety/compound to the N-terminal side or C-terminal side of
the antibody or subunit thereof, an appropriate substituent group
or side chain or to a sugar chain in the antibody (see, e.g.,
Antibody Engineering Handbook, edited by Osamu Kanemitsu, published
by Chijin Shokan (1994)). Derivatives may also be generated by
conjugation at internal residues or sugars, where appropriate.
[0226] In one embodiment, the present invention uses an antibody
that is conjugated to a second molecule that is selected from a
radionuclide, an enzyme, an enzyme substrate, a cofactor, a
fluorescent marker, a chemiluminescent marker, a peptide tag, or a
magnetic particle. In one embodiment, an antibody may be conjugated
to one or more antibody fragments, nucleic acids
(oligonucleotides), nucleases, hormones, immunomodulators,
chelators, boron compounds, photoactive agents, dyes, and the like.
These and other suitable agents may be coupled either directly or
indirectly to an antibody. One example of indirect coupling of a
second agent is coupling by a spacer moiety. These spacers, in
turn, may be either insoluble or soluble (see for instance Diener
et al., Science 231, 148 (1986)) and may be selected to enable drug
release from the antibody at a target site and/or under particular
conditions. Additional examples of therapeutic agents that may be
coupled to an antibody include lectins and fluorescent
peptides.
[0227] In one embodiment, antibody derivatives comprising one or
more radiolabeled amino acids are used. Methods for preparing
radiolabeled amino acids and related peptide derivatives are known
in the art (see for instance Junghans et al., in Cancer
Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo,
eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581, U.S.
Pat. No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No.
5,102,990 (U.S. RE35,500), U.S. Pat. No. 5,648,471 and U.S. Pat.
No. 5,697,902. For example, a radioisotope may be conjugated by a
chloramine T method.
[0228] In one embodiment, the present invention uses molecules
comprising an antibody, such as an anti-EGFR antibody, conjugated
to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic
drug, an immunosuppressant, or a radioisotope. Such conjugates are
referred to herein as "immunoconjugates". Immunoconjugates which
include one or more cytotoxins are referred to as
"immunotoxins".
[0229] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to (e.g., kills) cells. For a description of these
classes of drugs which are well known in the art, and their
mechanisms of action, see Goodman et al., Goodman and Gilman's The
Pharmacological Basis Of Therapeutics, 8th Ed., Macmillan
Publishing Co., 1990. Additional techniques relevant to the
preparation of antibody immunotoxins are provided in for instance
Vitetta, Immunol. Today 14, 252 (1993) and U.S. Pat. No.
5,194,594.
[0230] Suitable therapeutic agents for forming immunoconjugates of
the present invention include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin, antimetabolites (such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine), alkylating agents (such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C, cisplatin and other platinum derivatives, such as
carboplatin), antibiotics (such as dactinomycin (formerly
actinomycin), bleomycin, daunorubicin (formerly daunomycin),
doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,
plicamycin, anthramycin (AMC)), diphtheria toxin and related
molecules (such as diphtheria A chain and active fragments thereof
and hybrid molecules), ricin toxin (such as ricin A or a
deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like
toxin (SLT-I, SLT-II, SLT-IIV), 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
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, and enomycin toxins. Therapeutic agents,
which may be administered in combination with antibody as described
elsewhere herein, may also be candidates for therapeutic moieties
useful for conjugation to an antibody.
[0231] Other examples of therapeutic cytotoxins that may be
conjugated to an antibody used in the present invention include
calicheamicins and duocarmycins. As indicated above, the drug
moiety need not be construed as limited to classical chemical
therapeutic agents. For example, the drug moiety may be a protein
or polypeptide possessing a desired biological activity. Such
proteins may include, for example, an agent active at the cell
surface, such as phospholipase enzymes, e.g. phospholipase C.
[0232] The lysing portion of a toxin typically may be readily
joined to the Fab fragment of an antibody or antibody fragment of
the present invention. Other suitable conjugated molecules include
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, diphtherin toxin, and Pseudomonas
endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and
Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994).
Additional toxins suitable for use in the present invention are
known to those of skill in the art (see for instance U.S. Pat. No.
6,077,499).
[0233] Techniques for conjugating such therapeutic moieties to
antibodies, are well known, see for instance Amon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al., (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985), Hellstrom et
al., "Antibodies For Drug Delivery", in Controlled Drug Delivery
(2nd Ed.), Robinson et al., (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987), Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al., (eds.), pp. 475-506 (1985),
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al.,
(eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62, 119-58 (1982).
[0234] In one embodiment, the antibody used in the present
invention is attached to a linker-chelator, e.g., tiuxetan, which
allows for the antibody to be conjugated to a radioisotope.
[0235] Additionally useful conjugate substituents include
anti-cancer retinoids. Taxane conjugates (see for instance Jaime et
al., Anticancer Res. 21 (2A), 1119-28 (2001), cisplatin conjugates,
thapsigargin conjugates, linoleic acid conjugates, calicheamicin
conjugates (see for instance Damle et al., Curr Opin Pharmacol.
3(4), 386-90 (2003), doxorubicin conjugates, geldanamycin
conjugates, and the like, also may be useful in promoting the
treatment of cancer (see, generally, Trail et al., Cancer Immunol
Immunother. 52(5), 328-37 (2003)).
[0236] Antibodies used in the present invention may be prepared by
recombinant expression in any suitable type of cells or animals.
Recombinant antibodies, such as recombinant human antibodies also
include antibodies isolated from a recombinant, combinatorial human
antibody library, antibodies isolated from an animal, such as a
transgenic animal, or antibodies prepared, expressed, created or
isolated by any other means that involves splicing of human
immunoglobulin-encoding nucleic acid sequences to other nucleic
acid sequences exogenous to the human immunoglobulin-encoding
nucleic acids and human immunoglobulin-encoding genes. Recombinant
human antibodies typically have variable and constant regions
derived from human germ line immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies are
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and,
thus, the amino acid sequences of the V.sub.H and V.sub.L regions
of the recombinant antibodies may be sequences that, while derived
from and related to human germ line V.sub.H and V.sub.L sequences,
may not naturally exist within the human antibody germ line
repertoire in vivo. Suitable methods for antibody production are
known in the art and include those described in for instance Harlow
et al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1988), Harlow and
Lane: Using Antibodies: A Laboratory Manual (Cold Spring Harbor
Laboratory Press (1999)), U.S. Pat. No. 4,376,110 and Ausubel et
al., eds., Current Protocols In Molecular Biology, Greene
Publishing Assoc. and Wiley InterScience N.Y., (1987, 1992).
Monoclonal antibodies may be made using the hybridoma method first
described by Kohler et al., Nature 256, 495 (1975), or by other
well-known, subsequently-developed methods (see, e.g., Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). Transformed immortalized B cells may also
be used to efficiently produce antibodies used in the present
invention. Such cells may be produced by standard techniques, such
as transformation with an Epstein Barr Virus, or a transforming
gene. (See, e.g., "Continuously Proliferating Human Cell Lines
Synthesizing Antibody of Predetermined Specificity," Zurawaki, V.
R. et al., in Monoclonal Antibodies, ed. by Kennett R. H. et al.,
Plenum Press, N.Y. 1980, pp 19-33).
[0237] Recombinant cells comprising exogenous nucleic acids
encoding antibodies used in the present invention may be prepared
by any suitable technique (e.g., transfection/transformation with a
naked DNA plasmid vector, viral vector, invasive bacterial cell
vector or other whole cell vector, etc., comprising an
antibody-encoding sequence (or sequences) delivered into the cell
by calcium phosphate-precipitation facilitated transfection,
receptor-mediated targeting and transfection, biolistic delivery,
electroporation, dextran-mediated transfection, liposome-mediated
transformation, protoplast fusion, direct microinjection, etc.).
Methods of transforming/transfecting cells are well known in the
art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press (2d Edition, 1989 and
3rd Edition, 2001) and F. Ausubel et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley InterScience New
York (1987). Such recombinant cells are a feature of the present
invention.
[0238] Cell lines available as hosts for recombinant protein
expression are well known in the art and include many immortalized
cell lines available from the American Type Culture Collection
(ATCC). These include, inter alia, Chinese hamster ovary (CHO)
cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), A549 cells, and a number of other cell lines. Other
cell lines that may be used are insect cell lines, such as Sf9
cells, or bacterial cells or eukaryotic unicellular microorganisms,
such as yeast.
[0239] Human antibodies of the present invention may also be
produced in a host cell transfectoma using, for example, a
combination of recombinant DNA techniques and gene transfection
methods as is well known in the art, see for instance Morrison, S.,
Science 229, 1202 (1985).
Dosage Regimens
[0240] In the method and use of the invention, the antibodies are
given in an effective amount, i.e. in an amount effective, at
dosages and for periods of time necessary, to achieve a desired
result.
[0241] A therapeutically effective amount may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the agent to elicit a desired
response in the individual.
[0242] An effective amount for tumor therapy may also be measured
by its ability to stabilize the progression of disease. The ability
of a compound to inhibit cancer may be evaluated in an animal model
system predictive of efficacy in human tumors. Alternatively, this
property of a composition may be evaluated by examining the ability
of the compound to inhibit cell growth or to induce apoptosis by in
vitro assays known to the skilled practitioner. A therapeutically
effective amount of a therapeutic compound may decrease tumor size,
or otherwise ameliorate symptoms in a subject. One of ordinary
skill in the art would be able to determine such amounts based on
such factors as the subject's size, the severity of the subject's
symptoms, and the particular composition or route of administration
selected.
[0243] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation.
[0244] In a preferred embodiment of the method of the invention,
the dosage regimen is such that substantial CDC is obtained at the
tumor site. This may e.g. be tested using the methods described in
Di Gaetano et al. (2003) J. Immunol. 171(3):1581-7, Kennedy et al.
(2004) J Immunol. 2004 Mar. 1; 172(5):3280-8 and Gelderman et al.
(2004) TRENDS in Immunology 25:158 and references mentioned
therein,
[0245] As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of the first and/or
second antibody in an amount of about 0.1-100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
[0246] In one embodiment of the method of the invention, the dosage
regimen of said first antibody comprises administration, at least
once per 14 days, of a dosage of antibody of at least 0.1 mg/kg,
such as at least 0.25 mg/kg, e.g. at least 0.5 mg/kg, such as at
least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2 mg/kg,
e.g. at least 3 mg/kg, such as at least 4 mg/kg, e.g. at least 5
mg/kg, such as at least 6 mg/kg, e.g. at least 7 mg/kg, such as at
least 8 mg/kg, e.g. at least 9 mg/kg, such as at least 10 mg/kg,
e.g. at least 12 mg/kg, such as at least 15 mg/kg, e.g. at least 20
mg/kg. In a further embodiment, the dosage of the first antibody is
at most 100 mg/kg, such as at most 50 mg/kg.
[0247] In another embodiment of the method of the invention, the
dosage regimen of said second antibody comprises administration, at
least once per 14 days, of a dosage of antibody of at least 0.1
mg/kg, such as at least 0.25 mg/kg, e.g. at least 0.5 mg/kg, such
as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2
mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg, e.g. at
least 5 mg/kg, such as at least 6 mg/kg, e.g. at least 7 mg/kg,
such as at least 8 mg/kg, e.g. at least 9 mg/kg, such as at least
10 mg/kg, e.g. at least 12 mg/kg, such as at least 15 mg/kg, e.g.
at least 20 mg/kg. In a further embodiment, the dosage of the
second antibody is at most 100 mg/kg, such as at most 50 mg/kg.
[0248] In a further embodiment, the administration of said first
and/or second antibody is at least once per week.
[0249] In another embodiment of the invention, the dosage regimen
of the first and/or second antibody a comprises administration, at
least once per 14 days, of a dose of antibody of at least 5 mg,
such as at least 10 mg, e.g. at least 25 mg, such as at least 50
mg, e.g. at least 75 mg, such as at least 100 mg, e.g. at least 150
mg, such as at least 200 mg, e.g. at least 250 mg, such as at least
300 mg e.g. at least 350 mg, such as at least 400 mg, e.g. at least
500 mg, such as at least 750 mg, e.g. at least 1000 mg, such as at
least 1250 mg, e.g. at least 1500 mg, such as at least 2000 mg.
