U.S. patent application number 16/342275 was filed with the patent office on 2019-09-05 for anti-edb antibodies and antibody-drug conjugates.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Pfizer Inc.. Invention is credited to Hans-Peter GERBER, Andrea Therese HOOPER, Kimberly Ann MARQUETTE, Chad Michael MAY, Chakrapani SUBRAMANYAM.
Application Number | 20190269791 16/342275 |
Document ID | / |
Family ID | 60117722 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190269791 |
Kind Code |
A1 |
HOOPER; Andrea Therese ; et
al. |
September 5, 2019 |
ANTI-EDB ANTIBODIES AND ANTIBODY-DRUG CONJUGATES
Abstract
The present invention provides antibodies and antibody-drug
conjugates that bind to the extra domain B splice variant of
fibronectin 1 and methods for preparing and using the same.
Inventors: |
HOOPER; Andrea Therese;
(Port Chester, NY) ; MARQUETTE; Kimberly Ann;
(Somerville, MA) ; SUBRAMANYAM; Chakrapani; (South
Glastonbury, CT) ; GERBER; Hans-Peter; (San Carlos,
CA) ; MAY; Chad Michael; (Belmont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc. |
New York |
NY |
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
|
Family ID: |
60117722 |
Appl. No.: |
16/342275 |
Filed: |
October 3, 2017 |
PCT Filed: |
October 3, 2017 |
PCT NO: |
PCT/IB2017/056093 |
371 Date: |
April 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62409081 |
Oct 17, 2016 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6843 20170801;
A61K 38/08 20130101; C07K 2317/52 20130101; C07K 2317/33 20130101;
C07K 2317/565 20130101; C07K 2317/21 20130101; C07K 2317/92
20130101; A61K 47/6811 20170801; A61P 35/00 20180101; A61K 47/6803
20170801; A61P 35/02 20180101; C07K 16/18 20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61P 35/00 20060101 A61P035/00; A61K 38/08 20060101
A61K038/08; C07K 16/18 20060101 C07K016/18 |
Claims
1. An antibody-drug conjugate comprising (a) an antibody, or
antigen binding fragment thereof, that binds to extra domain B of
fibronectin, (b) a linker and (c) a drug.
2. The antibody-drug conjugate of claim 1, wherein the antibody, or
antigen binding fragment, comprises a heavy chain variable region
comprising three CDRs comprising SEQ ID NOs: 3, 5 and 7, and a
light chain variable region comprising three CDRs comprising SEQ ID
NOs: 12, 13 and 14.
3. The antibody-drug conjugate of claim 1 or 2, wherein the
antibody, or antigen binding fragment, comprises a heavy chain
variable region comprising SEQ ID NO: 1 or 21, and a light chain
variable region comprising SEQ ID NO: 10.
4. The antibody-drug conjugate of any one of claims 1-3, wherein
the antibody, or antigen binding fragment, comprises a heavy chain
variable region comprising SEQ ID NO: 1 and a light chain variable
region comprising SEQ ID NO: 10; or a heavy chain variable region
comprising SEQ ID NO: 21 and a light chain variable region
comprising SEQ ID NO: 10.
5. The antibody-drug conjugate of any one of claims 1-4, wherein
the antibody, or antigen binding fragment, comprises a heavy chain
comprising SEQ ID NO: 8, 17, 19, 23, 25, 27 or 29, and a light
chain comprising SEQ ID NO: 15 or 31.
6. The antibody-drug conjugate of any one of claims 1-5, wherein
the antibody, or antigen binding fragment, comprises a heavy chain
comprising SEQ ID NO: 8 and a light chain comprising SEQ ID NO: 15;
a heavy chain comprising SEQ ID NO: 8 and a light chain comprising
SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 17 and a light
chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO:
17 and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO:
15; a heavy chain comprising SEQ ID NO: 19 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 23
and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 23 and a light chain comprising SEQ ID NO:
31; a heavy chain comprising SEQ ID NO: 25 and a light chain
comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 25
and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 27 and a light chain comprising SEQ ID NO:
15; a heavy chain comprising SEQ ID NO: 27 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 29
and a light chain comprising SEQ ID NO: 15; or a heavy chain
comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO:
31.
7. The antibody-drug conjugate of claim 1, wherein the antibody, or
antigen binding fragment, comprises a heavy chain and/or light
chain constant region comprising an engineered cysteine residue for
site-specific conjugation.
8. The antibody-drug conjugate of claim 7, wherein the heavy chain
constant region comprises an engineered cysteine residue at
position 290 (K290C), according to the numbering of the EU index of
Kabat.
9. The antibody-drug conjugate of claim 7, wherein the light chain
constant region comprises an engineered cysteine residue at
position 183 (.kappa.K183C), according to the numbering of
Kabat.
10. The antibody-drug conjugate of claim 1, wherein the heavy chain
constant region comprises an engineered cysteine residue at
position 290 (K2900), according to the numbering of the EU index of
Kabat, and wherein the light chain constant region comprises an
engineered cysteine residue at position 183 (.kappa.K183C),
according to the numbering of Kabat.
11. The antibody-drug conjugate of claim 1, wherein the antibody,
or antigen binding fragment, comprises a heavy chain constant
region comprising an engineered glutamine-containing tag inserted
in the antibody or replaces one or more endogenous amino acids in
the antibody.
12. The antibody-drug conjugate of claim 11, wherein the engineered
glutamine-containing tag is inserted in the antibody at position
E294-N297.
13. The antibody-drug conjugate of claim 12, wherein the
glutamine-containing tag comprises an amino acid sequence LLQG (SEQ
ID NO: 40).
14. The antibody-drug conjugate of claim 11, wherein the heavy
chain constant region further comprises a lysine (K) substituting
an arginine (R) at position 222 (K222R), according to the numbering
of the EU index of Kabat.
15. The antibody-drug conjugate of claim 1, wherein the antibody,
or antigen binding fragment, comprises a heavy chain variable
region comprising a lysine (K) substituting an arginine (R) at
position 94 (K94R), according to the numbering of Kabat.
16. The antibody-drug conjugate of any one of claims 1-15, wherein
the linker is a cleavable linker.
17. The antibody-drug conjugate of claim 16, wherein the cleavable
linker is selected from the group consisting of vc, diS,
diS-C.sub.2OCO and AcLys-vc.
18. The antibody-drug conjugate of any one of claims 1-17, wherein
the drug is a cytotoxic agent.
19. The antibody-drug conjugate of claim 18, wherein the cytotoxic
agent is an auristatin.
20. The antibody-drug conjugate of claim 19, wherein the auristatin
is selected from the group consisting of 0101, 1569, 9411 and
4574.
21. The antibody-drug conjugate of any one of claims 1-18, wherein
the cytotoxic agent is a CPI dimer.
22. The antibody-drug conjugate of claim 21, wherein the CPI dimer
is CPI-8314 or CPI-0326.
23. An antibody-drug conjugate comprising (a) an antibody, or
antigen binding fragment thereof, comprising a heavy chain variable
region comprising three CDRs comprising SEQ ID NOs: 3, 5 and 7, and
a light chain variable region comprising three CDRs comprising SEQ
ID NOs: 12, 13 and 14, (b) a vc linker and (c) a 0101 drug.
24. An antibody-drug conjugate comprising (a) an antibody, or
antigen binding fragment thereof, comprising a heavy chain variable
region comprising SEQ ID NO: 21 and a light chain variable region
comprising SEQ ID NO: 10; (b) a vc linker and (c) a 0101 drug.
25. An antibody-drug conjugate comprising (a) an antibody, or
antigen binding fragment thereof, comprising a heavy chain
comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO:
31; (b) a vc linker and (c) a 0101 drug.
26. A pharmaceutical composition comprising the antibody-drug
conjugate of any of claims 1-25 and a pharmaceutically acceptable
carrier.
27. A composition comprising a plurality of an antibody-drug
conjugates of any one of claims 1-25, and optionally a
pharmaceutical carrier, wherein the composition has an average DAR
of ranging from 3 to 5.
28. A composition comprising a plurality of an antibody-drug
conjugates of any one of claims 1-25, and optionally a
pharmaceutical carrier, wherein the composition has an average DAR
of ranging from 1 to 3.
29. A nucleic acid encoding a heavy chain or a light chain of the
antibody of any one of claims 1-25.
30. A nucleic acid of any of SEQ ID NOs: 9, 18, 20, 24, 26, 28 or
30 encoding a heavy chain or any of SEQ ID NOs: 16 or 32 encoding a
light chain.
31. A vector comprising the nucleic acid of claim 29 or 30.
32. A host cell comprising the nucleic acid of claim 29 or 30.
33. A process for producing an antibody-drug conjugate of any one
of claims 1-25 comprising: (a) linking the linker to the drug; (b)
conjugating the linker and drug to the antibody; and (c) purifying
the antibody-drug conjugate.
34. The process of claim 33, wherein the conjugating is
site-specific on one or more engineered cysteine residue and/or
engineered glutamine residues on the antibody.
35. A method of treating an EDB+ FN-expressing disorder or disease,
comprising administering an effective amount of a composition
comprising an antibody-drug conjugate of any one of claims 1-25 to
a subject in need thereof.
36. The method of claim 35, wherein EDB+ FN-expressing disorder or
disease is cancer.
37. The method of claim 36, wherein the cancer is a solid tumor or
blood cancer.
38. The method of claim 37, wherein the solid tumor is thyroid
cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma,
gallbladder cancer, kidney cancer, skin cancer, uterine cancer,
mesothelioma, colorectal cancer, head and neck cancer, ovarian
cancer, bladder cancer, testicular cancer, prostate cancer, liver
cancer, endocrine cancer, thymus cancer, brain cancer, adrenal
cancer, eye cancer cervical cancer and lung cancer.
39. The method of claim 36, wherein the blood cancer is leukemia,
lymphoma or myeloma.
40. Use of the antibody-drug conjugate of any one of claims 1-25,
in the manufacture of a medicament for the treatment of an EDB+
FN-expressing disorder or disease in a subject.
41. The use according to claim 40, wherein the EDB+ FN-expressing
disorder or disease is cancer.
42. The use according to claim 41, wherein the cancer is a solid
tumor or blood cancer.
43. The use according to claim 42, wherein the solid tumor is
thyroid cancer, sarcoma, breast cancer, pancreatic cancer,
glioblastoma, gallbladder cancer, kidney cancer, skin cancer,
uterine cancer, mesothelioma, colorectal cancer, head and neck
cancer, ovarian cancer, bladder cancer, testicular cancer, prostate
cancer, liver cancer, endocrine cancer, thymus cancer, brain
cancer, adrenal cancer, eye cancer cervical cancer and lung
cancer.
44. The use according to claim 43, wherein the blood cancer is
leukemia, lymphoma or myeloma.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to anti-EDB antibodies and EDB
antibody-drug conjugates (ADCs). The present invention further
relates to the methods of using such antibodies and ADCs for the
treatment of EDB+ FN-expressing disorders, such as cancer.
BACKGROUND OF THE INVENTION
[0002] Fibronectins are high-molecular-weight adhesive
glycoproteins present in soluble form in plasma and other body
fluids, and in insoluble form in the extracellular matrix (ECM).
The extra domain B splice variant of fibronectin 1 (EDB+FN or EDB)
is a non-internalizing ECM protein. EDB is a 91 amino acid type III
homology domain that is inserted into the fibronectin molecule by a
mechanism of alternative splicing at the level of the primary
transcript whenever tissue remodeling takes place. EDB+ FN has been
shown to selectively accumulate in the stroma around new blood
vessels in tumors and other pathologies, but to be largely absent
in normal adult vasculature. Zardi et al., Embo J. 6(8): 2337-42
(1987). EDB+ FN is expressed in many aggressive tumors and
depending on the tumor type displays either predominantly vascular
or diffuse stromal patterns of expression. Carnemolla et al., J.
Cell Biol. 108(3): 1139-48 (1989).
[0003] An antibody that specifically binds to the EDB domain of
fibronectin (FN), the L19 antibody, has been isolated by phage
display technology. Carnemolla et al., Int. J. Cancer 68(3):
397-405 (1996); Neri et al., Nat. Biotechnol. 15(12): 1271-5.
(1997); Pini et al., J. Biol. Chem. 273(34): 21769-76 (1998). The
L19 antibody is able to stain tumor blood vessels in a wide range
of experimental tumor models and on sections of human tumors and
other angiogenic disorders. Carnemolla et al., J. Cell Biol.
108(3): 1139-48 (1989); Kaczmarek et al., Int. J. Cancer 59(1):
11-6 (1994); Berndt et al., Histochem. Cell Biol. 109(3): 249-55
(1998).
[0004] Various targeting strategies have been explored using
different formats of the L19 antibody in the treatment of cancer.
For example, a scFv(L19) monoclonal antibody fragment, Birchler et
al. Nat Biotechnol. 17: 984-8 (1999), fusion proteins including
interleukin-12 (IL-12) and tumor necrosis factor (TNF-alpha) fused
with scFv(L19), Halin C. et al. Cancer Res. 63(12):3202-10 (2003)
and L19 small immune protein (SIP) alone and conjugated to a
photosensitizer, Fabbrini M. et al. Int J Cancer 118(7):1805-13
(2006).
[0005] Although various L19 antibody based therapies have been
disclosed, there remains a significant clinical need for the
development of further improved and optimized EDB+ FN-targeting
therapies, such as antibody-drug conjugates, for those patients
with EDB+ FN-expressing disorders or diseases, such as cancers
associated with EDB+ FN expression and/or EDB+ FN-expressing
cancers.
SUMMARY OF THE INVENTION
[0006] The present invention provides for, an antibody-drug
conjugate comprising (a) an antibody, or antigen binding fragment
thereof, that binds to extra domain B (EDB) of fibronectin (FN),
(b) a linker and (c) a drug. In some aspects, an antibody-drug
conjugate comprises an antibody, or antigen binding fragment, may
comprise a heavy chain variable region comprising three CDRs
comprising SEQ ID NOs: 3, 5 and 7, and a light chain variable
region comprising three CDRs comprising SEQ ID NOs: 12, 13 and 14.
In some aspects, an antibody-drug conjugate comprises an antibody,
or antigen binding fragment, may comprise a heavy chain variable
region comprising SEQ ID NO: 1 or 21, and a light chain variable
region comprising SEQ ID NO: 10.
[0007] The present invention also provides for an antibody-drug
conjugate comprising an antibody, or antigen binding fragment, may
comprise a heavy chain variable region comprising SEQ ID NO: 1 and
a light chain variable region comprising SEQ ID NO: 10; or a heavy
chain variable region comprising SEQ ID NO: 21 and a light chain
variable region comprising SEQ ID NO: 10. In some aspects, an
antibody-drug conjugate comprises an antibody, or antigen binding
fragment, comprises a heavy chain comprising SEQ ID NO: 8, 17, 19,
23, 25, 27 or 29, and a light chain comprising SEQ ID NO: 15 or
31.
[0008] The present invention also provides for an antibody-drug
conjugate comprising an antibody, or antigen binding fragment,
comprising a heavy chain comprising SEQ ID NO: 8 and a light chain
comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 8 and
a light chain comprising SEQ ID NO: 31; a heavy chain comprising
SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 15; a heavy
chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID
NO: 31; a heavy chain comprising SEQ ID NO: 19 and a light chain
comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 19
and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 23 and a light chain comprising SEQ ID NO:
15; a heavy chain comprising SEQ ID NO: 23 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 25
and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO:
31; a heavy chain comprising SEQ ID NO: 27 and a light chain
comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 27
and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO:
15; or a heavy chain comprising SEQ ID NO: 29 and a light chain
comprising SEQ ID NO: 31.
[0009] The present invention also provides for an antibody-drug
conjugate comprising an antibody, or antigen binding fragment,
having a heavy chain and/or light chain constant region comprising
an engineered cysteine residue for site-specific conjugation. In
some aspects, an antibody-drug conjugate has a heavy chain constant
region comprising an engineered cysteine residue at position 290
(K290C), according to the numbering of the EU index of Kabat. In
some aspects, an antibody-drug conjugate has a light chain constant
region comprising an engineered cysteine residue at position 183
(.kappa.K183C), according to the numbering of Kabat. In some
aspects, an antibody-drug conjugate has a heavy chain constant
region comprising an engineered cysteine residue at position 290
(K290C), according to the numbering of the EU index of Kabat, and a
light chain constant region comprises an engineered cysteine
residue at position 183 (.kappa.K183C), according to the numbering
of Kabat.
[0010] The present invention further provides for an antibody-drug
conjugate having an antibody, or antigen binding fragment,
comprising a heavy chain constant region comprising an engineered
glutamine-containing tag inserted in the antibody or replacing one
or more endogenous amino acids in the antibody. In some aspects, an
antibody-drug conjugate has an engineered glutamine-containing tag
inserted in the antibody at position E294-N297. In some aspects, an
antibody-drug conjugate has a glutamine-containing tag comprising
an amino acid sequence LLQG (SEQ ID NO: 40). In some aspects, an
antibody-drug conjugate having a heavy chain constant region
further comprising a lysine (K) substituting an arginine (R) at
position 222 (K222R), according to the numbering of the EU index of
Kabat.
[0011] The present invention also provides for an antibody-drug
conjugate of having an antibody, or antigen binding fragment,
comprising a heavy chain variable region comprising a lysine (K)
substituting an arginine (R) at position 94 (K94R), according to
the numbering of Kabat.
[0012] The present invention further provides for an antibody-drug
conjugate having a linker that is a cleavable linker. In some
aspects, the cleavable linker is selected from the group consisting
of vc, diS, diS-C.sub.2OCO and AcLys-vc.
[0013] The present invention further provides for an antibody-drug
conjugate having a drug that is a cytotoxic agent. In some aspects,
the cytotoxic agent is an auristatin. In some aspects, the
auristatin is selected from the group consisting of 0101, 1569,
9411 and 4574. In some aspects the cytotoxic agent is a CPI dimer.
In some aspects, the CPI dimer is CPI-8314 or CPI-0326.
[0014] The present invention also provides for an antibody-drug
conjugate comprising (a) an antibody, or antigen binding fragment
thereof, comprising a heavy chain variable region comprising three
CDRs comprising SEQ ID NOs: 3, 5 and 7, and a light chain variable
region comprising three CDRs comprising SEQ ID NOs: 12, 13 and 14,
(b) a vc linker and (c) a 0101 drug.
[0015] The present invention also provides for an antibody-drug
conjugate comprising (a) an antibody, or antigen binding fragment
thereof, comprising a heavy chain variable region comprising SEQ ID
NO: 21 and a light chain variable region comprising SEQ ID NO: 10;
(b) a vc linker and (c) a 0101 drug.
[0016] The present invention also provides for an antibody-drug
conjugate comprising (a) an antibody, or antigen binding fragment
thereof, comprising a heavy chain comprising SEQ ID NO: 25 and a
light chain comprising SEQ ID NO: 31; (b) a vc linker and (c) a
0101 drug.
[0017] The present invention further provides for a pharmaceutical
composition comprising an antibody-drug conjugate of the invention
and a pharmaceutically acceptable carrier. The present invention
also provides for a composition comprising a plurality of an
antibody-drug conjugates of the invention, and optionally a
pharmaceutical carrier, wherein the composition has an average DAR
of ranging from 3 to 5. The present invention also provides for a
composition comprising a plurality of an antibody-drug conjugates
of any one of claims 1-25, and optionally a pharmaceutical carrier,
wherein the composition has an average DAR of ranging from 1 to
3.
[0018] The present invention provides for a nucleic acid encoding a
heavy chain or a light chain of an antibody of the invention. In
some aspects the nucleic acid may comprise SEQ ID NOs: 9, 18, 20,
24, 26, 28 or 30 encoding a heavy chain or may comprise SEQ ID NOs:
16 or 32 encoding a light chain. The present invention further
provides for a vector comprising any nucleic acid of the invention.
Also, the present invention provides for a host cell comprising any
nucleic acid of the invention.
[0019] The present invention provides a process for producing an
antibody-drug conjugate of the invention comprising: (a) linking
the linker to the drug; (b) conjugating the linker and drug to the
antibody; and (c) purifying the antibody-drug conjugate. In some
aspects, the conjugating is site-specific on one or more engineered
cysteine residue and/or engineered glutamine residues on the
antibody.
[0020] The present invention also provides a method of treating an
EDB+ FN-expressing disorder or disease, comprising administering an
effective amount of a composition comprising an antibody-drug
conjugate of the invention to a subject in need thereof. In some
aspects, the EDB+ FN-expressing disorder or disease is cancer. In
some aspects, the cancer is a solid tumor or blood cancer. In some
aspects, the solid tumor is thyroid cancer, sarcoma, breast cancer,
pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer,
skin cancer, uterine cancer, mesothelioma, colorectal cancer, head
and neck cancer, ovarian cancer, bladder cancer, testicular cancer,
prostate cancer, liver cancer, endocrine cancer, thymus cancer,
brain cancer, adrenal cancer, eye cancer cervical cancer and lung
cancer. In some aspects, the blood cancer is leukemia, lymphoma or
myeloma.
[0021] The present invention further provides for the use of an
antibody-drug conjugate of the invention, in the manufacture of a
medicament for the treatment of an EDB+ FN-expressing disorder or
disease in a subject. In some aspects, the EDB+ FN-expressing
disorder or disease is cancer. In some aspects, the cancer is a
solid tumor or blood cancer. In some aspects, the solid tumor is
thyroid cancer, sarcoma, breast cancer, pancreatic cancer,
glioblastoma, gallbladder cancer, kidney cancer, skin cancer,
uterine cancer, mesothelioma, colorectal cancer, head and neck
cancer, ovarian cancer, bladder cancer, testicular cancer, prostate
cancer, liver cancer, endocrine cancer, thymus cancer, brain
cancer, adrenal cancer, eye cancer cervical cancer and lung cancer.
In some aspects, the blood cancer is leukemia, lymphoma or
myeloma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A and 1B show binding properties of [A] EDB-L19,
EDB-PFE and EDB-(K94R) antibodies; and [B] EDB-(K94R) and
EDB-(.kappa.K183C-K94R-2900) antibodies.