More preferably, the administration of the antibody or antibodies
is at least once per week.
[0250] In one embodiment, the antibodies used in the present
invention may be administered by infusion in a weekly dosage of
from 10 to 500 mg/m.sup.2, such as of from 200 to 400 mg/m.sup.2.
Such administration may be repeated, e.g., 1 to 8 times, such as 3
to 5 times. The administration may be performed by continuous
infusion over a period of from 2 to 24 hours, such as of from 2 to
12 hours.
[0251] As explained above, it has surprisingly been found that
combinations of non-cross-blocking anti-EGFR antibodies very
potently deposit complement components C1q and C4c on tumor cells,
leading to highly effective complement-mediated cell killing (CDC).
This induction of CDC leads to a very potent anti-tumor effect,
allowing a reduction in the antibody dosage during treatment.
[0252] Thus, in preferred embodiments of the method of the
invention, the dosage regimens of the first and/or second antibody
each individually, or the total dosage for both together, comprise
lower dosages than standard dosages for antibody therapy with the
same antibody, e.g. lower dosages than standard monotherapy dosages
for antibody therapy with the same antibody. Accordingly, in some
embodiments, the dosage regimen for said first and/or second
antibody is lower than a standard monotherapy dosage regimen for
said first and/or second antibody.
[0253] When used herein, the term "standard dosage regimen" for a
given antibody refers to the dosage regimen recommended for
antibodies that have received marketing approval or to a dosage
regimen used for phase III clinical studies for an antibody that is
in clinical development. For example, a standard dosage regimen for
zalutumumab is a dosage of between 4 and 16 mg/kg once weekly.
[0254] As explained in more detail below, anti-EGFR therapy is
known to cause undesired side-effects, such as rash, due to
expression of wild-type EGFR in healthy, non-tumor, tissues. Thus,
in particular, when a combination of non-cross-blocking anti-EGFR
antibodies is used wherein both antibodies bind an epitope of
wild-type EGFR (such as an EGFR epitope which is found on all
wild-type-EGFR-expressing cells), then it may be necessary to
reduce the dosages of the antibodies as compared to standard
therapy in order to avoid toxicity for healthy tissues, due to
CDC.
[0255] Accordingly, in a preferred embodiment of the method of the
invention:
[0256] the dosage regimen of said first antibody comprises
administration of a total dosage per 14 days of between 0.01 mg/kg
and 2 mg/kg, such as between 0.01 mg/kg and 1 mg/kg, e.g. between
0.01 mg/kg and 0.5 mg/kg, such as between 0.01 mg/kg and 0.25
mg/kg, e.g. between 0.01 mg/kg and 0.1 mg/kg, such as between 0.01
mg/kg and 0.05 mg/kg;
and/or
[0257] the dosage regimen of said second antibody comprises
administration of a total dosage per 14 days of between 0.01 mg/kg
and 2 mg/kg, such as between 0.01 mg/kg and 1 mg/kg, e.g. between
0.01 mg/kg and 0.5 mg/kg, such as between 0.01 mg/kg and 0.25
mg/kg, e.g. between 0.01 mg/kg and 0.1 mg/kg, such as between 0.01
mg/kg and 0.05 mg/kg.
[0258] As explained above, in a particularly interesting embodiment
of the method of the invention, [0259] a) the first antibody is an
antibody which binds an EGFR epitope which is found in all
wild-type-EGFR-expressing cells, [0260] b) the second antibody is
an antibody, such as ch806, which binds an EGFR epitope which is
found in tumor cells, but is not detectable in normal cells, and
[0261] c) the dosage regimen is such that substantial CDC is
obtained at tumor sites, but substantially no CDC is obtained at
non-tumor sites.
[0262] In a further embodiment hereof, the first antibody in a) is
selected from the group consisting of: zalutumumab, cetuximab,
panitumumab, nimotuzumab, matuzumab, 528, LC1006-003, LC1006-005,
LC1006-008, LC1006-011 and LC1006-018.
[0263] In an even further embodiment hereof, the dosage regimen in
c) for said first antibody is a dosage regimen which comprises an
equal or a higher dosage than a standard monotherapy dosage regimen
for said first antibody, such as a dosage regimen which ensures
efficient inhibition of ligand binding at tumor sites. This can
e.g. be tested as described in Bleeker, et al (2004) J Immunol,
173, 4699-4707.
[0264] In one preferred embodiment, said first antibody is
administered at an at least 2 times higher dose than said second
antibody, such as an at least 4 times higher dose, e.g. an at least
10 times higher dose, such as an at least 25 times higher dose,
e.g. an at least 50 times higher dose than said second
antibody.
[0265] In another preferred embodiment, said first antibody is
administered at a between 2 and 50 times higher dose than said
second antibody, such as a between 5 and 20 times higher dose than
said second antibody.
[0266] In an even further preferred embodiment:
[0267] the dosage regimen of the first antibody comprises
administration, at least once per 14 days, of a dose of antibody of
at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg,
e.g. at least 5 mg/kg, such as at least 6 mg/kg, e.g. at least 7
mg/kg, such as at least 8 mg/kg, e.g. at least 9 mg/kg, such as at
least 10 mg/kg, e.g. at least 12 mg/kg, such as at least 15 mg/kg,
e.g. at least 20 mg/kg, and
[0268] the dosage regimen of the second antibody comprises
administration of a total dosage per 14 days of between 0.1 mg/kg
and 1 mg/kg, such as a dose of antibody of between 0.2 mg/kg and 1
mg/kg, e.g. such as a dose of antibody of between 0.1 mg/kg and 0.5
mg/kg, such as a dose of antibody of between 0.2 mg/kg and 0.5
mg/kg.
[0269] In another even further preferred embodiment:
[0270] the dosage regimen of the first antibody comprises
administration, at least once per 14 days, of a dose of antibody of
at least 4 mg/kg, e.g. at least 5 mg/kg, such as at least 6 mg/kg,
e.g. at least 7 mg/kg, such as at least 8 mg/kg, e.g. at least 9
mg/kg, such as at least 10 mg/kg, e.g. at least 12 mg/kg, such as
at least 15 mg/kg, e.g. at least 20 mg/kg, and
[0271] the dosage regimen of the second antibody comprises
administration of a total dosage per 14 days of between 0.1 mg/kg
and 2 mg/kg, such as a dose of antibody of between 0.2 mg/kg and 1
mg/kg, e.g. such as a dose of antibody of between 0.1 mg/kg and 2
mg/kg, such as a dose of antibody of between 0.1 mg/kg and 0.5
mg/kg.
[0272] The first and second antibody may be given simultaneously or
sequentially in any order. In one embodiment, both agents are
administered on the same day. In a preferred embodiment, the second
antibody, e.g. an antibody that binds the same epitope as ch806, is
administered at least 15 minutes, such as at least one hour, e.g.
at least two hours, such as at least eight hours before the first
antibody, preferably between 15 minutes and 6 hours, such as
between 1 hour and 4 hours before the first antibody.
[0273] In a further embodiment, the total duration of the treatment
is at least one month, such as at least two months, e.g. at least
four months, such as at least six months.
[0274] In some embodiments of the invention, the method of
treatment is repeated after an interval of two months or more, such
as three months or more, e.g. after six months or more.
[0275] In another particularly interesting embodiment of the method
of the invention, [0276] a) the first antibody is an antibody which
binds an EGFR epitope which is found in all
wild-type-EGFR-expressing cells, [0277] b) the second antibody is
an antibody which is specific for EGFR-vIII, e.g. binds to
EGFR-vIII with a K.sub.D which is at least 10 fold lower, such as
at least 50 fold lower, e.g. at least 100 fold lower than the
K.sub.D for binding to wild-type EGFR.
[0278] Preferably, the dosage regimen is such that substantial CDC
is obtained at tumor sites, but substantially no CDC is obtained at
non-tumor sites.
Bispecific Antibodies
[0279] In a further main aspect, the invention relates to a
bispecific antibody comprising a first binding specificity which
binds an EGFR epitope which is found on all
wild-type-EGFR-expressing cells and a second binding specificity
which preferentially binds an EGFR epitope which is found in tumor
cells.
[0280] In one embodiment, the second binding specificity binds an
EGFR epitope which is located within the region comprising residues
273-501 of EGFR, preferably the same EGFR epitope as bound by
ch806, wherein said first and second binding specificity are
non-cross-blocking.
[0281] In another embodiment, the second binding specificity is
specific for EGFR-vIII, i.e. does not bind wild-type EGFR.
[0282] In a further embodiment, the antibody comprises a first
binding specificity which binds an epitope selected from the group
consisting of:
[0283] the EGFR epitope bound by zalutumumab,
[0284] the EGFR epitope bound by cetuximab,
[0285] the EGFR epitope bound by panitumumab,
[0286] the EGFR epitope bound by nimotuzumab,
[0287] the EGFR epitope bound by matuzumab,
[0288] the EGFR epitope bound by 528,
[0289] the EGFR epitope bound by LC1006-003,
[0290] the EGFR epitope bound by LC1006-005,
[0291] the EGFR epitope bound by LC1006-008,
[0292] the EGFR epitope bound by LC1006-011, and
[0293] the EGFR epitope bound by LC1006-018.
[0294] In a further aspect, the invention relates to a bispecific
antibody as defined above for use as a medicament.
[0295] In an even further aspect, the invention relates to a
bispecific antibody as defined above for use as a medicament for
the treatment of cancer.
[0296] In a yet even further aspect, the invention relates to the
use of a bispecific antibody as defined above for the preparation
of a medicament for the treatment of cancer.
[0297] In a similar aspect, the invention relates to a method for
the treatment of cancer comprising administration of a bispecific
antibody as defined above.
[0298] In one embodiment, said cancer is selected from the group
consisting of: breast cancer, bladder cancer, uterine/cervical
cancer, esophageal cancer, pancreatic cancer, colorectal cancer,
kidney cancer, ovarian cancer, prostate cancer, head and neck
cancer, non-small cell lung cancer and stomach cancer.
[0299] As non-limiting examples, treatment with the bispecific
antibody according to the present invention may be provided as a
daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9,
1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50,
60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
[0300] In a preferred embodiment of the method of the invention,
the dosage regimen of the bispecific anti-EGFR antibody comprises
administration, at least once per 14 days, of a dose of antibody of
at least 0.1 mg/kg, such as at least 0.25 mg/kg, e.g. at least 0.5
mg/kg, such as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as
at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg,
e.g. at least 5 mg/kg, such as at least 6 mg/kg, e.g. at least 7
mg/kg, such as at least 8 mg/kg, e.g. at least 9 mg/kg, such as at
least 10 mg/kg, e.g. at least 12 mg/kg, such as at least 15 mg/kg,
e.g. at least 20 mg/kg. More preferably, the administration of the
bispecific anti-EGFR antibody is at least once per week.
[0301] In another embodiment of the invention, the dosage regimen
of the bispecific anti-EGFR antibody comprises administration, at
least once per 14 days, of a dose of antibody of at least 5 mg,
such as at least 10 mg, e.g. at least 25 mg, such as at least 50
mg, e.g. at least 75 mg, such as at least 100 mg, e.g. at least 150
mg, such as at least 200 mg, e.g. at least 250 mg, such as at least
300 mg e.g. at least 350 mg, such as at least 400 mg, e.g. at least
500 mg, such as at least 750 mg, e.g. at least 1000 mg, such as at
least 1250 mg, e.g. at least 1500 mg, such as at least 2000 mg.
More preferably, the administration of the bispecific anti-EGFR
antibody is at least once per week.
Novel Anti-EGFR Antibodies
[0302] In a further aspect, the invention relates to an isolated
monoclonal antibody which binds to human EGFR, wherein the antibody
binds to the same epitope on EGFR as an antibody selected from the
group consisting of:
[0303] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 7 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
8;
[0304] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
10;
[0305] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
11;
[0306] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 12 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
13;
[0307] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
15;
[0308] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
16; and
[0309] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 17 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
18.
[0310] In one embodiment, the antibody is selected from the group
consisting of:
[0311] an antibody having a heavy chain CDR3 region identical to
the CDR3 region of the heavy chain sequence shown in SEQ ID NO:
7;
[0312] an antibody having a heavy chain CDR3 region identical to
the CDR3 region of the heavy chain sequence shown in SEQ ID NO:
9;
[0313] an antibody having a heavy chain CDR3 region identical to
the CDR3 region of the heavy chain sequence shown in SEQ ID NO:
12;
[0314] an antibody having a heavy chain CDR3 region identical to
the CDR3 region of the heavy chain sequence shown in SEQ ID NO: 14;
and
[0315] an antibody having a heavy chain CDR3 region identical to
the CDR3 region of the heavy chain sequence shown in SEQ ID NO:
17.
[0316] In a further embodiment, the antibody is selected from the
group consisting of:
[0317] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 7 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 8;
[0318] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 9 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 10;
[0319] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 9 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 11;
[0320] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 12 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 13;
[0321] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 14 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 15;
[0322] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 14 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 16; and
[0323] an antibody comprising CDR sequences that are identical to
the CDR sequences of an antibody comprising a heavy chain variable
region having the amino acid sequence shown in SEQ ID NO: 17 and a
light chain variable region having the amino acid sequence shown in
SEQ ID NO: 18.
[0324] In an even further embodiment, the antibody is selected from
the group consisting of:
[0325] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 7 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
8;
[0326] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
10;
[0327] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 9 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
11;
[0328] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 12 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
13;
[0329] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
15;
[0330] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 14 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
16; and
[0331] an antibody having a heavy chain variable region having the
amino acid sequence shown in SEQ ID NO: 17 and a light chain
variable region having the amino acid sequence shown in SEQ ID NO:
18.
[0332] In an even further embodiment, the antibody is an IgG1, IgA,
IgE, IgM, IgG4 or IgD antibody. In an even further embodiment, the
antibody is a human antibody. In an even further embodiment, the
antibody inhibits EGFR ligand binding to human EGFR, preferably by
at least about 50%. In an even further embodiment, the antibody
binds to human EGFR with an equilibrium association constant
(K.sub.A) of at least about 10.sup.8 M.sup.-1, preferably an
equilibrium associated constant (K.sub.A) of at least 10.sup.9
M.sup.-1.
[0333] In a further aspect, the invention relates to a transfectoma
comprising nucleic acids encoding a human heavy chain and a human
light chain, wherein the transfectoma produces a detectable amount
of the antibody described herein above.
[0334] In a further aspect, the invention relates to a composition
comprising the antibody described herein above and a
pharmaceutically acceptable carrier.
[0335] In an even further aspect, the invention relates to the
antibody described herein above for use as a medicament, preferably
for use a medicament for the treatment of cancer.
[0336] In an even further aspect, the invention relates to method
of treating or preventing a disease mediated by expression of EGFR,
comprising administration to a subject the antibody of the
invention in an amount effective to treat or prevent the
EGFR-mediated disease. In one embodiment, the disease is cancer. In
another embodiment, the method further comprises the
co-administration of a therapeutic agent.
Undesired Side-Effects
[0337] Undesired side-effects common to anti-EGFR agents include
dermatological side-effects, such as papulopustolar rash, usually
on the face, upper back and upper torso. Rash generally develops in
a dose-dependent manner.
[0338] In some of the above described embodiments of the method of
the invention, anti-EGFR antibodies are given at a lower dose than
what is usual for anti-EGFR antibody therapy. In such embodiments,
rash may be reduced.
[0339] Rash can be quantified using the grades defined in the
Common Terminology Criteria for Adverse Events (CTCAE), e.g.
version 3.0, under the term "Rash/desquamation". As desquamation is
not a common side effect of treatment with EGFR inhibition therapy,
the patient's skin rash may be scored based on rash only. The CTCAE
criterion for rash/desquamation can therefore suitably be adjusted
as follows:
TABLE-US-00002 CTCAE Description Grade Term Rash 1 Macular or
papular eruption or erythema without associated symptoms 2 Macular
or papular eruption or erythema with pruritus or other associated
symptoms 3 Widespread and confluent erythroderma or macular,
papular, or vesicular eruption 4 Generalized exfoliative,
ulcerative, or bullos dermatitis 5 Death
[0340] A reduction in rash, e.g. of 10%, when used herein indicates
is a statistically significant reduction of 10% in the total CTCAE
score of a representative population, as compared to standard
therapy.
[0341] In a preferred embodiment of the method or use of the
invention, the rash is reduced by at least 10%, such as at least
20%, e.g. at least 30%, such as at least 40%, e.g. at least 50%,
such as at least 60%, e.g. at least 70%, such as at least 80%, e.g.
at least 90%, such as least 95%, as compared to standard therapy
with the first anti-EGFR antibody, e.g. zalutumumab, cetuximab or
panitumumab, and/or compared to standard therapy for the second
anti-EGFR antibody.
Compositions
Formulation, Additives and Mode-of-Administration
[0342] The antibodies used in the present invention may be
formulated with pharmaceutically acceptable carriers or diluents as
well as any other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack
Publishing Co., Easton, Pa., 1995.
[0343] The pharmaceutically acceptable carriers or diluents as well
as any other known adjuvants and excipients should be suitable for
the chosen composition of the present invention and the chosen mode
of administration. Suitability for carriers and other components of
pharmaceutical compositions is determined based on the lack of
significant negative impact on the desired biological properties of
the chosen compound or pharmaceutical composition of the present
invention (e.g., less than a substantial impact (10% or less
relative inhibition, 5% or less relative inhibition, etc.) on
antigen binding.
[0344] A pharmaceutical composition of the present invention may
also include diluents, fillers, salts, buffers, detergents (e.g., a
nonionic detergent, such as Tween-80), stabilizers, stabilizers
(e.g., sugars or protein-free amino acids), preservatives, tissue
fixatives, solubilizers, and/or other materials suitable for
inclusion in a pharmaceutical composition.
[0345] The actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0346] The pharmaceutical composition may be administered by any
suitable route and mode. Suitable routes of administering a
composition in vivo and in vitro are well known in the art and may
be selected by those of ordinary skill in the art.
[0347] The antibodies may be administered via any suitable route,
such as an oral, nasal, inhalable, topical (including buccal,
transdermal and sublingual), rectal, vaginal and/or parenteral
route.
[0348] In one embodiment of the method of the present invention,
one or both antibodies are administered parenterally, preferably
intravenously.
[0349] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
include intratumoral, epidermal, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, intratendinous,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, intracranial,
intrathoracic, epidural and intrasternal injection and infusion. In
a preferred embodiment, the pharmaceutical composition is
administered by intravenous or subcutaneous injection or
infusion.
[0350] In another embodiment, the antibodies used in the present
invention are administered in crystalline form by subcutaneous
injection, cf. Yang et al., PNAS USA 100(12), 6934-6939 (2003).
[0351] The pharmaceutical compositions of the present invention may
be administered with medical devices known in the art. For example,
in one embodiment, a pharmaceutical composition of the present
invention may be administered with a needleless hypodermic
injection device, such as the devices disclosed in U.S. Pat. No.
5,399,163, U.S. Pat. No. 5,383,851, U.S. Pat. No. 5,312,335, U.S.
Pat. No. 5,064,413, U.S. Pat. No. 4,941,880, U.S. Pat. No.
4,790,824, or U.S. Pat. No. 4,596,556. Examples of well-known
implants and modules useful in the present invention include: U.S.
Pat. No. 4,487,603, which discloses an implantable micro-infusion
pump for dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicaments through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system.
Many other such implants, delivery systems, and modules are known
to those skilled in the art.
[0352] The pharmaceutical compositions containing the antibodies
may conveniently be presented in unit dosage form and may be
prepared by any methods known in the art of pharmacy. The amount of
active ingredient which may be combined with a carrier material to
produce a single dosage form will vary depending upon the subject
being treated, and the particular mode of administration. The
amount of active ingredient which may be combined with a carrier
material to produce a single dosage form will generally be that
amount of the composition which produces a therapeutic effect.
Generally, out of one hundred percent, this amount will range from
about 0.01% to about 99% of active ingredient, such as from about
0.1% to about 70%, for instance from about 1% to about 30%.
[0353] Regardless of the route of administration selected, the
compositions used in the present invention, which may be used in
the form of a pharmaceutically acceptable salt or in a suitable
hydrated form are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art. A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see for
instance Berge, S. M. et al., 3. Pharm. Sci. 1-19 (1977)). Examples
of such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous acids and the like, as well as
from nontoxic organic acids such as aliphatic mono- and
dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy
alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic
acids and the like. Base addition salts include those derived from
alkaline earth metals, such as sodium, potassium, magnesium,
calcium and the like, as well as from nontoxic organic amines, such
as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylene-diamine, procaine and the
like.
[0354] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and absorption
delaying agents, and the like that are physiologically compatible
with a compound used in the present invention.
[0355] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed
oil, and sesame oil, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
[0356] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the present invention is
contemplated.
[0357] Proper fluidity may be maintained, for example, by the use
of coating materials, such as lecithin, by the maintenance of the
required particle size in the case of dispersions, and by the use
of surfactants.
[0358] Pharmaceutical compositions used in the present invention
may also comprise pharmaceutically acceptable antioxidants for
instance (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0359] Pharmaceutical compositions used in the present invention
may also comprise isotonicity agents, such as sugars, polyalcohols
such as mannitol, sorbitol, glycerol or sodium chloride in the
compositions
[0360] Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions. The pharmaceutical compositions of the
present invention may also contain one or more adjuvants
appropriate for the chosen route of administration such as
preservatives, wetting agents, emulsifying agents, dispersing
agents, preservatives or buffers, which may enhance the shelf life
or effectiveness of the pharmaceutical composition. Compounds used
in the present invention may for instance be admixed with lactose,
sucrose, powders (e.g., starch powder), cellulose esters of
alkanoic acids, stearic acid, talc, magnesium stearate, magnesium
oxide, sodium and calcium salts of phosphoric and sulphuric acids,
acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or
polyvinyl alcohol. Other examples of adjuvants are QS21, GM-CSF,
SRL-172, histamine dihydrochloride, thymocartin, Tio-TEPA,
monophosphoryl-lipid A/micobacteria compositions, alum, incomplete
Freund's adjuvant, montanide ISA, ribi adjuvant system, TiterMax
adjuvant, syntex adjuvant formulations, immune-stimulating
complexes (ISCOMs), gerbu adjuvant, CpG oligodeoxynucleotides,
lipopolysaccharide, and polyinosinic:polycytidylic acid.
[0361] Prevention of presence of microorganisms may be ensured both
by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0362] Pharmaceutical compositions used in the present invention
may also include a suitable salt therefore. Any suitable salt, such
as an alkaline earth metal salt in any suitable form (e.g., a
buffer salt), may be used in the stabilization of the compound used
in the present invention. Suitable salts typically include sodium
chloride, sodium succinate, sodium sulfate, potassium chloride,
magnesium chloride, magnesium sulfate, and calcium chloride. In one
embodiment, an aluminum salt is used to stabilize a compound used
in the present invention in a pharmaceutical composition of the
present invention, which aluminum salt also may serve as an
adjuvant when such a composition is administered to a patient.
[0363] Pharmaceutical compositions used in the present invention
may be in a variety of suitable forms. Such forms include, for
example, liquid, semi-solid and solid dosage forms, such as liquid
solutions (e.g., injectable and infusible solutions), dispersions
or suspensions, emulsions, microemulsions, gels, creams, granules,
powders, tablets, pills, powders, liposomes, dendrimers and other
nanoparticles (see for instance Baek et al., Methods Enzymol. 362,
240-9 (2003), Nigavekar et al., Pharm Res. 21(3), 476-83 (2004),
microparticles, and suppositories.
[0364] The compounds used in the present invention may be prepared
with carriers that will protect the compound against rapid release,
such as a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems. Such
carriers may include gelatin, glyceryl monostearate, glyceryl
distearate, biodegradable, biocompatible polymers such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid alone or with a wax, or other
materials well known in the art. Methods for the preparation of
such formulations are generally known to those skilled in the art.