[0023] FIG. 2 shows EDB+ FN expression using RNA-Seq analysis in
human patient derived xenograft (PDX) cancer models.
[0024] FIGS. 3A and 3B show ELISA binding curves for [A] EDB-L19
antibody and EDB-L19-vc-0101 ADC, and EDB-(.kappa.K1830-K94R-2900)
antibody and EDB-(.kappa.K183C-K94R-2900)-vc-0101 ADC; and [B]
EDB-(K94R) antibody and EDB-(K94R)-vc-0101 ADC, and
EDB-(.kappa.K183C-K290C) antibody and
EDB-(.kappa.K183C-K2900)-vc0101 ADC.
[0025] FIG. 4 shows EDB+ FN expression by western blot in WI38-VA13
and HT-29 cells.
[0026] FIGS. 5A-5F show anti-tumor efficacy in PDX-NSX-11122, a
high EDB+ FN expressing NSCLC patient derived xenograft (PDX) model
of human cancer, of [A] EDB-L19-vc-0101 at 0.3, 0.75, 1.5 and 3
mg/kg; [B] EDB-L19-vc-0101 at 3 mg/kg and 10 mg/kg of disulfide
linked EDB-L19-diS-DM1; [C] EDB-L19-vc-0101 at 1 and 3 mg/kg and 5
mg/kg of disulfide linked EDB-L19-diS-C.sub.2OCO-1569; [D]
site-specific conjugated EDB-(.kappa.K183C+K290C)-vc-0101 and
conventionally conjugated EDB-L19-vc-0101 (ADC1) at the doses of
0.3, 1 and 3 mg/kg and 1.5 mg/kg, respectively; [E] site-specific
conjugated EDB-(.kappa.K1830-K94R-K2900)-vc-0101 at the doses of
0.3, 1 and 3 mg/kg, and [F] EDB-(.kappa.K183C-K94R-K290C)-vc-0101
group dosed at 3 mg/kg as tumor growth inhibition curves for each
individual tumor bearing mouse.
[0027] FIGS. 6A-6F show anti-tumor efficacy in H-1975, a moderate
to high EDB+ FN expressing NSCLC cell line xenograft (CLX) model of
human cancer, of [A] EDB-L19-vc-0101 at 0.3, 0.75, 1.5 and 3 mg/mg;
[B] EDB-L19-vc-0101 and EDB-L19-vc-1569 at 0.3, 1 and 3 mg/kg, [C]
EDB-L19-vc-0101 and EDB-(H16-K222R)-AcLys-vc-CPI-8314 at 0.5, 1.5
and 3 mg/kg and 0.1, 0.3 and 1 mg/kg, respectively; [D]
site-specific conjugated EDB-(.kappa.K183C+K290C)-vc-0101 and
conventionally conjugated EDB-L19-vc-0101 at 0.5, 1.5 and 3 mg/kg;
[E] EDB-L19-vc-0101 and EDB-(K94R)-vc-0101 at 1 and 3 mg/kg; and
[F] EDB-(.kappa.K183C+K290C)-vc-0101 and
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 at 1 and 3 mg/kg.
[0028] FIG. 7 shows anti-tumor efficacy in HT29, a moderate EDB+ FN
expressing colon CLX model of human cancer, of EDB-L19-vc-0101 and
EDB-L19-vc-9411 at 3 mg/kg.
[0029] FIGS. 8A and 8B show anti-tumor efficacy of EDB-L19-vc-0101
at 0.3, 1 and 3 mg/kg in [A] PDX-PAX-13565, a moderate to high EDB+
FN expressing pancreatic PDX; and [B] PDX-PAX-12534, a low to
moderate EDB+ FN expressing pancreatic PDX.
[0030] FIG. 9 shows anti-tumor efficacy of EDB-L19-vc-0101 at 1 and
3 mg/kg in Ramos, a moderate EDB+ FN expressing lymphoma CLX model
of human cancer.
[0031] FIGS. 10A and 10B show the anti-tumor efficacy in EMT-6, a
mouse syngeneic breast carcinoma model, of [A]
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 at 4.5 mg/kg; and [B]
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 group dosed at 4.5 mg/kg as
tumor growth inhibition curves for each individual tumor bearing
mouse.
[0032] FIG. 11 shows absolute neutrophil counts for conventionally
conjugated EDB-L19-vc-0101 at 5 mg/kg compared to site-specific
conjugated EDB-(.kappa.K183C-K94R-K290C)-vc-0101 (ADC4) at 6
mg/kg.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides antibodies and antibody drug
conjugates (ADCs) that bind to the extra-domain B (EDB) of
fibronectin (FN), referred to as "EDB+ FN" or "EDB",
interchangeably. The invention also provides processes for
preparing the ADCs using anti-EDB antibodies, linkers, and drugs
(payloads). The invention further provides for ADCs generated using
conventional and/or site-specific conjugation technology. The
antibodies and ADCs of the invention are useful for the preparation
and manufacture of compositions, such as medicaments, that may be
used in the diagnosis, prophylaxis, and/or treatment of
hyperproliferative disorders characterized by or associated with
EDB+ FN expression, such as cancer. The invention also provides for
nucleic acids encoding the anti-EDB antibodies used in making the
EDB ADCs.
[0034] ADCs comprise an antibody component conjugated to a drug,
typically through the use of a linker. ADCs generated by
conventional conjugation technology randomly link the drug to the
antibody through lysine or cysteine residues that are endogenously
on the antibody heavy and/or light chain. Accordingly, such ADCs
are a heterogeneous mixture of species having different
drug:antibody ratios (DAR). ADCs generated by site-specific
conjugation technology link the drug to the antibody at particular
engineered residues on the antibody heavy and/or light chain. As
such, the site-specific conjugated ADCs are a homogeneous mixture
of ADCs comprised of a species with a defined drug:antibody ratio
(DAR). Thus, site-specific conjugated ADCs demonstrate uniform
stoichiometry resulting in improved pharmacokinetics,
biodistribution and safety profile.
[0035] ADCs of the present invention include anti-EDB antibodies
conjugated to one or more drugs via a linker (i.e. forming
linker-drug moieties). The present invention provides for ADCs
having (a) an antibody, or antigen binding fragment thereof, that
binds to EDB; (b) a linker and (c) a drug. The present invention
further provides for ADCs of the formula Ab-(L-D), wherein (a) Ab
is an antibody, or antigen-binding fragment thereof, that binds to
EDB, and (b) L-D is a linker-drug moiety, wherein L is a linker,
and D is a drug. In another aspect, the present invention provides
for ADCs of the formula Ab-(L-D)p, wherein (a) Ab is an antibody,
or antigen-binding fragment thereof, that binds to EDB, (b) L-D is
a linker-drug moiety, wherein L is a linker, and D is a drug and
(c) p is the number of linker-drug moieties attached to the
antibody.
[0036] The number of linker-drug moieties attached to an antibody
can be any number preferred for development of an ADC. In some
aspects, the number of linker-drug moieties per antibody is 4. In
other aspects, the number of linker-drug moieties per antibody is
3. In another aspect, the number of linker-drug moieties per
antibody is 2. In another aspect, the number of linker-drug
moieties per antibody is 1. In other aspects, the number of
linker-drug moieties per antibody is greater than 4, such as 5, 6,
7, 8, 9, 10, 11, 12 or greater than 12 linker-drug moieties per
antibody.
[0037] Further the present invention provides for ADCs, wherein the
linker-drug moieties are attached to the antibody via conventional
or site-specific conjugation technology. In some aspects, the
anti-EDB antibodies, or antigen-binding fragments thereof, are
conjugated or linked to a drug such as a cytotoxic, cytostatic,
and/or therapeutic agent, as described further herein. For example,
a cytotoxic agent can be linked or conjugated to an anti-EDB
antibody as described herein for targeted local delivery of the
cytotoxic agent. Also provided are methods of preparing and
manufacturing such ADCs, and use of the same in clinical
applications.
[0038] In contrast to other ADCs being developed to target
internalizing cell surface expressed proteins, the ADCs of the
present invention target EDB, a protein expressed in the
extracellular matrix (ECM). Targeting a protein expressed in the
ECM may provide benefits over targeting a protein expressed on the
tumor cells. The ADC may directly access the target without having
to penetrate through the stromal and ECM barriers common in many
difficult-to-treat human cancers. Further, targeting EDB in the ECM
with an ADC provides a specific mechanism to access many difficult
to target cell types in the tumor microenvironment. This may result
in the extracellular release of a cytotoxic payload or drug,
resulting in the killing of a variety of cells, via mechanisms such
as cell death/cell-cycle arrest of tumor cells and/or stromal cells
by bystander mechanism. In addition, further mechanisms include,
but are not limited to disregulated angiogenesis or cytotoxic
vascular targeting/collapse, vascular normalization,
immunomodulation and induction of cellular differentiation and/or
impediment of the epithelial to mesenchymal transition.
[0039] The Examples provided herein demonstrate the improved
characteristics obtained during anti-EDB antibody and EDB ADC
generation, such as allotype optimization to reduce immunogenicity,
removal of COOH-terminal lysine to increase product homogeneity,
and introduction of mutations to mitigate potential glycation
liability and decrease heterogeneity (see Examples 1 and 2).
Further, as shown in the Examples, EDB ADCs generated using various
conventional and site-specific conjugation technologies (i.e.
cysteines, lysines and/or acyl donor glutamine-containing ("Q")
tags) and various linker-drug moieties demonstrate robust in vitro
and in vivo efficacy (see Examples 6 to 8). Examples provided
herein also showed that EDB ADCs generated using site-specific
conjugation via engineered cysteine residues demonstrated improved
characteristics compared to EDB ADCs generated using conventional
conjugation via cysteine residues, such as improved pharmacokinetic
(PK) profile (i.e. increased exposure and conjugation stability
leading to less off-target toxic effects), favorable thermal
stability and nonclinical safety profiled (i.e. alleviation of
myelosuppression) (see Examples 9, 10 and 11, respectively).
Further, the improved characteristic of the EDB ADCs generated with
site-specific conjugation technologies may allow higher dosages in
human treatment and thus provide increased efficacy. In some
aspects, the EDB ADCs may comprise a substitution of the lysine (K)
at position 290 (according to the EU index of Kabat) in the human
IgG1 heavy chain constant region with a reactive cysteine (C)
(K290C) and/or a substitution of the lysine (K) at position 183
(according to Kabat) in the human Kappa light chain constant region
with a reactive cysteine (C) (.kappa.K1830) to enable site-specific
conjugation.
Extra-Domain B of Fibronectin
[0040] As used herein "EDB+ FN" and "EDB" are used interchangeable
and refer to fibronectin (FN) containing the extra-domain B (EDB).
Further, "anti-EDB antibodies" and "anti-EDB+ FN antibodies" are
used interchangeable and refer to antibodies that bind to EDB.