See e.g., Sustained and Controlled Release Drug Delivery Systems,
J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0365] In one embodiment, the compounds of the present invention
may be formulated to ensure proper distribution in vivo. For
example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds of
the present invention cross the BBB (if desired), they may be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see for instance U.S. Pat. No. 4,522,811, U.S. Pat. No.
5,374,548 and U.S. Pat. No. 5,399,331. The liposomes may comprise
one or more moieties which are selectively transported into
specific cells or organs, thus enhance targeted drug delivery (see
for instance V. V. Ranade J. Clin. Pharmacol. 29, 685 (1989)).
Exemplary targeting moieties include folate or biotin (see for
instance U.S. Pat. No. 5,416,016), mannosides (Umezawa et al.,
Biochem. Biophys. Res. Commun. 153, 1038 (1988)), antibodies (P. G.
Bloeman et al., FEBS Lett. 357, 140 (1995), M. Owais et al.,
Antimicrob. Agents Chemother. 39, 180 (1995)), surfactant protein A
receptor (Briscoe et al., Am. J. Physiol. 1233, 134 (1995)),
different species of which may comprise the pharmaceutical
compositions of the present inventions, as well as components of
the invented molecules, p120 (Schreier et al., J. Biol. Chem. 269,
9090 (1994)), see also K. Keinanen, M. L. Laukkanen, FEBS Lett.
346, 123 (1994) and J. J. Killion, I. J. Fidler, Immunomethods 4,
273 (1994).
[0366] In one embodiment of the present invention, the compounds of
the present invention are formulated in liposomes. In a further
embodiment, the liposomes include a targeting moiety. In a further
embodiment, the compounds in the liposomes are delivered by bolus
injection to a site proximal to the desired area, e.g., the site of
inflammation or infection, or the site of a tumor. The composition
should be fluid to the extent that easy syringability exists. It
should be stable under the conditions of manufacture and storage
and should be preserved against the contaminating action of
microorganisms such as bacteria and fungi.
[0367] In one embodiment, the compounds of the present invention
may be formulated to prevent or reduce their transport across the
placenta. This may be done by methods known in the art, e.g., by
PEGylation of the compounds or by use of F(abt).sub.2 fragments.
Further reference can be made to Cunningham-Rundles C et al., J
Immunol Methods. 152, 177-190 (1992) and to Landor M., Ann Allergy
Asthma Immunol 74, 279-283 (1995).
Tumors to be Treated
[0368] In one embodiment of the method or use of the invention,
said tumor is selected from the group consisting of: breast cancer
tumor, bladder cancer tumor, uterine/cervical cancer tumor,
esophageal cancer tumor, pancreatic cancer tumor, colon cancer
tumor, colorectal cancer tumor, kidney cancer tumor, ovarian cancer
tumor, prostate cancer tumor, renal cancer, head and neck cancer
(SCCHN) tumor, non-small cell lung cancer (NSCLC) tumor, stomach
cancer tumor, glioblastoma, pons glioma, high grade astrocytoma and
other EGFR-expressing tumors.
[0369] In a further embodiment of the method or use of the
invention, said tumor is a resistant or relapsed high-grade glioma,
such as a diffuse, intrinsic or pontine glioma.
[0370] In one embodiment of the method or use of the invention, the
EGFR levels in the tumor cells to be treated are not below
threshold for obtaining ADCC when treated with the first antibody
without co-administration of the second antibody. For example, in
one embodiment, the EGFR levels in the tumor or tumor cells may be
sufficient for obtaining ADCC in vivo when treated with zalutumumab
with a standard dosage of between 2 and 20 mg/kg once per week or
per 14 days without co-administration of a second antibody.
[0371] In a further embodiment of the method or use of the
invention, said tumor is an EGFRvIII-expressing tumor.
[0372] In a further embodiment of the method or use of the
invention, the human being in need of the treatment is a human
being who is likely to have tumors that exhibit EGFRvIII expression
or has been diagnosed to have such tumors. This may be determined
using standard diagnostic procedures well-known in the art, e.g.
similar those described on pages 26-31 of WO 2007123661
(incorporated herein by reference).
Combination Therapy
[0373] In further embodiments, the present invention provides
methods which comprise administration of anti-EGFR antibodies
combined with one or more additional therapeutic agents as
described below.
[0374] In one such embodiment, the method comprises administration
of a third antibody, such as an anti-EGFR antibody, wherein said
third antibody is not cross-blocking with either of said first and
second antibody.
[0375] In a further embodiment hereof:
[0376] said first antibody is an antibody which binds the same EGFR
epitope as LC1006-018,
and
[0377] said second antibody is selected from the group consisting
of: [0378] an antibody which binds the same EGFR epitope as
zalutumumab, [0379] an antibody which binds the same EGFR epitope
as cetuximab, [0380] an antibody which binds the same EGFR epitope
as panitumumab, [0381] an antibody which binds the same EGFR
epitope as 528, and
[0382] said third antibody is selected from the group consisting
of: [0383] an antibody which binds the same EGFR epitope as
LC1006-003, [0384] an antibody which binds the same EGFR epitope as
LC1006-005, [0385] an antibody which binds the same EGFR epitope as
LC1006-008, and [0386] an antibody which binds the same EGFR
epitope as LC1006-011.
[0387] In a further embodiment:
[0388] said first antibody is an antibody which binds the same EGFR
epitope as LC1006-018,
and
[0389] said second antibody is selected from the group consisting
of: [0390] an antibody which binds the same EGFR epitope as
zalutumumab, [0391] an antibody which binds the same EGFR epitope
as cetuximab, [0392] an antibody which binds the same EGFR epitope
as panitumumab, [0393] an antibody which binds the same EGFR
epitope as 528, and
[0394] said third antibody is an antibody which binds the same EGFR
epitope as ch806.
[0395] In a further embodiment:
[0396] said first antibody is an antibody which binds the same EGFR
epitope as LC1006-018,
and
[0397] said second antibody is selected from the group consisting
of: [0398] an antibody which binds the same EGFR epitope as
zalutumumab, [0399] an antibody which binds the same EGFR epitope
as cetuximab, [0400] an antibody which binds the same EGFR epitope
as panitumumab, [0401] an antibody which binds the same EGFR
epitope as 528, and
[0402] said third antibody is an antibody which binds the same EGFR
epitope as MR1-1.
[0403] In a further embodiment, the method of the invention
comprises administration of one or more further therapies selected
from chemotherapeutic agents, immunosuppressive agents,
anti-inflammatory agents, anti-psoriasis agents, radiation therapy,
hyperthermia, transplantation, surgery, sunlight therapy and
phototherapy.
[0404] In a further embodiment, the method comprises administration
of one or more further therapies selected from the group consisting
of nitrogen mustards, aziridines, alkyl sulfonates, nitrosoureas,
platinum complexes, non-classical alkylating agents, folate
analogs, purine analogs, adenosine analogs, pyrimidine analogs,
substituted ureas, antitumor antibiotics, epipodophyllotoxins,
microtubule agents, camptothecin analogs, enzymes, cytokines,
monoclonal antibodies, recombinant toxins and immunotoxins, cancer
gene therapies and cancer vaccines.
[0405] In an even further embodiment, the method comprises
administration of one or more further therapies selected from the
group consisting of immunosuppressive antibodies against MHC, CD2,
CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma, TNF-alpha, IL-4,
IL-5, IL-6R, IL-7, Il-8, IL-10, CD11a, CD20, and CD58 or antibodies
against their ligands, soluble IL-15R, and IL-10.
[0406] In a yet further embodiment, the method comprises
administration of one or more further therapies selected from the
group consisting of cyclosporine, azathioprine, mycophenolic acid,
mycophenolate mofetil, corticosteroids, methotrexate, gold salts,
sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,
tacrolimus (FK-506), OKT3, anti-thymocyte globulin,
transplantation.
[0407] In an even further embodiment, the method comprises
administration of one or more further therapies selected from the
group consisting of aspirin, other salicylates, steroidal drugs,
NSAIDs (nonsteroidal anti-inflammatory drugs), Cox-2 inhibitors,
and DMARDs (disease modifying antirheumatic drugs).
[0408] In another embodiment, the method comprises administration
of one or more further therapies selected from the group consisting
of coal tar, A vitamin, anthralin, calcipotrien, tarazotene,
corticosteroids, methotrexate, retinoids, cyclosporine, etanercept,
alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil,
tacrolimus (FK-506), hydroxyurea, sunlight therapy, and
phototherapy.
[0409] In a preferred embodiment, the method comprises
administration of one or more further therapies selected from:
platinum derivatives, such as cisplatin or carboplatin;
fluorouracil; paclitaxel, docetaxel and radiotherapy.
[0410] In a further preferred embodiment of the method of the
invention, in particular when one or both of the antibodies sued
are anti-EGFR antibodies, the method further comprises
administration one or more tyrosine kinase inhibitors, such as
gefitinib, erlotinib, XL-647, JNJ-26483327, vandetanib, BMS-599626,
AZD-9935, AEE-788, BIBW-2992, ISU-101, HMPL-010, ON-012380,
EKI-785, TX-2036, EHT-102, KI-6783, KI-6896 and LFM-A12.
Further Aspects and Embodiments of the Invention
[0411] 1. A method for the treatment of a tumor comprising combined
administration, to a human being in need thereof, of a first
antibody and a second antibody, wherein
[0412] said first antibody binds EGFR,
[0413] said second antibody binds EGFR, and
[0414] said first and second antibody are non-cross-blocking.
[0415] 2. The method of embodiment 1, wherein the dosage regimen is
such that substantial CDC is obtained at the tumor site.
[0416] 3. The method of any of the preceding embodiments, wherein
said first antibody is an antibody which is capable of binding an
EGFR epitope which is found on all wild-type-EGFR-expressing
cells.
[0417] 4. The method of any of the preceding embodiments, wherein
said first antibody binds to human EGFR with an equilibrium
dissociation constant (K.sub.D) of at most 10.sup.-8 M, preferably
at most 10.sup.-10 M.
[0418] 5. The method of any of the preceding embodiments, wherein
said first antibody is an antibody which is capable of inducing
ADCC at the tumor site in the absence of said second antibody.
[0419] 6. The method of any of the preceding embodiments, wherein
said first antibody is selected from the group consisting of:
[0420] an antibody which binds the same EGFR epitope as
zalutumumab,
[0421] an antibody which binds the same EGFR epitope as
cetuximab,
[0422] an antibody which binds the same EGFR epitope as
panitumumab,
[0423] an antibody which binds the same EGFR epitope as
nimotuzumab,
[0424] an antibody which binds the same EGFR epitope as matuzumab,
and
[0425] an antibody which binds the same EGFR epitope as 528.
[0426] 7. The method of any of the preceding embodiments, wherein
said first antibody is selected from the group consisting of:
[0427] an antibody which comprises the same heavy chain CDR3
sequence as zalutumumab and binds the same EGFR epitope as
zalutumumab,
[0428] an antibody which comprises the same heavy chain CDR3
sequence as cetuximab and binds the same EGFR epitope as
cetuximab,
[0429] an antibody which comprises the same heavy chain CDR3
sequence as panitumumab and binds the same EGFR epitope as
panitumumab,
[0430] an antibody which comprises the same heavy chain CDR3
sequence as nimotuzumab and binds the same EGFR epitope as
nimotuzumab,
[0431] an antibody which comprises the same heavy chain CDR3
sequence as matuzumab and binds the same EGFR epitope as matuzumab,
and
[0432] an antibody which comprises the same heavy chain CDR3
sequence as 528 and binds the same EGFR epitope as 528.
[0433] 8. The method of any of the preceding embodiments, wherein
said first antibody is selected from the group consisting of:
[0434] an antibody which comprises the same 6 CDR sequences as
zalutumumab,
[0435] an antibody which comprises the same 6 CDR sequences as
cetuximab,
[0436] an antibody which comprises the same 6 CDR sequences as
panitumumab,
[0437] an antibody which comprises the same 6 CDR sequences as
nimotuzumab,
[0438] an antibody which comprises the same 6 CDR sequences as
matuzumab, and
[0439] an antibody which comprises the same 6 CDR sequences as
528.
[0440] 9. The method of any of the preceding embodiments, wherein
said first antibody is selected from the group consisting of:
zalutumumab, cetuximab, panitumumab, nimotuzumab, matuzumab and
528.
[0441] 10. The method of embodiment 6, wherein said first antibody
is an antibody which binds the same EGFR epitope as zalutumumab and
said second antibody is selected from the group consisting of:
[0442] an antibody which binds the same EGFR epitope as
nimotuzumab, and
[0443] an antibody which binds the same EGFR epitope as
matuzumab.
[0444] 11. The method of embodiment 6, wherein said first antibody
is an antibody which binds the same EGFR epitope as cetuximab and
said second antibody is an antibody which binds the same EGFR
epitope as matuzumab.
[0445] 12. The method of embodiment 6, wherein said first antibody
is an antibody which binds the same EGFR epitope as panitumumab and
said second antibody is an antibody which binds the same EGFR
epitope as matuzumab.
[0446] 13. The method of embodiment 6, wherein said first antibody
is an antibody which binds the same EGFR epitope as nimotuzumab and
said second antibody is an antibody which binds the same EGFR
epitope as matuzumab.
[0447] 14. The method of embodiment 13, wherein said second
antibody is capable of binding an EGFR epitope which is found in
tumor cells, but is not detectable in normal cells.
[0448] 15. The method of embodiment 14, wherein said EGFR epitope
does not demonstrate any amino acid sequence alterations or
substitutions as compared to wild-type EGFR.
[0449] 16. The method of embodiment 14 or 15, wherein said second
antibody binds an EGFR epitope which is located within the region
comprising residues 273-501 of EGFR.
[0450] 17. The method of embodiments 14 to 16, wherein said second
antibody binds an EGFR epitope, which is located within the region
comprising residues 287-302 of EGFR.
[0451] 18. The method of any of embodiments 14 to 17, wherein said
second antibody is cross-blocking with ch806.
[0452] 19. The method of any of embodiments 14 to 18, wherein said
second antibody binds the same EGFR epitope as ch806.
[0453] 20. The method of embodiment 19, wherein the second antibody
comprises SEQ ID NO:3 and optionally one or more or all of SEQ ID
NO:1, 2, 4, 5 and 6.
[0454] 21. The method of embodiment 19, wherein the second antibody
is ch806.
[0455] 22. The method of embodiment 13, wherein the second antibody
is MR1-1.
[0456] 23. The method of any of embodiments 1 to 9, wherein said
second antibody is specific for EGFR-vIII.
[0457] 24. The method of any of the preceding embodiments, wherein
the first and/or the second antibody is a human antibody.
[0458] 25. The method of any of the preceding embodiments, wherein
the dosage regimen of said first antibody comprises administration,
at least once per 14 days, of a dosage of antibody of at least 0.1
mg/kg, such as at least 0.25 mg/kg, e.g. at least 0.5 mg/kg, such
as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such as at least 2
mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg, e.g. at
least 5 mg/kg, such as at least 6 mg/kg, e.g. at least 7 mg/kg,
such as at least 8 mg/kg, e.g. at least 9 mg/kg, such as at least
10 mg/kg, e.g. at least 12 mg/kg, such as at least 15 mg/kg, e.g.
at least 20 mg/kg.
[0459] 26. The method of any of the preceding embodiments, wherein
the dosage regimen of said second antibody comprises
administration, at least once per 14 days, of a dosage of antibody
of at least 0.1 mg/kg, such as at least 0.25 mg/kg, e.g. at least
0.5 mg/kg, such as at least 1 mg/kg, e.g. at least 1.5 mg/kg, such
as at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4
mg/kg, e.g. at least 5 mg/kg, such as at least 6 mg/kg, e.g. at
least 7 mg/kg, such as at least 8 mg/kg, e.g. at least 9 mg/kg,
such as at least 10 mg/kg, e.g. at least 12 mg/kg, such as at least
15 mg/kg, e.g. at least 20 mg/kg.
[0460] 27. The method of any of embodiments 25 or 26, wherein the
administration of said first and second antibody is at least once
per week.
[0461] 28. The method of any of embodiments 1 to 24, wherein the
dosage regimen for said first antibody is lower than a standard
dosage regimen for said first antibody.
[0462] 29. The method of any of embodiments 1 to 24, wherein the
dosage regimen of said first antibody comprises administration of a
total dosage per 14 days of between 0.01 mg/kg and 2 mg/kg, such as
between 0.01 mg/kg and 1 mg/kg, e.g. between 0.01 mg/kg and 0.5
mg/kg, such as between 0.01 mg/kg and 0.25 mg/kg, e.g. between 0.01
mg/kg and 0.1 mg/kg, such as between 0.01 mg/kg and 0.05 mg/kg.
[0463] 30. The method of any of embodiments 1 to 24 or 28 or 29,
wherein the dosage regimen for said second antibody is lower than a
standard dosage regimen for said second antibody.
[0464] 31. The method of any of embodiments 1 to 24 or 28 or 29,
wherein the dosage regimen of said second antibody comprises
administration of a total dosage per 14 days of between 0.01 mg/kg
and 2 mg/kg, such as between 0.01 mg/kg and 1 mg/kg, e.g. between
0.01 mg/kg and 0.5 mg/kg, such as between 0.01 mg/kg and 0.25
mg/kg, e.g. between 0.01 mg/kg and 0.1 mg/kg, such as between 0.01
mg/kg and 0.05 mg/kg.
[0465] 32. The method of any of the preceding embodiments, wherein
the dosage regimen is such that substantially no CDC is obtained at
non-tumor sites.
[0466] 33. The method of any of the preceding embodiments, wherein
the dosage regimen ensures efficient inhibition of ligand binding
at tumor sites.
[0467] 34. The method of any of embodiments 13 to 23 or 30 to 34,
wherein the dosage regimen for said first antibody is a dosage
regimen which comprises an equal or a higher dosage than a standard
dosage regimen for said first antibody.
[0468] 35. The method of any of embodiments 13 to 23 or 30 to 34,
wherein said first antibody is administered at an at least 2 times
higher dose than said second antibody, such as an at least 4 times
higher dose, e.g. an at least 10 times higher dose, such as an at
least 25 times higher dose, e.g. an at least 50 times higher dose
than said second antibody.
[0469] 36. The method of embodiment 35, wherein said first antibody
is administered at a between 2 and 50 times higher dose than said
second antibody, such as a between 5 and 20 times higher dose than
said second antibody.
[0470] 37. The method of any of embodiments 13 to 23 or 30 to 34,
wherein:
[0471] the dosage regimen of the first antibody comprises
administration, at least once per 14 days, of a dose of antibody of
at least 2 mg/kg, e.g. at least 3 mg/kg, such as at least 4 mg/kg,
e.g. at least 5 mg/kg, such as at least 6 mg/kg, e.g. at least 7
mg/kg, such as at least 8 mg/kg, e.g. at least 9 mg/kg, such as at
least 10 mg/kg, e.g. at least 12 mg/kg, such as at least 15 mg/kg,
e.g. at least 20 mg/kg, and
[0472] the dosage regimen of the second antibody comprises
administration of a total dosage per 14 days of between 0.1 mg/kg
and 1 mg/kg, such as a dose of antibody of between 0.2 mg/kg and 1
mg/kg, e.g. such as a dose of antibody of between 0.1 mg/kg and 0.5
mg/kg, such as a dose of antibody of between 0.2 mg/kg and 0.5
mg/kg.
[0473] 38. The method of any of embodiments 13 to 23 or 30 to 34,
wherein:
[0474] the dosage regimen of the first antibody comprises
administration, at least once per 14 days, of a dose of antibody of
at least 4 mg/kg, e.g. at least 5 mg/kg, such as at least 6 mg/kg,
e.g. at least 7 mg/kg, such as at least 8 mg/kg, e.g. at least 9
mg/kg, such as at least 10 mg/kg, e.g. at least 12 mg/kg, such as
at least 15 mg/kg, e.g. at least 20 mg/kg, and
[0475] the dosage regimen of the second antibody comprises
administration of a total dosage per 14 days of between 0.1 mg/kg
and 2 mg/kg, such as a dose of antibody of between 0.2 mg/kg and 1
mg/kg, e.g. such as a dose of antibody of between 0.1 mg/kg and 2
mg/kg, such as a dose of antibody of between 0.1 mg/kg and 0.5
mg/kg.
[0476] 39. The method of any of the preceding embodiments, wherein
said second antibody is administered at least 15 minutes, such as
at least one hour, e.g. at least two hours, such as at least eight
hours before the first antibody, preferably between 15 minutes and
6 hours, such as between 1 hour and 4 hours before said first
antibody.
[0477] 40. The method of any of the preceding embodiments, wherein
the total duration of the treatment is at least one month, such as
at least two months, e.g. at least four months, such as at least
six months.
[0478] 41. The method of any of the preceding embodiments, wherein
said first and/or second antibody is administered parenterally,
preferably intravenously.
[0479] 42. The method of any of the preceding embodiments, further
comprising administration of a third antibody, wherein said third
antibody is not cross-blocking with either of said first and second
antibody.
[0480] 43. The method of any of the preceding embodiments, wherein
said tumor is selected from the group consisting of: breast cancer
tumor, bladder cancer tumor, uterine/cervical cancer tumor,
esophageal cancer tumor, pancreatic cancer tumor, colorectal cancer
tumor, kidney cancer tumor, ovarian cancer tumor, prostate cancer
tumor, head and neck cancer tumor, non-small cell lung cancer
tumor, stomach tumor, glioblastoma and other EGFR-expressing
tumors.
[0481] 44. The method of any of the preceding embodiments, wherein
the EGFR levels in the tumor cells to be treated are not below
threshold for obtaining ADCC when treated with the first antibody
without co-administration of the second antibody.
[0482] 45. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from chemotherapeutic agents, immunosuppressive agents,
anti-inflammatory agents, anti-psoriasis agents, radiation therapy,
hyperthermia, transplantation, surgery, sunlight therapy, and
phototherapy.
[0483] 46. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from the group consisting of nitrogen mustards, aziridines, alkyl
sulfonates, nitrosoureas, platinum complexes, non-classical
alkylating agents, folate analogs, purine analogs, adenosine
analogs, pyrimidine analogs, substituted ureas, antitumor
antibiotics, epipodophyllotoxins, microtubule agents, camptothecin
analogs, enzymes, cytokines, monoclonal antibodies, recombinant
toxins and immunotoxins, cancer gene therapies, and cancer
vaccines.
[0484] 47. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from the group consisting of immunosuppressive antibodies against
MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma,
TNF-alpha, IL-4, IL-5, IL-6R, IL-7, IL-10, CD11a, CD20, and CD58 or
antibodies against their ligands, soluble IL-15R, and IL-10.
[0485] 48. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from the group consisting of cyclosporine, azathioprine,
mycophenolic acid, mycophenolate mofetil, corticosteroids,
methotrexate, gold salts, sulfasalazine, antimalarials, brequinar,
leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine,
cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3,
anti-thymocyte globulin, transplantation, and surgery.
[0486] 49. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from the group consisting of aspirin, other salicylates, steroidal
drugs, NSAIDs (nonsteroidal anti-inflammatory drugs), Cox-2
inhibitors, and DMARDs (disease modifying antirheumatic drugs).
[0487] 50. The method of any of the preceding embodiments,
comprising administration of one or more further therapies selected
from the group consisting of coal tar, A vitamin, anthralin,
calcipotrien, tarazotene, corticosteroids, methotrexate, retinoids,
cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine,
mycophenolate mofetil, tacrolimus (FK-506), hydroxyurea, sunlight
therapy, and phototherapy.
[0488] 51. The method of any of the preceding embodiments,
comprising administration of one or more tyrosine kinase
inhibitors, such as gefitinib, erlotinib, XL-647, JNJ-26483327,
vandetanib, BMS-599626, AZD-9935, AEE-788, BIBW-2992, ISU-101,
HMPL-010, ON-012380, EKI-785, TX-2036, EHT-102, KI-6783, KI-6896
and LFM-A12.
[0489] 52. The method of any of the preceding embodiments, wherein
said tumor is an EGFRvIII-expressing tumor.