"Anti-EDB antibody-drug conjugates", "EDB antibody-drug
conjugates", "anti-EDB ADCs", "EDB ADCs" are used interchangeable
and refer to ADCs comprising antibodies, or antigen-binding
fragments thereof, that bind to EDB and are conjugated or linked to
a drug. FN is a high-molecular-weight glycoprotein present in the
extracellular matrix (ECM) and is involved in cell adhesion and
migration processes including embryogenesis, wound healing, blood
coagulation, host defense, and metastasis. FN typically exists as a
dimer formed by two nearly identical .about.250 kDa subunits
covalently linked near their C-terminus by a pair of disulfide
bonds. Each monomer consists of three types of repeating units:
type I, type II and type III FN repeats. A single 75-kb gene
encodes FN, however there are twenty protein variants observed in
humans. Alternative splicing of the FN gene occurs in three regions
resulting in the inclusion or exclusion of either one of the two
type III repeats, called extra domain A (EDA) and extra domain B
(EDB), and of a segment connecting two other type III repeats,
called type III connecting segment (IICS). EDB is a 91 amino acid
sequence that is 100% identical in mice, rats, rabbits, dogs,
cynomologus monkey and humans. A representative EDB+ FN nucleotide
sequence is provided under Accession No. NM_001306129.1 and
corresponding amino acid sequence is provided under Accession No.
NP_001293058.1. EDB and recombinant human 7-EDB-8-9 amino acid
sequences are provided in Table 1. Recombinant human 7-EDB-8-9
comprises EDB flanked by domain 7 on the amino terminus and and
domain 9 at the carboxy terminus of EDB.
TABLE-US-00001 TABLE 1 EDB and 7-EDB-8-9 sequences SEQ ID NO.
Description Sequence 33 EDB
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFV
DSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQT 34 Human FN-
VVTQLSPPTNLHLEANPDTGVLAVSWERSTTPDITGYRITTTPTNGQQ 7-EDB-89-
GNSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIP HIS protein
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFV ##STR00001##
##STR00002## ANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNL
TPGTEYVVSIVALNGREESPLLIGRSRSHHHHHH 35 Cynomolgus
VVTPLSPPTNLHLETNPDTGVLTVSWERSTTPDITGYRITTTPTNGQQ FN-7-EDB-
GYSLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIP 89-HIS
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFV protein
##STR00003## ##STR00004## ##STR00005##
ANSFTVHWIAPRATITGYRIRHHPEHMSGRPREDRVPPSRNSITLTNL
TPGTEYVVSIVALNGREESPLLIGRSRSHHHHHH 36 Rat FN-7-
VVTPLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQ EDB-89-HIS
GTALEEVVHADQSSCTFENLNPGLEYNVSVYTVKDDKESAPISDTVIP protein
EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFV ##STR00006##
##STR00007## ##STR00008##
TANSFTVHWVAPRAPITGYIIRHHAEHSAGRPRQDRVPPSRNSITLTN
LNPGTEYIVTIIAVNGREESPPLIGRSRSHHHHHH
Anti-EDB Antibodies
[0041] Antibodies of the present invention specifically bind to
EDB. For preparation of ADCs of the invention, an antibody, or
antigen-binding fragment thereof, may be any antibody (including
antibodies described herein), or antigen-binding fragment thereof,
that specifically binds to EDB. The antibody, or antigen-binding
fragment thereof, may be isolated, purified, or derivatized for use
in preparation of an EDB ADC.
[0042] As used herein, "antibody" or "Ab" refers to an
immunoglobulin molecule capable of recognizing and binding to a
specific target or antigen, such as a carbohydrate, polynucleotide,
lipid, polypeptide, etc., through at least one antigen recognition
site, located in the variable region of the immunoglobulin
molecule. The term can encompass any type of antibody, including
but not limited to monoclonal antibodies, polyclonal antibodies,
"antigen-binding fragments" (or portion), such as Fab, Fab',
F(ab').sub.2, Fd, Fv, Fc, etc., of intact antibodies that retain
the ability to specifically bind to a given antigen (e.g. EDB), an
isolated complementarity determining region (CDR), bispecific
antibodies, heteroconjugate antibodies, mutants thereof, fusion
proteins having an antibody, or antigen-binding fragment thereof,
(e.g., a domain antibody), single chain (ScFv) and single domain
antibodies (e.g., shark and camelid antibodies), maxibodies,
minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR
and bis-scFv (see, e.g., Holliger and Hudson, 2005, Nature
Biotechnology 23(9): 1126-1136), humanized antibodies, chimeric
antibodies and any other modified configuration of the
immunoglobulin molecule that includes an antigen recognition site
of the required specificity, including glycosylation variants of
antibodies, amino acid sequence variants of antibodies, and
covalently modified antibodies. The antibodies may be murine, rat,
human, or any other origin (including chimeric or humanized
antibodies). In some aspects of the invention, the antibody, or
antigen-binding fragment thereof, of the disclosed EDB ADCs is a
chimeric, humanized, or a recombinant human antibody, or
EDB-binding fragment thereof.
[0043] Native or naturally occurring antibodies and native
immunoglobulins are typically heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light chains (LC)
and two identical heavy chains (HC). Each heavy chain has a
variable domain (VH) followed by a number of constant domains or
regions (e.g. hinge, CH1, CH2 or CH3), referred to as "CH domains".
Each light chain has a variable domain (VL) and a constant domain,
referred to as "CL domain". The term "constant region" or "constant
domain" of an antibody refers to the constant region of the
antibody light chain or the constant region of the antibody heavy
chain, either alone or in combination. The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as Fc receptor (FcR) binding,
participation of the antibody in antibody-dependent cellular
toxicity (ADCC), opsonization, initiation of complement dependent
cytotoxicity, and mast cell degranulation. The constant regions of
the EDB antibodies may be derived from constant regions of any one
of IgA, IgD, IgE, IgG, IgM, any isotypes thereof (e.g., IgG1, IgG2,
IgG3, or IgG4 isotypes of IgG), as well as subclasses and mutated
versions thereof.
[0044] CH1 domain includes the first (most amino terminal) constant
region domain of an immunoglobulin heavy chain that extends, e.g.
from about positions 118-215 according to the EU index of Kabat.
The CH1 domain is adjacent to the VH domain and amino terminal to
the hinge region of an immunoglobulin heavy chain molecule, and
does not form a part of the Fc region of an immunoglobulin heavy
chain.
[0045] The hinge region includes the portion of a heavy chain
molecule that joins the CH1 domain to the CH2 domain. This hinge
region comprises approximately 25 residues and is flexible, thus
allowing the two N-terminal antigen binding regions to move
independently. Hinge regions can be subdivided into three distinct
domains: upper, middle, and lower hinge domains.
[0046] CH2 domain includes the portion of a heavy chain
immunoglobulin molecule that extends, e.g. from about positions
231-340 according to the EU index of Kabat. The CH2 domain is
unique in that it is not closely paired with another domain.
Rather, two N-linked branched carbohydrate chains are interposed
between the two CH2 domains of an intact native IgG molecule. In
some aspects, the antibody (or fragment thereof) of the invention
comprises a CH2 domain derived from an IgG molecule, such as IgG1,
IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.
[0047] CH3 domain includes the portion of a heavy chain
immunoglobulin molecule that extends approximately 110 residues
from N-terminus of the CH2 domain, e.g. from about positions
341-447 according to the EU index of Kabat. The CH3 domain
typically forms the C-terminal portion of the antibody. In some
immunoglobulins, however, additional domains may extend from CH3
domain to form the C-terminal portion of the molecule (e.g. the CH4
domain in the p chain of IgM and the E chain of IgE). In some
aspects, the antibody (or fragment thereof) of the invention
comprises a CH3 domain derived from an IgG molecule, such as IgG1,
IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.
[0048] CL domain includes the constant region domain of an
immunoglobulin light chain that extends, e.g. from about positions
108-214 according to the EU index of Kabat. The CL domain is
adjacent to the VL domain. In some aspects, the antibody (or
fragment thereof) of the invention comprises a kappa light chain
constant domain (CL.kappa.). In some aspects, the antibody (or
fragment thereof) comprises a lambda light chain constant domain
(CL.lamda.). CL.kappa. has known polymorphic loci CL.kappa.-V/A45
and CL.kappa.-LA/83 (using Kabat numbering) thus allowing for
polymorphisms Km(1): CL.kappa.-V45/L83; Km(1,2): CL.kappa.-A45/L83;
and Km(3): CL.kappa.-A45/V83. Polypeptides, antibodies and ADCs of
the invention may have antibody components with any of these light
chain constant regions.
[0049] The Fc region generally comprises a CH2 domain and a CH3
domain. Although the boundaries of the Fc region of an
immunoglobulin heavy chain might vary, the human IgG heavy chain Fc
region is usually defined to stretch from an amino acid residue at
position Cys226, or from Pro230 (according to the EU index of
Kabat), to the carboxyl-terminus thereof. A Fc region may be a
native sequence Fc region or a variant Fc region. (Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991).
[0050] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FR) connected by three
complementarity determining regions (CDRs) also known as
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen binding site of
antibodies.
[0051] A CDR of a variable domain may be identified in accordance
with the definitions of the Kabat (Kabat et al., 1992, Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
NIH, Washington D.C.), Chothia (Chothia et al., Nature 342:877-883,
(1989)), the accumulation of both Kabat and Chothia, AbM definition
(derived using Oxford Molecular's AbM antibody modeling software
(now Accelrys.RTM.)), contact definition (based on observed antigen
contacts, set forth in MacCallum et al., J. Mol. Biol.,
262:732-745, (1996)), and/or conformational definition (Makabe et
al., Journal of Biological Chemistry, 283:1156-1166, 2008) or any
method of CDR determination well known in the art. As used herein,
a CDR may refer to CDRs defined by any approach known in the art,
including combinations of approaches. For the present invention,
the CDRs set forth in Table 2 below were derived using Kabat and
Chothia definitions. The anti-EDB antibodies, or antigen-binding
fragment thereof, of the present invention include one or more
CDR(s) (such as one, two, three, four, five, or all six CDRs).
[0052] An antibody, an ADC, or a polypeptide that "specifically
binds" or "preferentially binds" (used interchangeably herein) to a
target or antigen (e.g., EDB protein) is a term well understood in
the art, and methods to determine such specific or preferential
binding are also well known in the art. A molecule is said to
exhibit "specific binding" or "preferential binding" if it reacts
or associates more frequently, more rapidly, with greater duration
and/or with greater affinity with a particular cell or substance
than it does with alternative cells or substances. An antibody
"specifically binds" or "preferentially binds" to a target or
antigen if it binds with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other substances. For
example, an antibody that specifically or preferentially binds to
an EDB epitope is an antibody that binds this epitope with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other EDB epitopes or non-EDB epitopes.
[0053] The term "binding affinity" or "K.sub.D" as used herein, is
intended to refer to the equilibrium dissociation constant of a
particular antigen-antibody interaction. The K.sub.D is the ratio
of the rate of dissociation, also called the "off-rate" or
"k.sub.d", to the rate of association, or "on-rate" or "k.sub.a".
Thus, K.sub.D equals k.sub.d/k.sub.a and is expressed as a molar
concentration (M). It follows that the smaller the K.sub.D, the
stronger the binding affinity. Therefore, a K.sub.D of 1 .mu.M
indicates weak binding affinity compared to a K.sub.D of 1 nM.