[0490] 53. The method of any of the preceding embodiments, wherein
the human being in need of the treatment is a human being who has
been diagnosed to have tumors that exhibit EGFRvIII expression.
[0491] 54. A first antibody for use in the treatment of a tumor in
combination with a second antibody, wherein
[0492] said first antibody binds EGFR,
[0493] said second antibody binds EGFR, and
[0494] said first and second antibody are non-cross-blocking.
[0495] 55. The first antibody of embodiment 54, wherein the first
antibody, the second antibody and/or the treatment comprises one or
more of the further features of any one of embodiment 2 to 53.
[0496] 56. A second antibody for use in the treatment of a tumor in
combination with a first antibody, wherein
[0497] said first antibody binds EGFR,
[0498] said second antibody binds EGFR, and
[0499] said first and second antibody are non-cross-blocking.
[0500] 57. The second antibody of embodiment 56, wherein the first
antibody, the second antibody and/or the treatment comprises one or
more of the further features of any one of embodiment 2 to 53.
[0501] 58. Use of a first antibody and a second antibody for the
preparation of a medicament for the treatment of a tumor,
wherein
[0502] said first antibody binds EGFR,
[0503] said second antibody binds EGFR, and
[0504] said first and second antibody are non-cross-blocking.
[0505] 59. The use of embodiment 58, comprising one or more of the
further features of any one of embodiment 2 to 53.
[0506] 60. A bispecific antibody comprising a first binding
specificity which binds an EGFR epitope which is found on all
wild-type-EGFR-expressing cells and a second binding specificity
which binds an EGFR epitope which is found in tumor cells, but is
not detectable in normal cells.
[0507] 61. The bispecific antibody of embodiment 60, wherein the
second binding specificity binds an EGFR epitope is located within
the region comprising residues 273-501 of EGFR, preferably the same
EGFR epitope as bound by ch806, wherein said first and second
binding specificity are non-cross-blocking.
[0508] 62. The bispecific antibody of embodiment 61, wherein the
second binding specificity is specific for EGFR-vIII.
[0509] 63. The bispecific antibody of any one of embodiments 60 to
62, wherein the antibody comprises a first binding specificity
which binds an epitope selected from the group consisting of:
[0510] the EGFR epitope bound by zalutumumab,
[0511] the EGFR epitope bound by cetuximab,
[0512] the EGFR epitope bound by panitumumab,
[0513] the EGFR epitope bound by nimotuzumab,
[0514] the EGFR epitope bound by matuzumab, and
[0515] the EGFR epitope bound by 528.
[0516] 64. A bispecific antibody as defined in any one of
embodiments 60 to 63 for use as a medicament.
[0517] 65. A bispecific antibody as defined any one of embodiments
60 to 63 for use as a medicament for the treatment of cancer.
[0518] 66. Use of a bispecific antibody as defined any one of
embodiments 60 to 63 for the preparation of a medicament for the
treatment of cancer.
[0519] 67. A method for the treatment of cancer comprising
administration of a bispecific antibody as defined in embodiment 60
or 61.
[0520] 68. The bispecific antibody of embodiment 60 or 61, the use
of embodiment 66 or the method of embodiment 67, wherein said tumor
is selected from the group consisting of: breast cancer tumor,
bladder cancer tumor, uterine/cervical cancer tumor, esophageal
cancer tumor, pancreatic cancer tumor, colorectal cancer tumor,
kidney cancer tumor, ovarian cancer tumor, prostate cancer tumor,
head and neck cancer tumor, non-small cell lung cancer tumor,
stomach cancer tumor, glioblastoma and other EGFR-expressing
tumors.
[0521] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
EXAMPLES
Example 1
Generation and Source of Antibodies
[0522] Fully human IgG1,.kappa. antibody 2F8 (HuMax-EGFR,
zalutumumab) was generated as described previously (WO 02/100348).
2F8 F(ab).sub.2 fragments were produced by trypsin digestion of
parental antibody 2F8. An IgG4 isotype variant of 2F8 was generated
by genetic engineering, and produced in CHO cells as described
previously (Danish patent application PA 2007 00491 (Genmab)).
[0523] Fully human IgG1,.kappa. antibodies 003 (P- or LC1006-003),
005 (P- or LC1006-005), 008 (P- or LC1006-008), 011 (P- or
LC1006-011) and 018 (P- or LC1006-018) were generated by immunizing
HuMab mice (Medarex, Milpitas, Calif.) with alternating A431 cells
and purified EGFR (Sigma-Aldrich, St. Louis, Mo. cat E-3641). Mouse
spleens were fused with Sp2/0 mouse myeloma cells using Peg fusion
or electrofusion. In some instances hybridomas derived directly
from expansion of the fused cells were used in experiments. These
hybridomas were named P1006-003, P1006-005, P1006-005, P1006-008,
P1006-011 and P1006-018. The hybridomas were also cloned by
limiting dilution, and subsequently named LC1006-003, -005, -008,
-011 and -018. Culture supernatant was harvested and antibodies
were purified at Genmab BV. using protein A affinity
chromatography, followed by size exclusion chromatography on an
HR200 column (Pharmacia, Peapack, N.J.) and formulation in PBS.
[0524] DNA constructs expressing antibody ch806 (described in
WO02092771) were generated and expressed in HEK293 cells using
standard procedures. Purification was performed using protein A
affinity chromatography as described above.
[0525] DNA constructs expressing antibody MR1-1 (described in Beers
et al. (2000) Clin. Cancer Res. 6:2835) were generated using
standard procedures. Expression CHO cells and antibody purification
was performed using protein A affinity chromatography as described
above.
[0526] A human IgG1,.kappa. antibody specific for keyhole limpet
hemocyanin (Humab-KLH) developed using the same mouse strain,
served as isotype control IgG in most experiments.
[0527] Apart from these, also the commercially available antibodies
225 (HB-8508, murine IgG1) and 528 (HB-8509, murine IgG2a; both
from ATCC, Manassas, Va.), C225 (chimeric IgG1; cetuximab, Merck,
Dietikon, Switzerland), E7.6.3 (fully human IgG2; panitumumab,
Amgen, Thousand Oaks, Calif.) and matuzumab (h425, humanized IgG1,
Merck, Darmstadt, Germany) were used.
[0528] For direct immunfluorescence studies, EGFR antibodies were
fluorescein isothiocyanate (FITC)-- conjugated using the
EZ-label-kit (Pierce, Rockford, Ill.) according to the
manufacturer's instructions.
Example 2
Sequencing of VH and VL regions
[0529] Total RNA was prepared from 5.times.10.sup.6 cells of
hybridoma cell lines LC1006-003, -005, -008, -011 and -018 with the
RNeasy kit (Qiagen, The Netherlands) according to the
manufacturer's protocol. 5'-RACE-Complementary DNA (cDNA) of RNA
was prepared from 100 ng total RNA, using the SMART RACE cDNA
Amplification kit (Clontech), following the manufacturer's
protocol. Oligonucleotide primers were synthesized and quantified
by Isogen Bioscience (Maarssen, The Netherlands). Primers were
dissolved in H.sub.2O to 100 pmol/.mu.l and stored at -20.degree.
C. A summary of all PCR and sequencing primers is tabulated in the
table below. For PCR, PfuTurbo.RTM. Hotstart DNA polymerase
(Stratagene, Amsterdam, The Netherlands) was used according to the
manufacturer's instructions. Each reaction mix contained 200 .mu.M
mixed dNTPs (Roche Diagnostics, Almere, The Netherlands), 12
.mu.mol of the reverse primer (RACEG1A1 for the VH and RACEKA1 for
the VL), 7.2 pmol UPM-Mix (UPM-Mix: 2 .mu.M ShortUPMH3 and 0.4
.mu.M LongUPMH3), 0.6 .mu.l of the 5'RACE cDNA template, and 1.5
unit of PfuTurbo.RTM. Hotstart DNA polymerase in PCR reaction
buffer (supplied with polymerase) in a total volume of 30 .mu.l.
PCR reactions were carried out with a TGradient Thermocycler 96
(Whatman Biometra, Goettingen, Germany) using a 35-cycle program:
denaturing at 95.degree. C. for 2 min; 35 cycles of 95.degree. C.
for 30 sec, a 50.degree. C. for 30 sec, and 72.degree. C. for 1
min; final extension at 72.degree. C. for 10 min. If appropriate,
the PCR mixes were stored at 4.degree. C. until further analysis or
processing. The reaction products were separated by electrophoresis
on a 1% TAE agarose gel and stained with ethidium bromide. Bands of
the correct size were cut from the gels and the DNA was isolated
from the agarose using the MiniElute Reaction Cleanup kit (Qiagen).
Gel isolated PCR fragments were cloned into the pCR4BIunt-Topo
vector (Invitrogen) using the Zero Blunt.RTM.TOPO.RTM. PCR Cloning
Kit for Sequencing (Invitrogen) and protocol. 5 .mu.l of the
ligation mixture was transformed into OneShot DH5.alpha.T1R
competent E. coli (Invitrogen) and plated on LB/Ampicillin plates.
The V-regions of the antibodies were sequenced by AGOWA (Berlin,
Germany) after picking more than 20 colonies of each specificity,
isolating plasmid and sequencing with the M13 reverse primer.
Analysis revealed a close similarity between the VHs of LC1006-003
and 008 and between LC1006-005 and 011 (see alignment in FIG. 17).
All VLs were closely related. LC1006-003 and 008 had an identical
VL and LC1006-005 and 011 both had an identical pair of two VLs,
differing in a single amino acid. All sequences were in accordance
with the results of the molecular weights of the antibodies as
determined by ESI-MS.
TABLE-US-00003 Primers Name Length Oligo Sequence ShortUPMH3 31
TGAAAGCTTCTAATACGACTCACTATAGGGC RACEKA1 22 TATCCACCTTCCACTGTACTTT
RACEG1A1 22 GGGAGTAGAGTCCTGAGGACTG M13reverse 20
GGATAACAATTTCACACAGG LongUPMH3 54 TGAAAGCTTCTAATACGACTCACTATAGGGCAA
GCAGTGGTATCAACGCAGAGT
Example 3
Functional characterization of EGFR antibodies 1006-003, -005,
-008, -011 and -018
[0530] Binding to purified EGFR: ELISA plates (Greiner Bioscience,
Frickenhausen, Germany, cat no: 655092) were coated with purified
EGFR (Sigma-Aldrich, cat no. E3641) 0.4 .mu.g/ml diluted in PBS 100
.mu.l/well. The plates were incubated over night at 4.degree.
C.
[0531] After incubation the plates were emptied, and PBSC (PBS, 2%
chicken serum) block solution was added 100 .mu.l/well for 1 hour
at room temperature (RT). The anti-EGFR clones were five fold
serial diluted (40 .mu.g/ml to 0.003 .mu.g/ml) in PBSTC (PBS, 2%
chicken serum, 0.05% tween-20) and incubated for 1 h at RT.
[0532] Subsequently, the plates were incubated with
peroxidase-labeled goat anti-human IgG Fc-specific antibodies
(Jackson Immunoresearch Laboratories, West Grove, Pa., cat no:
109-035-098). Next, the plates were incubated with ABTS (Roche,
Mannheim, Germany, ABTS tablets cat no: 1112422, ABTS buffer cat
no: 1112597). Absorbance was measured using a microplate reader
(Bio-Tek Instruments, Winooski, Vt. cat no: EL808) at 405 nm. The
binding was analysed using Graphpad Prism for fitting the data to a
four parameter logistic curve.
TABLE-US-00004 EC50 (.mu.g/ml) 2F8 0.019 P1006-003 0.117 P1006-005
0.089 P1006-008 2.459 P1006-011 0.053 P1006-018 0.081
[0533] The above table shows the concentrations of half-maximal
binding (EC50) of clones P1006-003, -005, -008, -011, -018 and 2F8
to purified EGFR as observed in ELISA. The EC50 values were
determined from a four-parameter logistic curve fit and are
expressed in .mu.g/ml. All antibodies bound to purified EGFR.
[0534] Binding to EGFR expressing cells of LC1006-003, -005, -008,
-011 and -018 is shown in example 4.
[0535] Inhibition of EGFR signalling: Inhibition of EGFR signaling
was tested by a ligand binding inhibition assay, an assay measuring
inhibition of ligand-induced EGFR phosphorylation, and a cell
proliferation assay.
[0536] A431 cells, an EGFR overexpressing epidermoid cancer cell
line, were from the Deutsche Sammlung von Mikroorganismen and
Zellkulturen (Braunschweig, Germany; cell line number ACC 91).
Cells were cultured in RPMI 1640 medium (BioWhittaker, Verviers,
Belgium, cat no: BE12-115F), supplemented with 10% heat-inactivated
CCS (Hyclone Perbio. Logan, Utah, cat no: SH30087.03) and 50 IU/ml
penicillin, 50 .mu.g/ml streptomycin (BioWhittaker, cat no:
DE17-603E). Cells were detached by using trypsin-EDTA (10.times.
stock Gibco BRL, cat no: 35400-027) in PBS.
[0537] For comparison of EGFR antibodies' capacity to block ligand
binding, 1.5.times.10.sup.5 A431 cells were co-incubated with 2.5
.mu.g/ml FITC-conjugated EGF (Invitrogen) and 200 .mu.g/ml
antibodies for 30 minutes. After washing, cells were analyzed by
flow cytometry. Blockade of ligand binding was calculated by the
formula: % inhibition of EGF-binding=(RFI without-RFI with
antibody)/(RFI without antibody).times.100. All experimental steps
were performed at 4.degree. C. Data are presented as mean.+-.SEM of
three independent experiments. FIG. 1 shows that C225, E7.6.3 2F8,
and 528 are strong inhibitors of EGF binding to A431 cells, that
LC1006-003, LC1006-005, LC1006-008, and LC1006-011 are week
inhibitors, and that LC1006-018 has no blocking activity.
[0538] To confirm that EGF binding inhibition resulted in
inhibition of receptor auto-phosphorylation, we evaluated the
potency of the different clones to inhibit ligand-induced EGFR
phosphorylation in vitro. This was measured in a two-step assay
using the epidermoid cell line, A431 (ATCC, American Type Culture
Collection, Manassas, USA). The cells were cultured overnight in
96-wells plates in serum-free medium containing 0.5% human albumin
(human albumin 20%, Sanquin, the Netherlands). Next, mAb were added
in serial dilution. After 60 minutes incubation at 37.degree. C.,
50 ng/ml recombinant human EGF (Biosource) was added to induce
activation of non-blocked EGFR. Following an additional 30 minutes
incubation, cells were solubilized with lysis buffer (Cell
Signaling Technology, Beverly, Mass.), and the lysates were
transferred to ELISA plates coated with 1 .mu.g/ml of mouse
anti-EGFR antibodies (mAb EGFR1, BD Pharmingen, San Diego, Calif.).
After 2 hours incubation at RT, the plates were washed and binding
of phosphorylated EGFR was detected using a europium-labelled mouse
mAb, specific for phosphorylated tyrosines (mAb Eu-N1 P-Tyr-100,
PerkinElmer). Finally, DELFIA enhancement solution was added, and
time-resolved fluorescence was measured by exciting at 315 nm and
measuring emission at 615 nm on an EnVision plate reader
(PerkinElmer). Sigmoidal dose-response curves were calculated using
non-linear regression (GraphPad Prism 4).
[0539] FIG. 2 shows that LC1006-003, -005, -008, and -011 partially
inhibited EGF-induced EGFR auto-phosphorylation, while clone 2F8
(HuMax-EGFR) gave complete inhibition and LC1006-018 did not.
[0540] The ability of EGFR antibodies to inhibit tumor cell
proliferation was tested in an A431 proliferation assay. A431 cells
were seeded at a density of 500 cells per well in a 96 wells
culture plate (White, 96-well, TC, sterile, with lid, PerkinElmer,
Boston, Mass., cat no: 6005680). A 3 fold serial dilution (100
.mu.g/ml to 0.005 .mu.g/ml) of each anti-EGFR clone in culture
medium (RPMI 1640, 3% fetal clone II (Hyclone, cat no: SH30066.03),
1% penicillin/streptomycin) was added. Next, cells were incubated
in a humidified incubator at 37.degree. C./5% CO.sub.2 for five
days. Subsequently, 20 .mu.l AlamarBlue (BioSource, Camarillo
Calif. cat no: DAL1100) was added to each well and incubated at
37.degree. C./5% CO.sub.2 for 4 hours. Next, the fluorescence of
the wells was measured at excitation wavelength of 528 nm and an
emission wavelength of 590 nm using an ELISA plate reader, (BIO-Tek
Synergy HT, Beun de Ronde, cat no: 7091000).
[0541] FIGS. 3 and 4 show that only clone 2F8 inhibited A431
proliferation.
[0542] Antibody-dependent cell-mediated cytotoxicity: Peripheral
blood mononuclear cells (PBMC) were isolated from standard blood
donations (Sanquin Blood Bank, Utrecht, The Netherlands). Buffy
coats were diluted by adding PBS and transferred to 50 ml tubes. 10
ml Lymfocyte Separation Medium (Bio Whittaker, cat no: US17-829E)
was carefully placed under the diluted buffy coats. Tubes were
centrifuged at 800.times.g for 20 min at RT. Thereafter, the PBMC
were recovered from the plasma-medium interface and were washed
several times with culture medium until the supernatant was clear.
A431 target cells (2-5.times.10.sup.6 cells) were labelled with 100
.mu.Ci Na.sub.2.sup.51CrO.sub.4 (Amersham Biosciences, Uppsala,
Sweden, cat no: CSJ11) under shaking conditions at 37.degree. C.
for 1 hour. After incubation cells were washed thrice with PBS and
resuspended in culture medium (1.times.10.sup.5 cells/ml). Labelled
cells were pipetted in 96 well plates (5.times.10.sup.3, in 50
.mu.l/well) and preincubated with a 5 fold serial dilution (20
.mu.g/ml to 0.0003 .mu.g/ml) of anti-EGFR clones for 30 min at RT.
Culture medium was added instead of mAb to determine the
spontaneous .sup.51Cr release, Triton X-100 (1% final
concentration, Riedel de Haen, cat no: 56029) was added to
determine the maximal .sup.51Cr release. Subsequently, PBMC's were
dispensed into the plate (5.times.10.sup.5/well) and the cells were
incubated at 37.degree. C. over night. The next day, supernatants
were collected for measurement of .sup.51Cr release by
determination of the cpm in a gamma counter. Specific lysis is
calculated with formula below:
% specific lysis=(experimental release (cpm)-spontaneous release
(cpm))/(maximal release (cpm)-spontaneous release
(cpm)).times.100
[0543] FIG. 5 shows that P1006-003, -005, -008, -011 and -018
stimulated PBMC-induced ADCC of A431 target cells. The ability of
LC1006-003, -005, -008, -011 and -018 to induce
complement-dependent cytotoxicity is described below.
Example 4
Binding of EGFR Antibodies to EGFR-Expressing Cells
[0544] The human epidermoid carcinoma cell line A431 (DSMZ,
Braunschweig, Germany) and human glioblastoma cell line A1207
(originally established by Dr. Aaronson, National Cancer Institute,
National Institutes of Health, Bethesda, Md.) were kept in RPMI
1640 or DMEM, respectively. Both media were supplemented with 10%
heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin, 100
U/ml streptomycin, and 4 mM L-glutamine (all from Invitrogen,
Carlsbad, Calif.). Viability of cells was tested by trypan blue
exclusion.
[0545] Cell lines were characterised for quantitative surface
expression of EGFR and complement regulatory proteins CD46, CD55
and CD59 by indirect immunofluorescence. 1.times.10.sup.5 target
cells were incubated with murine monoclonal antibodies 225 (EGFR),
J 4-48 (CD46, Immunotech, Marseille, France), IA10 (CD55) or p282
(CD59, both from BD Pharmingen, Franklin Lakes, N.J.),
respectively, at saturating concentrations for 30 minutes at
4.degree. C. After washing, cells were stained with FITC-conjugated
polyclonal goat anti-mouse Ig (DAKO, Glostrup, Denmark) for 30
minutes at 4.degree. C., washed and analysed by flow cytometry. For
calculation of antigens' surface expression, the Qifikit (DAKO) was
used according to the manufacturer's instructions.
[0546] The table below shows the characterization of the cell
lines:
TABLE-US-00005 Cell line Antigen Calculated molecules/cell A431
EGFR 1,782,006 .+-. 196,146 CD46 220,087 .+-. 17,811 CD55 175,691
.+-. 25,906 CD59 708,530 .+-. 81,880 A1207 EGFR 1,673,737 .+-.
87,269 CD46 179,972 .+-. 15,654 CD55 66,077 .+-. 5,063 CD59 355,082
.+-. 46,611
[0547] Binding of the panel of EGFR-antibodies to A431 cells, as
measured by FACS analysis is shown in FIG. 6.
Example 5
Cross-Blocking of EGFR Antibodies
[0548] The EGFR binding epitopes of different EGFR antibodies were
analyzed by competitive immunofluorescence binding assays.
2.times.10.sup.5 EGFR expressing target cells were incubated for 30
minutes at 4.degree. C. with FITC-conjugated EGFR antibodies at
non-saturating concentrations in combination with 200-fold excess
of different unconjugated antibodies. After washing, samples were
analysed by flow cytometry. Level of competition was calculated
with the following formula: % competition=(experimental
MFI-background MFI)/(maximal MFI-background MFI).times.100, with
maximal MFI defined by the combination of FITC-conjugated EGFR
antibody with isotype control antibody KLH.
[0549] FIG. 7 shows the results of competitive immunofluorescence
studies. FIG. 8 exemplifies the cross-blocking of the different
antibodies graphically. To further investigate epitope differences,
the antibodies were also compared in their ability to bind to Ba/F3
cells transfected with EGFR-vIII. EGFR-vIII is characterized by
deletion of exons 2 to 7, involving nucleotides 275 to 1075. Thus,
EGFR-vIII lacks a major portion of the Cys-rich ligand-binding
domain, near the NH2 terminus of the extracellular portion of the
molecule. In this experiment, 2.times.10.sup.5 EGFR-vIII
transfected cells were incubated with EGFR antibodies at saturating
concentrations for 30 minutes, washed and stained with
FITC-conjugated F(ab').sub.2-fragments of polyclonal anti-human IgG
antibodies (DAKO) for 30 minutes. After washing again, cells were
analyzed by flow cytometry. All experimental steps were performed
at 4.degree. C. Data are presented as mean.+-.SEM of four
independent experiments. FIG. 9 shows that C225 and 2F8 bound to
EGFR-vIII, whereas LC1006-003, 005, 008, 011 and 018 did not.
Example 6
Complement Deposition
[0550] 1.times.10.sup.5 target cells were incubated for 15 minutes
at room temperature either with individual EGFR antibodies, or with
antibody combinations at additive antibody concentrations of 10
.mu.g/ml/well, followed by addition of 1% (vol/vol) NHS and
incubation at 37.degree. C. for 10 minutes. After washing, samples
were stained with polyclonal FITC-conjugated C1q or C4c antibodies
(both from DAKO) for 30 minutes at 4.degree. C., and analyzed by
flow cytometry (FIG. 10a: Coulter EPICS XL-MCL, Beckman Coulter,
Fullerton, Calif., FIG. 10b: FACSCanto II, Becton Dickinson, Aalst,
Belgium).
[0551] FIG. 10 shows that while individual EGFR antibodies did not
trigger C1q deposition, all examined non cross-blocking
combinations led to C1q deposition (except for certain combinations
that include 008, which is presumably due to the low affinity of
008 (see FIG. 6)). As shown in FIG. 11, the combination of three
non-blocking antibodies is superior to individual dual combinations
in C1q deposition.
Example 7
Complement Dependent Cytotoxicity (CDC)
[0552] Target cells were labeled with 200 .mu.Ci (7.4 MBq)
.sup.51Cr for 2 hours. After washing three times with RPMI 1640
medium, cells were adjusted to 10.sup.5/ml. 50 .mu.l freshly drawn
human serum, sensitizing antibodies and RPMI 1640 (10% FCS) were
added to round-bottomed microtiter plates (Nunc, Rochester, N.Y.).