K.sub.D values for antibodies can be determined using methods well
established in the art. One method for determining the K.sub.D of
an antibody is by using surface plasmon resonance, typically using
a biosensor system such as a BIAcore.RTM. system. Other standard
assays to evaluate the binding ability of ligands such as
antibodies towards targets are known in the art, including for
example, ELISAs, Western blots, RIAs, and flow cytometry
analysis.
[0054] An "isolated antibody", as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody that
specifically binds EDB is substantially free of antibodies that
specifically bind antigens other than EDB). Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals. It is also understood that by reading this
definition, for example, an antibody (or moiety or epitope) that
specifically or preferentially binds to a first target may or may
not specifically or preferentially bind to a second target.
[0055] In some aspects of the invention, an EDB ADC includes an
antibody that competes for binding to human EDB with, and/or binds
the same epitope as, an antibody, or antigen-binding fragment
thereof, described herein.
[0056] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
fragment thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
fragment thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0057] The "L19" antibody, herein also referenced as "EDB-L19"
antibody, is a human antibody that binds EDB. The L19 antibody is
disclosed and characterized in PCT International Publication Nos.
WO1997/045544, WO1999/058570 and WO2001/062800, which are
incorporated herein by reference in their entirety, and the L19-EDB
sequences are provided herein in Table 2 (SEQ ID NOs. 1-16).
[0058] In some aspects of the invention, antibodies used to prepare
EDB ADCs may be monoclonal antibodies. The term "monoclonal
antibody" or "mAb" refers to an antibody obtained from a population
of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
polyclonal antibody preparations, which typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method.
[0059] In some aspects of the invention, antibodies used to prepare
ADCs of the invention may be monovalent, i.e., having one antigen
binding site per molecule (e.g., IgG or Fab). In some instances, a
monovalent antibody can have more than one antigen binding sites,
but the binding sites are from different antigens. In some aspects
of the invention, the antibody, or antigen-binding fragment
thereof, of an ADC of the invention may include a "bivalent
antibody", i.e., having two antigen binding sites per molecule
(e.g., IgG). In some instances, the two binding sites have the same
antigen specificities. Alternatively, bivalent antibodies may be
bispecific. A "bispecific," "dual-specific" or "bifunctional"
antibody is a hybrid antibody having two different antigen binding
sites. The two antigen binding sites of a bispecific antibody bind
to two different epitopes, which may reside on the same or
different protein targets.
[0060] The term "chimeric antibody" is intended to refer to
antibodies in which part or all of the variable region sequences
are derived from one species and the constant region sequences are
derived from another species, such as an antibody in which the
variable region sequences are derived from a mouse antibody and the
constant region sequences are derived from a human antibody.
[0061] As used herein, "humanized" or "CDR grafted" antibody refers
to forms of non-human (e.g. murine) antibodies that are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen binding
subsequences of antibodies) that contain minimal sequence derived
from a non-human immunoglobulin. Preferably, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from one or more CDRs of the recipient are replaced by residues
from one or more CDRs of a non-human species (donor antibody) such
as mouse, rat, or rabbit having the desired specificity, affinity,
and capacity.
[0062] Antibodies of the invention can be produced using techniques
well known in the art, e.g., recombinant technologies, phage
display technologies, synthetic technologies or combinations of
such technologies or other technologies readily known in the art
(see, for example, Jayasena, S. D., Clin. Chem., 45: 1628-50 (1999)
and Fellouse, F. A., et al, J. Mol. Biol., 373(4):924-40 (2007)).
Additional guidance may be found in Sambrook J. & Russell D.
Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al.,
Short Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, Wiley, John & Sons,
Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1998); and Coligan et al., Short Protocols in Protein Science,
Wiley, John & Sons, Inc. (2003).
[0063] Nucleic acids encoding the heavy and light chains of the
antibodies used to prepare the ADCs of the invention can be cloned
into a vector for expression or propagation. The sequence encoding
the antibody of interest may be maintained in vector in a host cell
and the host cell can then be expanded and frozen for future use.
Production of recombinant monoclonal antibodies in cell culture can
be carried out through cloning of antibody genes from B cells by
means known in the art. See, e.g. Tiller et al., J. Immunol.
Methods 329:112-124, 2008; U.S. Pat. No. 7,314,622.
[0064] As used herein, the term "vector" refers to a construct,
which is capable of delivering, and, preferably, expressing, one or
more gene(s) or sequence(s) of interest in a host cell. Examples of
vectors include, but are not limited to, viral vectors, naked DNA
or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or
RNA expression vectors associated with cationic condensing agents,
DNA or RNA expression vectors encapsulated in liposomes, and
certain eukaryotic cells, such as producer cells.
[0065] As used herein, the term "host cell" includes an individual
cell or cell culture that can be or has been a recipient for
vector(s) for incorporation of polynucleotide inserts. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in genomic
DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a polynucleotide(s) of this invention.
[0066] As known in the art, "polynucleotide," "nucleic
acid/nucleotide," and "oligonucleotide" are used interchangeably
herein, and include polymeric forms of nucleotides of any length,
either deoxyribonucleotides or ribonucleotides, analogs thereof, or
any substrate that can be incorporated into a chain by DNA or RNA
polymerase. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown.
Polynucleotides may be naturally-occurring, synthetic, recombinant
or any combination thereof.
[0067] For all heavy chain constant region amino acid positions
discussed in the present invention, numbering is according to the
Eu index first described in Edelman et al., 1969, Proc. Natl. Acad.
Sci. USA 63(1):78-85, describing the amino acid sequence of myeloma
protein Eu, which is the first human IgG1 sequenced. The Eu index
of Edelman et al. is also set forth in Kabat et al., 1991,
Sequences of Proteins of Immunological Interest, 5th Ed., United
States Public Health Service, National Institutes of Health,
Bethesda. Thus, the "EU index as set forth in Kabat" or "EU index
of Kabat" refers to the residue numbering system based on the human
IgG1 Eu antibody of Edelman et al. as set forth in Kabat 1991.
[0068] The numbering system used for the light chain constant
region amino acid sequence is that set forth in Kabat 1991.
[0069] The EDB ADCs of the present invention may be conjugated to
the drug/payload using conventional cysteine technology or
site-specific conjugation technology. To accommodate site-specific
conjugation via engineered cysteines, the constant domain may be
modified to provide for a reactive cysteine residue engineered at
one or more specific sites (sometimes referred to as "Cys"
mutants). To accommodate site-specific conjugation via
transglutaminase-based conjugation, an acyl donor
glutamine-containing ("Q") tag or an endogenous glutamine is made
reactive by polypeptide engineering in the presence of
transglutaminase and an amine.
[0070] The present invention provides for optimization of the
L19-EDB antibody by generation of a non-immunogenic antibody. In
some aspects, the L19-EDB human IgG1 constant region comprising a
G1m(a) allotype having aspartic acid (D) at position 356 and
leucine (L) at position 358, may be substituted with a non-G1m(a)
allotype having glutamic acid (E) at position 356 and methionine
(M) at position 358 (according to the numbering of the EU index of
Kabat).
[0071] Further, to reduce potential chemical liabilities and
antigen binding a putative protein glycation site, anti-EDB
antibodies of the present invention may have a heavy chain variable
region comprising a mutation of the lysine (K) at position 94
(according to the numbering of the EU index of Kaba) to an arginine
(R), e.g. (K94R).
[0072] For site-specific conjugation via engineered cysteines, the
anti-EDB antibody heavy chain constant domain may comprise a
reactive engineered cysteine residue at position 290 (K290C),
according to the numbering of the EU index of Kabat. Additional
cysteine substitutions may be introduced. In another aspect, the
anti-EDB antibody light chain constant domain may comprise a
reactive engineered cysteine residue at position 183
(.kappa.K1830), according to the numbering of Kabat. Additional
cysteine substitutions may be introduced.
[0073] For site-specific conjugation via engineered glutamine
residues, the anti-EDB antibody heavy chain constant domain may
comprise an engineered H16-glutamine-containing tag LLQG (SEQ ID
NO: 40). Further, to optimize this site-specific conjugation the
lysine (K) amino acid at position 222 (according to the EU index of
Kabat) on the heavy chain may be substituted with an arginine (R),
e.g. (K222R).
[0074] Amino acid modifications can be made by any method known in
the art and many such methods are well known and routine for the
skilled artisan, e.g. mutations, substitutions, deletions, and/or
additions. For example, but not by way of limitation, amino acid
substitutions, deletions and insertions may be accomplished using
any well-known PCR-based technique. Amino acid substitutions may be
made by site-directed mutagenesis (see, for example, Zoller and
Smith, 1982, Nucl. Acids Res. 10:6487-6500; and Kunkel, 1985, PNAS
82:488).
[0075] In some aspects of the invention, the EDB ADCs include an
antibody, or antigen binding fragment thereof, having a heavy chain
and/or a light chain comprising an amino acid sequence that is at
least 90%, 95%, 98%, or 99% identical to any of the heavy or light
chains disclosed herein. Residues that have been altered can be in
the variable region or in the constant region of the antibody. In
some aspects, there are no more than 1, 2, 3, 4 or 5 residues that
have been altered as compared to any of the heavy or light chains
disclosed herein.
[0076] The term "percent identical" in the context of amino acid
sequences means the number of residues in two sequences that are
the same when aligned for maximum correspondence. There are a
number of different algorithms known in the art which can be used
to measure amino acid percent identity (i.e., the Basic Local
Alignment Tool or BLAST.RTM.). Unless otherwise specified, default
parameters for a particular program or algorithm are used.
[0077] For use in preparation of EDB ADCs, antibodies described
herein may be substantially pure, i.e., at least 50% pure (i.e.,
free from contaminants), more preferably, at least 90% pure, more
preferably, at least 95% pure, yet more preferably, at least 98%
pure, and most preferably, at least 99% pure.
[0078] Tables 2 and 3 provide the amino acid (protein) sequences
and associated nucleic acid (DNA) sequences of anti-EDB antibodies
of the present invention. The CDRs are as defined by Kabat and
Chothia. The shaded residues identify amino acid mutations,
substitutions and/or insertions relating to antibody optimization
and underlined residues identify amino acid mutations,
substitutions and/or insertions relating to site-specific
conjugation technology.
TABLE-US-00002 TABLE 2 Anti-EDB antibody sequences SEQ ID NO.