Assays were started by adding target cells (50 .mu.l), resulting in
a final volume of 200 .mu.l/well and a final concentration of 25%
serum (unless otherwise indicated). After 3 hours at 37.degree. C.,
assays were centrifuged, and .sup.51Cr release from the
supernatants was measured in triplicates as counts per minute
(cpm). Percentage of cytotoxicity was calculated with the following
formula: % specific lysis=(experimental cpm-basal cpm)/(maximal
cpm-basal cpm).times.100, with maximal .sup.51Cr release determined
by adding perchloric acid (3% final concentration) to target cells
and basal release measured in the absence of sensitizing antibodies
and serum. Antibody-independent cytotoxicity (serum without target
antibodies) was not observed.
[0553] FIG. 12 shows that while none of the individual EGFR
antibodies and none of the cross-blocking combinations triggered
CDC, most of the non cross-blocking combinations led to significant
CDC on A431 cells (12a) and all non cross-blocking combinations led
to significant CDC on A1207 cells (12b).
[0554] FIG. 13 shows CDC of A431 (13a) and A1207 (13b) cells in
correlation to EGFR antibody concentration.
Example 8
Contribution of the Alternative and Classical Pathway of Complement
Activation in EGFR Antibody Mediated CDC
[0555] To determine the contribution of different complement
activation pathways in EGFR antibody mediated CDC, final
concentrations of 5 mM MgCl.sub.2 and 10 mM
ethylene-glycol-bis(.beta.-aminoethylether)-tetraacetic-acid
(Mg-EGTA) were added to selectively inhibit the classical pathway,
or 10 mM ethylenediamine tetraacetic acid (EDTA; both from Roth,
Karlsruhe, Germany) for complete blockade of complement activation,
respectively. Furthermore, for some experiments serum was heated to
50.degree. C. for 15 minutes to selectively inactivate the
alternative pathway, or to 56.degree. C. for 30 minutes for
complete heat-inactivation of the complement system. A431 cells
were used as target cells, antibodies at a concentration of 2
.mu.g/ml.
[0556] FIG. 14 shows that CDC by EGFR antibody combinations is
mediated by the classical complement pathway.
Example 9
Requirement of IgG1 Fc Regions for EGFR Antibody Mediated CDC
[0557] Combinations of EGFR antibody LC1006-003 with 2F8-IgG1,
2F8-IgG4 or 2F8-F(ab).sub.2 were tested in their ability to bind
C1q and C4c, and to induce CDC. A431 cell line was used as target.
It was found that relevant complement-deposition and -lysis was
only observed in the presence of two human IgG1 Fc fragments (FIGS.
15 and 16).
Example 10
Specific Lysis of EGFR-vIII Expressing A431 Cells
[0558] The human epidermoid carcinoma cell lines A431 and Ba/F3
cells (DSMZ, The German Resource Centre for Biological Material,
Braunschweig, Germany) were cultured in RPMI 1640-Glutamax-I medium
(Invitrogen Life Technologies) containing 10% heat-inactivated
fetal calf serum (FCS), 100 U/ml penicillin, and 100 U/ml
streptomycin (R10+). For the Ba/F3 cell line, murine (m) IL-3 was
either added as recombinant mIL-3 (R&D Systems) at 10 ng/ml or
as supernatant from WEHI-3B cells (DSMZ; concentration 10% v/v).
Medium for transfected cells additionally contained 1 mg/ml
geneticin (Invitrogen Life Technologies). Viability of cells was
tested by trypan blue exclusion.
[0559] The extracellular EGFR-vIII mutation was generated by
splicing by overlapping extension PCR (SOE-PCR) of the plasmid
vector pUSE-EGFR (Upstate Biotechnology) harboring wt EGFR.
EGFR-vIII coding DNA was amplified by PCR using pUse-EGFR as a
template with the following primers: exon 1;
5'ACCCACTGCTTACTGGCTTATCG-3' and 5'-CCGTGATCTGTCACCA
CATAATTACCTTTCTTTTCCTCCAGAGCCCGACTC-3', Exon 8; 5'-GAGTCGGG
CTCTGGAGGAAAAGAAAGGTAATTATGTGGTGACAGATCACGG-3' and 5'-CCTG
TGCAGGTGATGTTCATGG-3'. Introduction of the respective mutation and
the correctness of the EGFRvIII coding region was confirmed by
complete sequencing.
[0560] Ba/F3 cells were stably transfected by nucleofection of 2
.mu.g of plasmid DNA and 2.times.10.sup.6 cells using the Amaxa
transfection system according to the manufacturer's instructions.
Forty-eight hours after transfection, cells were put under
selection by adding 1 mg/ml geneticin.
[0561] A431 cells (3.5.times.10.sup.5) were transfected with
Lipofectamine2000 (Invitrogen Life Technologies) according to the
manufacturer's instructions. Transfected cells were selected with
0.8 mg/ml geneticin.
[0562] Twenty million transfected Ba/F3 cells were incubated with 4
ml of EGFR mAb m225 (mIgG1) at 20 .mu.g/ml in PBS containing 0.5%
BSA and 25% rabbit serum (to block nonspecific binding of the
primary Ab). After 15 min on ice, cells were washed twice with PBS
containing 0.5% BSA. The cell pellet was resuspended in 200 .mu.l
of PBS containing 0.5% BSA and 50% rabbit serum. Fifty microliters
of anti-mouse IgG1 magnetic beads (Miltenyi Biotec) was added, and
cells were incubated for another 10 min on ice. Cells were washed
twice and separated on LD depletion columns according to the
manufacturer's instructions (Miltenyi Biotec).
[0563] Transfected A431 cells (5.times.10.sup.6) were labeled with
MR1-1 IgG1 antibody and rabbit anti human IgG fluorescein
conjugated secondary antibody. Labeled cells were sorted with a
ARIA flow cytometer (Becton Dickinson) into 96 well plates and
cloned by limited dilution. Expression of EGFRvIII of individual
clones was detected by flow cytometry. High expression clones were
used for further analysis.
[0564] Target cells were labeled with 200 .mu.Ci (7.4 MBq)
.sup.51Cr for 2 hours. After washing three times with RPMI 1640
medium, cells were adjusted to 10.sup.5/ml. 50 .mu.l freshly drawn
human serum, sensitizing antibodies and RPMI 1640 (10% FCS) were
added to round-bottomed microtiter plates (Nunc, Rochester, N.Y.).
Assays were started by adding target cells (50 .mu.l), resulting in
a final volume of 200 .mu.l/well and a final concentration of 25%
serum (unless otherwise indicated). After 3 hours at 37.degree. C.,
assays were centrifuged, and .sup.51Cr release from the
supernatants was measured in triplicates as counts per minute
(cpm). Percentage of cytotoxicity was calculated with the following
formula: % specific lysis=(experimental cpm-basal cpm)/(maximal
cpm-basal cpm).times.100, with maximal .sup.51Cr release determined
by adding perchloric acid (3% final concentration) to target cells
and basal release measured in the absence of sensitizing antibodies
and serum.
[0565] FIG. 18 shows that antibodies ch806, MR1-1 and zalutumumab
all bound to wild-type EGFR on A431 cells as well as to EGFR-vIII
expressed on Ba/F3 and A431 cells.
[0566] FIG. 19 shows that neither the individual EGFR antibodies
tested nor combinations of these with ch806 or MR1-1 induced CDC on
untransfected A431 cells, expressing only wild-type EGFR. In
contrast, all combinations with ch806 or MR1-1 tested induced
significant CDC in EGFR-vIII expressing A431 cells.
[0567] FIG. 20 shows CDC induction by double and triple
combinations with ch806 or MR1-1. The triple combination of ch806,
zalutumumab and 018 induced even more lysis of EGFR-vIII expressing
cells than the double combinations of MR1-1 and zalutumumab or
MR1-1 and antibody 018.
TABLE-US-00006 SEQUENCE LISTING SEQ ID NO: 1 the heavy chain CDR1
sequence of antibody 806: GYSITSDFAWN SEQ ID NO: 2 the heavy chain
CDR2 sequence of antibody 806: GYISYSGNTRYNPSLK SEQ ID NO: 3 the
heavy chain CDR3 sequence of antibody 806: VTAGRGFPY SEQ ID NO: 4
the light chain CDR1 sequence of antibody 806: HSSQDINSNIG SEQ ID
NO: 5 the light chain CDR2 sequence of antibody 806: HGTNLDD SEQ ID
NO: 6 the light chain CDR3 sequence of antibody 806: VQYAQFPWT SEQ
ID NO: 7 VH 1006-003
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPGKGLEWVAN
IQQDGSEKNYLDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCARTY
SGFEDFWGQGTLVTVSS SEQ ID NO: 8 VL 1006-003
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIFD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFG QGTKVEIK SEQ ID
NO: 9 VH 1006-005
EVQLVESGGGLVQPGGSLRLSCAASRFTFSDYWMTWVRQAPGKGLEWVAH
IKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARGF
LIYFDYWGQGTLVTVSS SEQ ID NO: 10 VL1 1006-005
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPWTFGQ GTKVEIK SEQ ID
NO: 11 VL2 1006-005
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWWTFGQG TKVEIK SEQ ID
NO: 12 VH 1006-008
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVAN
IKQDGSEENYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCARTY
SGFEDYWGQGTLVTVSS SEQ ID NO: 13 VL 1006-008
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIFD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPWTFG QGTKVEIK SEQ ID
NO: 14 VH 1006-011
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMSWVRQAPGRGLEWVAH
INQDGSEKYYVDSVKGRFTLSRDTAKNSLYLQMNSLRAEDTAVYYCARGF
LIYFDYWGQGTLVTVSS SEQ ID NO: 15 VL1 1006-011
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWWTFGQG TKVEIK SEQ ID
NO: 16 VL2 1006-011
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPWTFGQ GTKVEIK SEQ ID
NO: 17 VH 1006-018
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVAN
IKKDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDL
GWGWGWYFDLWGRGTLVTVSS SEQ ID NO: 18 VL 1006-018
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQ GTKVEIK
Sequence CWU 1
1
18111PRTArtificialantibody variable region sequence 1Gly Tyr Ser
Ile Thr Ser Asp Phe Ala Trp Asn1 5 10216PRTArtificialantibody
variable region sequence 2Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg
Tyr Asn Pro Ser Leu Lys1 5 10 1539PRTArtificialantibody variable
region sequence 3Val Thr Ala Gly Arg Gly Phe Pro Tyr1
5411PRTArtificialantibody variable region sequence 4His Ser Ser Gln
Asp Ile Asn Ser Asn Ile Gly1 5 1057PRTArtificialantibody variable
region sequence 5His Gly Thr Asn Leu Asp Asp1
569PRTArtificialantibody variable region sequence 6Val Gln Tyr Ala
Gln Phe Pro Trp Thr1 57117PRTArtificialantibody variable region
sequence 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Asn Ile Gln Gln Asp Gly Ser Glu Lys Asn
Tyr Leu Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Ser65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Tyr Ser Gly Phe
Glu Asp Phe Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
1158108PRTArtificialantibody variable region sequence 8Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Phe Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 1059117PRTArtificialantibody variable region sequence 9Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala His Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Leu Tyr Tyr Cys 85 90 95Ala Arg Gly Phe Leu Ile Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11510107PRTArtificialantibody variable region sequence 10Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10511106PRTArtificialantibody variable region sequence 11Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10512117PRTArtificialantibody variable region sequence 12Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Glu Asn Tyr Val Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Tyr Ser Gly Phe Glu Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11513108PRTArtificialantibody variable region sequence 13Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Phe Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10514117PRTArtificialantibody variable region sequence
14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu
Trp Val 35 40 45Ala His Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Leu Ser Arg Asp Thr Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Phe Leu Ile Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11515106PRTArtificialantibody variable region sequence 15Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10516107PRTArtificialantibody variable region sequence 16Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 10517121PRTArtificialantibody variable region sequence 17Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Asn Ile Lys Lys Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Leu Gly Trp Gly Trp Gly Trp
Tyr Phe Asp Leu Trp Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser
Ser 115 12018107PRTArtificialantibody variable region sequence
18Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105
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References