Description Sequence 1 EDB-L19 VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG Protein
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
TAVYYCAKPFPYFDYWGQGTLVTVSS 2 EDB-L19 VH
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG DNA
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 3 EDB L19 VH SFSMS CDR1
Kabat 4 EDB-L19 VH GFTFSSF CDR1 Chothia 5 EDB-L19 VH
SISGSSGTTYYADSVKG CDR2 Kabat 6 EDB-L19 VH SGSSGT CDR2 Chothia 7
EDB-L19 VH PFPYFDY CDR3 Kabat/Chothia 8 EDB-L19 HC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG Human IgG1
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein
TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK ##STR00009##
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 9 EDB-L19 HC
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG DNA
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 10 EDB-L19 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPR Protein
LLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTG RIPPTFGQGTKVEIK 11
EDB-L19 VL GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC DNA
AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT
AGCAGCAGCTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACTGGC
ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA
CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT
TACTGTCAGCAGACGGGTCGTATTCCGCCGACGTTCGGCCAAG GGACCAAGGTGGAAATCAAA 12
EDB-L19 VL RASQSVSSSFLA CDR1 Kabat/Chothia 13 EDB-L19 VL YASSRAT
CDR2 Kabat/Chothia 14 EDB-L19 VL QQTGRIPPT CDR3 Kabat/Chothia 15
EDB-L19 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPR Human
LLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTG Kappa
RIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF Protein
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 16 EDB-L19 LC
GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC DNA
AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT
AGCAGCAGCTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACTGGC
ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA
CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT
TACTGTCAGCAGACGGGTCGTATTCCGCCGACGTTCGGCCAAG
GGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG
CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC
CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC
AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGA
AACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG
CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 17 EDB-PFE HC
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG Protein
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED
TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK ##STR00010##
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 18 EDB-PFE HC
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG DNA
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 19 EDB-(K290C)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG HC
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein
TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGK ##STR00011##
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 20 EDB-(K290C)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG HC
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT DNA
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACATGCC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 21 EDB-(K94R)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG VH
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein ##STR00012##
22 EDB-(K94R) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG VH
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT DNA
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAGACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 23 EDB-(K94R)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSVVVRQAPGKG HC
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein ##STR00013##
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 24 EDB-(K94R)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG HC
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT DNA
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAGACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 25 EDB-(K94R-
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG K290C) HC
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein ##STR00014##
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGK ##STR00015##
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 26 EDB-(K94R-
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG K290C) HC
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT DNA
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAGACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT
GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACATGCC
CGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA 27 EDB-(H16-
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSVVVRQAPGKG K222R) HC
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED Protein
TAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDRTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPRELLQGSTYRVVSVLTVLHQDWLNGKE ##STR00016##
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 28 EDB-(H16-
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG K222R) HC
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT DNA
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAAT
CTTGTGACCGCACTCACACATGCCCACCGTGCCCAGCACCTGAA
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGCTGCTGCAGGGGAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT 29 EDB-(K94R-
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKG H16-K222R)
LEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED HC ##STR00017##
Protein GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDRTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPRELLQGSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 30 EDB-(K94R-
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTG H16-K222R)
GGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT HC
AGCAGTTTTTCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG DNA
GGCTGGAGTGGGTCTCATCTATTAGTGGTAGTTCGGGTACCACA
TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGA
CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAG
CCGAAGACACGGCCGTATATTACTGTGCGAGACCGTTTCCGTAT
TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTG
CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC
CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGT
CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAAT
CTTGTGACCGCACTCACACATGCCCACCGTGCCCAGCACCTGAA
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGCTGCTGCAGGGGAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCCGGA 31 EDB-
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPR (.kappa.K183C) LC
LLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTG Protein
RIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF
YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSCA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 32 EDB-
GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC (.kappa.K183C) LC
AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT DNA
AGCAGCAGCTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGG
CTCCCAGGCTCCTCATCTATTATGCATCCAGCAGGGCCACTGGC
ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA
CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT
TACTGTCAGCAGACGGGTCGTATTCCGCCGACGTTCGGCCAAG
GGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTC
TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC
CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACA
GCCTCAGCAGCACCCTGACGCTGAGCTGCGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT
CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
[0079] In some aspects of the invention, an EDB ADC includes an
antibody, or antigen binding fragment thereof, that binds to extra
domain B (EDB) of fibronectin (FN).
[0080] In some aspects of the invention, an antibody of the present
invention, or antigen binding fragment thereof, has a heavy chain
variable region (VH) and a light chain variable region (VL),
wherein the VH has three CDRs comprising SEQ ID NOs: 3, 5 and 7. In
some aspects of the invention, an antibody, or antigen binding
fragment thereof, has a heavy chain variable region (VH) and a
light chain variable region (VL), wherein the VL has three CDRs
comprising SEQ ID NOs: 12, 13 and 14. An antibody, or
antigen-binding fragment thereof, may have a VH having three CDRs
comprising SEQ ID NOs: 3, 5 and 7; and a VL having three CDRs
comprising SEQ ID NOs: 12, 13 and 14.
[0081] In another aspect, an antibody of the present invention, or
antigen binding fragment thereof, may have a heavy chain variable
region (VH) comprising a VH CDR1 of SEQ ID NO: 3, a VH CDR2 of SEQ
ID NO: 5 and a VH CDR3 of SEQ ID NO: 7 (according to Kabat), or a
VH CDR1 of SEQ ID NO: 4, a VH CDR2 of SEQ ID NO: 6 and a VH CDR3 of
SEQ ID NO: 7 (according to Chothia), or a VH CDR1 of SEQ ID NO: 3
or 4, a VH CDR2 of SEQ ID NO: 5 or 6 and a VH CDR3 of SEQ ID NOs:
7. In another aspect, an antibody, or antigen binding fragment
thereof, may have a light chain variable region (VL) comprising a
VL CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13 and a VL CDR3
of SEQ ID NO: 14 (according to Kabat and Chothia). In a further
aspect, an antibody, or antigen binding fragment thereof, may have
a VH CDR1 of SEQ ID NO: 3 or 4, a VH CDR2 of SEQ ID NO: 5 or 6 and
a VH CDR3 of SEQ ID NOs: 7 and a VL CDR1 of SEQ ID NO: 12, a VL
CDR2 of SEQ ID NO: 13 and a VL CDR3 of SEQ ID NO: 14.
[0082] In some aspects of the invention, an antibody, or
antigen-binding fragment thereof, may heave a heavy chain variable
region comprising SEQ ID NOs: 1 or 21 and/or a light chain variable
region comprising SEQ ID NO: 10. An antibody, or antigen-binding
fragment thereof, may comprise: a heavy chain variable region
having an amino acid sequence that is at least 90% identical to SEQ
ID NO: 1 and a light chain variable region having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 10; a heavy
chain variable region having an amino acid sequence that is at
least 90% identical to SEQ ID NO: 21 and a light chain variable
region having an amino acid sequence that is at least 90% identical
to SEQ ID NO: 10; a heavy chain variable region comprising SEQ ID
NO: 1 and a light chain variable region comprising SEQ ID NO: 10;
or a heavy chain variable region comprising SEQ ID NO: 21 and a
light chain variable region comprising SEQ ID NO: 10.
[0083] In another aspect of the invention, an antibody, or
antigen-binding fragment thereof, may have a heavy chain comprising
any one of SEQ ID NOs: 8, 17, 19, 23, 25, 27 and 29, and/or a light
chain comprising SEQ ID NOs: 15 or 31.
[0084] An antibody of the present invention, or antigen-binding
fragment thereof, may comprise: a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 8 and a light
chain having an amino acid sequence that is at least 90% identical
to SEQ ID NO: 15; a heavy chain having an amino acid sequence that
is at least 90% identical to SEQ ID NO: 8 and a light chain having
an amino acid sequence that is at least 90% identical to SEQ ID NO:
31; a heavy chain having an amino acid sequence that is at least
90% identical to SEQ ID NO: 17 and a light chain having an amino
acid sequence that is at least 90% identical to SEQ ID NO: 15; a
heavy chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 17 and a light chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 31; a heavy
chain having an amino acid sequence that is at least 90% identical
to SEQ ID NO: 19 and a light chain having an amino acid sequence
that is at least 90% identical to SEQ ID NO: 15; a heavy chain
having an amino acid sequence that is at least 90% identical to SEQ
ID NO: 19 and a light chain having an amino acid sequence that is
at least 90% identical to SEQ ID NO: 31; a heavy chain having an
amino acid sequence that is at least 90% identical to SEQ ID NO: 23
and a light chain having an amino acid sequence that is at least
90% identical to SEQ ID NO: 15; a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 23 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 31; a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 25 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 15; a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 25 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 31; a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 27 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 15; a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 27 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 31; or a heavy chain having an amino acid
sequence that is at least 90% identical to SEQ ID NO: 29 and a
light chain having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 15.
[0085] An antibody of the present invention, or antigen-binding
fragment thereof, may comprise: a heavy chain comprising SEQ ID NO:
8 and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 8 and a light chain comprising SEQ ID NO: 31;
a heavy chain comprising SEQ ID NO: 17 and a light chain comprising
SEQ ID NO: 15; a heavy chain comprising SEQ ID NO:17 and a light
chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID
NO:19 and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO:
31; a heavy chain comprising SEQ ID NO: 23 and a light chain
comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 23
and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO:
15; a heavy chain comprising SEQ ID NO: 25 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 27
and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 27 and a light chain comprising SEQ ID NO:
31; a heavy chain comprising SEQ ID NO: 29 and a light chain
comprising SEQ ID NO: 15; or a heavy chain comprising SEQ ID NO: 29
and a light chain comprising SEQ ID NO: 31.
[0086] Representative DNAs encoding anti-EDB antibody heavy chain
and light chain variable regions comprise SEQ ID NOs: 2 and 22 and
SEQ ID NO: 11, respectively. Representative DNAs encoding anti-EDB
antibody heavy chains and light chains comprise SEQ ID NOs: 9, 18,
20, 24, 26, 28 and 30, and SEQ ID NOs: 16 and 32, respectively.
TABLE-US-00003 TABLE 3 SEQ ID NOs for various anti-EDB antibodies.
CDRs in Kabat and (Chothia). VH VH VH VL VL VL VH CDR1 CDR2 CDR3 HC
VL CDR1 CDR2 CDR3 LC EDB-L19 1 3 (4) 5 (6) 7 8 10 12 13 14 15
EDB-PFE 1 3 (4) 5 (6) 7 17 10 12 13 14 15 EDB- 1 3 (4) 5 (6) 7 19
10 12 13 14 31 (.kappa.K183C- K290C) EDB-(K94R) 21 3 (4) 5 (6) 7 23
10 12 13 14 15 EDB- 21 3 (4) 5 (6) 7 25 10 12 13 14 31
(.kappa.K183C- K94R- K290C) EDB-(H16- 1 3 (4) 5 (6) 7 27 10 12 13
14 15 K222R) EDB-(K94R- 21 3 (4) 5 (6) 7 29 10 12 13 14 15
H16-K222R)
Drugs
[0087] Drugs useful in preparation of the disclosed EDB ADCs
include any substance having biological or detectable activity, for
example, therapeutic agents, detectable labels, binding agents,
etc., and prodrugs, which are metabolized to an active agent in
vivo. A drug may also be a drug derivative, wherein a drug has been
functionalized to enable conjugation with an antibody of the
invention.
[0088] A therapeutic agent is an agent that exerts a cytotoxic,
cytostatic, and/or immunomodulatory effect on cancer cells or
activated immune cells. Examples of therapeutic agents include
cytotoxic agents, chemotherapeutic agents, cytostatic agents, and
immunomodulating agents. A cytotoxic effect refers to the
depletion, elimination and/or the killing of a target cell(s). A
cytotoxic agent refers to an agent that has a cytotoxic and/or
cytostatic effect on a cell. A cytostatic effect refers to the
inhibition of cell proliferation. A cytostatic agent refers to an
agent that has a cytostatic effect on a cell, thereby inhibiting
the growth and/or expansion of a specific subset of cells. A
chemotherapeutic agent refers to an agent that is a chemical
compound useful in the treatment of cancer. An immunomodulating
agent refers to an agent that stimulates the immune response though
the production of cytokines and/or antibodies and/or modulating T
cell function thereby inhibiting or reducing the growth of a subset
of cells (i.e., tumor cells) either directly or indirectly by
allowing another agent to be more efficacious.
[0089] In some aspects the drug is a membrane permeable drug. In
such aspects, the payload can elicit a bystander effect wherein
cells that may not express EDB+FN or have EDB+ FN bound to their
surface, but surround the cell that is bound by the ADC are killed
by the cell permeable payload. This occurs when the payload is
released from the antibody (i.e., by cleaving of a cleavable
linker) and crosses the cellular membrane and, upon diffusion,
induces the killing of surrounding cells.
[0090] In accordance with the disclosed methods, the EDB ADCs may
be produced or generated having (a) an antibody, or antigen binding
fragment thereof, that binds to EDB; (b) a linker and (c) a drug.
The drug-to-antibody ratio (DAR), or drug loading, indicates the
number of drug molecules conjugated per antibody. Compositions,
batches, and/or formulations of a plurality of ADCs may be
characterized by an average DAR. DAR and average DAR can be
determined by various conventional means such as UV spectroscopy,
mass spectroscopy, ELISA assay, radiometric methods, hydrophobic
interaction chromatography (HIC), electrophoresis and HPLC.
[0091] In aspects of the invention, an EDB ADC may have a DAR of 1,
a DAR of 2, a DAR of 3, a DAR of 4, a DAR of 5, a DAR of 6, a DAR
of 7, a DAR of 8, a DAR of 9, a DAR of 10, a DAR of 11, a DAR of 12
or a DAR greater than 12. In aspects of the invention, an EDB ADC
may have one drug molecule, or 2 drug molecules, or 3 drug
molecules, or 4 drug molecules, or 5 drug molecules, or 6 drug
molecules, or 7 drug molecules, or 8 drug molecules, or 9 drug
molecules, or 10 drug molecules, or 11 drug molecules, or 12 drug
molecules or greater than 12 molecules.
[0092] In aspects of the invention, an EDB ADC may have average DAR
in the range of about 2 to about 4, or an average DAR in the range
of about 3 to about 5, or an average DAR in the range of about 4 to
about 6, or an average DAR in the range of about 5 to about 7, or
an average DAR in the range of about 6 to about 8, or an average
DAR in the range of about 7 to about 9, or an average DAR in the
range of about 8 to about 10, or an average DAR in the range of
about 9 to about 11, or an average DAR in the range of about 10 to
about 12, etc. In some aspects the compositions, batches and/or
formulations of EDB ADCs may have an average DAR of about 1, or an
average DAR of about 2, an average DAR of about 3, or an average
DAR of about 4, or an average DAR of about 5, or an average DAR of
about 6, or an average DAR of about 7, or an average DAR of about
8, or an average DAR of about 9, or an average DAR of about 10, or
an average DAR of about 11, or an average DAR of about 12 or an
average DAR greater than 12. As used in the foregoing ranges of
average DAR, the term "about" means+/-0.5%.
[0093] A composition, batch, and/or formulation of EDB ADCs may be
characterized by a preferred range of average DAR, e.g., an average
DAR in the range of about 3 to about 5, an average DAR in the range
of about 3 to about 4, or an average DAR in the range of about 4 to
about 5. Further, a composition, batch, and/or formulation of EDB
ADCs may be characterized by a preferred range of average DAR,
e.g., an average DAR in the range of 3 to 5, an average DAR in the
range of 3 to 4, or an average DAR in the range of 4 to 5.
[0094] In some aspects of the invention, a composition, batch,
and/or formulation of EDB ADCs may be characterized by an average
DAR of about 1.0, or an average DAR of 1.0, or an average DAR of
1.1, or an average DAR of 1.2, or an average DAR of 1.3, or an
average DAR of 1.4, or an average DAR of 1.5, or an average DAR of
1.6, or an average DAR of 1.7, or an average DAR of 1.8, or an
average DAR of 1.9. In another aspect, a composition, batch, and/or
formulation of EDB ADCs may be characterized by an average DAR of
about 2.0, or an average DAR of 2.0, or an average DAR of 2.1, or
an average DAR of 2.2, or an average DAR of 2.3, or an average DAR
of 2.4, or an average DAR of 2.5, or an average DAR of 2.6, or an
average DAR of 2.7, or an average DAR of 2.8, or an average DAR of
2.9. In another aspect, a composition, batch, and/or formulation of
EDB ADCs may be characterized by an average DAR of about 3.0, or an
average DAR of 3.0, or an average DAR of 3.1, or an average DAR of
3.2, or an average DAR of 3.3, or an average DAR of 3.4, or an
average DAR of 3.5, or an average DAR of 3.6, or an average DAR of
3.7, or an average DAR of 3.8, or an average DAR of 3.9. In another
aspect, a composition, batch, and/or formulation of EDB ADCs may be
characterized by an average DAR of about 4.0, or an average DAR of
4.0, or an average DAR of 4.1, or an average DAR of 4.2, or an
average DAR of 4.3, or an average DAR of 4.4, or an average DAR of
4.5, or an average DAR of 4.6, or an average DAR of 4.7, or an
average DAR of 4.8, or an average DAR of 4.9, or an average DAR of
5.0.
[0095] In another aspect, a composition, batch, and/or formulation
of EDB ADCs may be characterized by an average DAR of 12 or less,
an average DAR of 11 or less, an average DAR of 10 or less, an
average DAR of 9 or less, an average DAR of 8 or less, an average
DAR of 7 or less, an average DAR of 6 or less, an average DAR of 5
or less, an average DAR of 4 or less, an average DAR of 3 or less,
an average DAR of 2 or less or an average DAR of 1 or less.
[0096] In other aspects, a composition, batch, and/or formulation
of EDB ADCs may be characterized by an average DAR of 11.5 or less,
an average DAR of 10.5 or less, an average DAR of 9.5 or less, an
average DAR of 8.5 or less, an average DAR of 7.5 or less, an
average DAR of 6.5 or less, an average DAR of 5.5 or less, an
average DAR of 4.5 or less, an average DAR of 3.5 or less, an
average DAR of 2.5 or less, an average DAR of 1.5 or less.
[0097] In some aspects of the present invention, the methods for
conventional conjugation via cysteine residues and purification
conditions disclosed herein provide a composition, batch, and/or
formulation of EDB ADCs with an optimized average DAR in the range
of about 3 to 5, preferably about 4.
[0098] In some aspects of the present invention, the methods for
site-specific conjugation via engineered cysteine residues and
purification conditions disclosed herein provide a composition,
batch, and/or formulation of EDB ADCs with an optimized average DAR
in the range of about 3 to 5, preferably about 4.
[0099] In some aspects of the present invention, the methods for
site-specific conjugation via transglutaminase-based conjugation
and purification conditions disclosed herein provide a composition,
batch, and/or formulation of EDB ADCs with an optimized average DAR
in the range of about 1 to 3, preferably about 2.
[0100] Examples of cytotoxic agents include, but are not limited to
an anthracycline, an auristatin, CC-1065, a dolastatin, a
duocarmycin, an enediyne, a geldanamycin, a maytansine, a
puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin,
hemiasterlin, and stereoisomers, isosteres, analogs or derivatives
thereof. Plant toxins, other bioactive proteins, enzymes (i.e.,
ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic
therapy) may also be used.
[0101] The anthracyclines are derived from bacteria Strepomyces and
have been used to treat a wide range of cancers, such as leukemias,
lymphomas, breast, uterine, ovarian, and lung cancers. Exemplary
anthracyclines include, but are not limited to, daunorubicin,
doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin,
and mitoxantrone.
[0102] Dolastatins and their peptidic analogs and derivatives,
auristatins, are highly potent antimitotic agents that have been
shown to have anticancer and antifungal activity. See, e.g., U.S.
Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother.
42:2961-2965, (1998). Exemplary dolastatins and auristatins
include, but are not limited to, dolastatin 10, auristatin E,
auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl
Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl
Auristatin F or
N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE
(Monomethyl Auristatin E or
N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine),
5-benzoylvaleric acid-AE ester (AEVB). and other novel
[0103] In some aspects, the drug/payload is an auristatin.
Auristatins inhibit cell proliferation by inhibiting the formation
of microtubules during mitosis through inhibition of tubulin
polymerization. PCT International Publication No. WO 2013/072813,
which is incorporated herein by reference in its entirety,
discloses auristatins that are useful in the EDB ADCs of the
present invention and provides methods of producing the
auristatins. For example, payload 0101 having the structure:
##STR00018##
[0104] payload 1569 having the structure:
##STR00019##
[0105] payload 9411 having the structure:
##STR00020##
[0106] payload 4574 having the structure:
##STR00021##
[0107] payload DM1 having the structure:
##STR00022##
and
[0108] payload Cemadotin having the structure:
##STR00023##
[0109] Duocarmycin and CC-1065 are CPI-based monomers that act as
DNA alkylating agents with cytotoxic potency. See Boger and
Johnson, PNAS 92:3642-3649, 1995. Exemplary dolastatins include,
but are not limited to, (+)-docarmycin A and (+)-duocarmycin SA,
and (+)-CC-1065.
[0110] In some aspects, the drug/payload is a CPI or CBI dimer. CPI
dimers induce inter-strand DNA crosslinking and potent
cytotoxicity. PCT International Publication No. WO2015/110935,
which is incorporated herein by reference in its entirety,
discloses CPI and CBI dimers that are useful in the EDB ADCs of the
present invention and provides methods of producing the CPI and CBI
dimers. For example, payload CPI-8314 dimer having the
structure:
##STR00024##
and
[0111] payload CPI-0326 having the structure:
##STR00025##
[0112] Enediynes are a class of anti-tumor bacterial products
characterized by either nine- and ten-membered rings or the
presence of a cyclic system of conjugated triple-double-triple
bonds. Exemplary enediynes include, but are not limited to,
calicheamicin, esperamicin, and dynemicin. Calicheamicin, also
called the LL-E33288 complex, for example, .beta.-calicheamicin,
.gamma.-calicheamicin or N-acetyl-.gamma.-calicheamicin
(gamma-calicheamicin (.gamma..sub.1)), is an enediyne antibiotic
that was originally isolated as a natural product from the soil
organism Micromonospora echinospora ssp. calichensis (Zein et al.
Science 27; 240(4856):1198-1201, 1988); it generates double-strand
DNA breaks and subsequently induces apoptosis in target cells (Zein
et al. Science 27, 240(4856):1198-1201, 1988; Nicolaou et al. Chem.
Biol. September; 1(1):57-66, 1994; Prokop et al. Oncogene
22:9107-9120, 2003). The disulfide analog is
N-acetyl-.gamma.-calicheamicin dimethyl hydrazide.
[0113] Geldanamycins are benzoquinone ansamycin antibiotic that
bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor
drugs. Exemplary geldanamycins include, but are not limited to,
17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG
(17-Dimethylaminoethylamino-17-demethoxygeldanamycin).
[0114] Maytansines or their derivatives maytansinoids inhibit cell
proliferation by inhibiting the microtubules formation during
mitosis through inhibition of polymerization of tubulin. See
Remillard et al., Science 189:1002-1005, 1975. Exemplary
maytansines and maytansinoids include, but are not limited to,
mertansine (DM1) and its derivatives as well as ansamitocin.
[0115] Taxanes are diterpenes that act as anti-tubulin agents or
mitotic inhibitors. Exemplary taxanes include, but are not limited
to, paclitaxel (e.g., TAXOL) and docetaxel (TAXOTERE.RTM.).
[0116] Vinca alkyloids are also anti-tubulin agents. Exemplary
vinca alkyloids include, but are not limited to, vincristine,
vinblastine, vindesine, and vinorelbine.
[0117] In some aspects of the invention, the agent is an
immunomodulating agent. Examples of an immunomodulating agent
include, but are not limited to, gancyclovier, etanercept,
tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin,
cyclophosphamide, azathioprine, mycophenolgate mofetil,
methotrextrate, glucocorticoid and its analogs, cytokines,
xanthines, stem cell growth factors, lymphotoxins, tumor necrosis
factor (TNF), hematopoietic factors, interleukins (e.g.,
interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and
IL-21), colony stimulating factors (e.g., granulocyte-colony
stimulating factor (G-CSF) and granulocyte macrophage-colony
stimulating factor (GM-CSF)), interferons (e.g.,
interferons-.alpha., -.beta. and -.gamma.), the stem cell growth
factor designated "S 1 factor," erythropoietin and thrombopoietin,
or a combination thereof.
[0118] Immunomodulatory agents useful in the invention also include
anti-hormones that block hormone action on tumors and
immunosuppressive agents that suppress cytokine production,
down-regulate self-antigen expression, or mask MHC antigens.
Representative anti-hormones include anti-estrogens including, for
example, tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapnstone, and toremifene; and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
anti-adrenal agents. Representative immunosuppressive agents
include 2-amino-6-aryl-5-substituted pyrimidines, azathioprine,
cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde,
anti-idiotypic antibodies for MHC antigens and MHC fragments,
cyclosporin A, steroids such as glucocorticosteroids, cytokine or
cytokine receptor antagonists (e.g., anti-interferon antibodies,
anti-IL10 antibodies, anti-TNF.alpha. antibodies, anti-IL2
antibodies), streptokinase, TGF.beta., rapamycin, T-cell receptor,
T-cell receptor fragments, and T cell receptor antibodies.
[0119] In some aspects of the invention, the drug is a therapeutic
protein including, but is not limited to, a toxin, a hormone, an
enzyme, and a growth factor.
[0120] Examples of a toxin protein (or polypeptide) include, but
are not limited to, dipththeria (e.g., diphtheria A chain),
Pseudomonas exotoxin and endotoxin, ricin (e.g., ricin A chain),
abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain),
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtherin toxin, Phytolaca
americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia
inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor,
mitogellin, restrictocin, phenomycin, enomycin, tricothecenes,
inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and
conotoxin (e.g., KIIIA or SmIIIa).
[0121] Examples of hormones include, but are not limited to,
estrogens, androgens, progestins and corticosteroids.
[0122] In some aspects of the invention, the drug is an
oligonucleotide, such as anti-sense oligonucleotides.
[0123] Additional drugs useful in the invention include
anti-angiogenic agents that inhibit blood vessel formation, for
example, farnesyltransferase inhibitors, COX-2 inhibitors, VEGF
inhibitors, bFGF inhibitors, steroid sulphatase inhibitors (e.g.,
2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE)),
interleukin-24, thrombospondin, metallospondin proteins, class I
interferons, interleukin 12, protamine, angiostatin, laminin,
endostatin, and prolactin fragments.
[0124] Anti-proliferative agents and pro-apoptotic agents include
activators of PPAR-gamma (e.g., cyclopentenone prostaglandins
(cyPGs)), retinoids, triterpinoids (e.g., cycloartane, lupane,
ursane, oleanane, friedelane, dammarane, cucurbitacin, and limonoid
triterpenoids), inhibitors of EGF receptor (e.g., HER4),
rampamycin, CALCITRIOL.RTM. (1,25-dihydroxycholecalciferol (vitamin
D)), aromatase inhibitors (FEMARA.RTM. (letrozone)), telomerase
inhibitors, iron chelators (e.g., 3-aminopyridine-2-carboxaldehyde
thiosemicarbazone (Triapine)), apoptin (viral protein 3-VP3 from
chicken aneamia virus), inhibitors of Bcl-2 and Bcl-X(L),
TNF-alpha, FAS ligand, TNF-related apoptosis-inducing ligand
(TRAIL/Apo2L), activators of TNF-alpha/FAS ligand/TNF-related
apoptosis-inducing ligand (TRAIL/Apo2L) signaling, and inhibitors
of PI3K-Akt survival pathway signaling (e.g., UCN-01 and
geldanamycin).
[0125] Representative chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziidines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlomaphazine, cholophosphamide, estramustine, ifosfamide,
mechiorethamine, mechiorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfarnide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, carminomycin,
carzinophilin, chromomycins, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic
acid, nogalamycin, olivomycins, peplomycin, potfiromycin,
puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,
tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such
as methotrexate and 5-fluorouracil (5-FU); folic acid analogues
such as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-EU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenal such as arninoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophospharnide glycoside; arninolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elfornithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside (Ara-C), cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology
of Princeton, N.J.) and doxetaxel (TAXOTERE.RTM., Rhone-Poulenc
Rorer of Antony, France); chiorambucil; gemcitabine; 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin 0; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aininopterin;
xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoic acid; esperamicins; and
capecitabine.
[0126] Additional therapeutic agents that may be used in accordance
with the present invention include photosensitizing agents, such as
U.S. Publication No. 20020197262 and U.S. Pat. No. 5,952,329, which
are incorporated herein by reference in its entirety, for
photodynamic therapy; magnetic particles for thermotherapy, such as
U.S. Publication No. 20030032995, which is incorporated herein by
reference in its entirety; binding agents, such as peptides,
ligands, cell adhesion ligands, etc., and prodrugs such as
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate containing prodrugs, peptide containing prodrugs,
.beta.-lectern-containing prodrugs, substituted
phenoxyacetamide-containing prodrugs or substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs that may be converted to the more active
cytotoxic free drug.
[0127] For diagnostic methods using anti-EDB antibodies, a drug may
include a detectable label used to detect the presence of EDB+
FN-expressing ECM or cells in vitro or in vivo. Radioisotopes that
are detectable in vivo, such as those labels that are detectable
using scintigraphy, magnetic resonance imaging, or ultrasound, may
be used in clinical diagnostic applications. Useful scintigraphic
labels include positron emitters and .gamma.-emitters.
Representative contrast agents for magnetic source imaging are
paramagnetic or superparamagnetic ions (e.g., iron, copper,
manganese, chromium, erbium, europium, dysprosium, holmium and
gadolinium), iron oxide particles, and water soluble contrast
agents. For ultrasonic detection, gases or liquids may be entrapped
in porous inorganic particles that are released as microbubble
contrast agents. For in vitro detection, useful detectable labels
include fluorophores, detectable epitopes or binding agents, and
radioactive labels.
[0128] Thus, in some aspects of the invention, the drug is an
imaging agent (e.g., a fluorophore or a PET (Positron Emission
Tomography) label, SPECT (Single-Photon Emission Computed
Tomorgraphy) label), or MRI (Magnetic Resonance Imaging) label.
[0129] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g., radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
that is detectable. Radionuclides that can serve as detectable
labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188,
Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label might also be
a non-detectable entity such as a toxin.
[0130] Examples of fluorophores include, but are not limited to,
fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein
amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein,
erythrosine, Alexa Fluor.RTM. (e.g., Alexa 350, 405, 430, 488, 500,
514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or
750), carboxytetramethylrhodamine (TAM RA) (e.g., 5,-TAMRA),
tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g.,
SR101).
[0131] Therapeutic or diagnostic radioisotopes or other labels
(e.g., PET or SPECT labels) can be incorporated in the agent for
conjugation to the anti-EDB antibodies as described herein. The
isotope may be directly bound to the antibody, for example, at a
cysteine residue present in the antibody, or a chelator may be used
to mediate the binding of the antibody and the radioisotope.
Radioisotopes suitable for radiotherapy include but are not limited
to .alpha.-emitters, .beta.-emitters, and auger electrons. For
diagnostic applications, useful radioisotopes include positron
emitters and .gamma.-emitters. An anti-EDB antibody of the
invention may further be iodinated, for example, on a tyrosine
residue of the antibody, to facilitate detection or therapeutic
effect of the antibody.
[0132] Examples of a radioisotope or other labels include, but are
not limited to, .sup.3H, .sup.11C, .sup.13N, .sup.14C, .sup.15N,
.sup.15O, .sup.35S, .sup.18F, .sup.32P, .sup.33P, .sup.47Sc,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.75Se, .sup.76Br, .sup.77Br,
.sup.86Y, .sup.89Zr, .sup.90Y, .sup.94Tc, .sup.95Ru, .sup.97Ru,
.sup.99Tc, .sup.103Ru, .sup.105Rh, .sup.105Ru, .sup.107Hg,
.sup.109Pd, .sup.111Ag, .sup.111In, .sup.113In, .sup.121Te,
.sup.122Te, .sup.123I, .sup.124I, .sup.125I, .sup.125Te, .sup.126I,
.sup.131I, .sup.131In, .sup.133I, .sup.142Pr, .sup.143Pr,
.sup.153Pd, .sup.153Sm, .sup.161Tb, .sup.165Tm, .sup.166Dy,
.sup.166H, .sup.167Tm, .sup.168Tm, .sup.169Yb, .sup.177Lu,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.197Pt, .sup.198Au,
.sup.199Au, .sup.201Tl, .sup.203Hg, .sup.211At, .sup.212Bi,
.sup.212Pb, .sup.213Bi, .sup.223Ra, .sup.224Ac, and .sup.225Ac.
Linkers
[0133] EDB ADCs of the present invention may be prepared using a
linker to directly or indirectly link or conjugate a drug to an
antibody. A linker is a bifunctional compound that links a drug and
an antibody to form an ADC. Such ADCs allow the selective delivery
of drugs via antibodies that bind to specific antigens or proteins.
Suitable linkers include, for example, cleavable and non-cleavable
linkers. A cleavable linker is typically susceptible to cleavage
and release of drug by specific intracellular and extracellular
conditions. Major mechanisms by which a conjugated drug may be
cleaved from an antibody intracellularly include hydrolysis in the
acidic pH of the lysosomes (hydrazones, acetals, and
cis-aconitate-like amides), peptide cleavage by lysosomal enzymes
(the cathepsins and other lysosomal enzymes), and reduction of
disulfides. A conjugated drug may be cleaved from an antibody
extracellulary by proteases in a tumor microenvironment (TME), such
as cathepsins. As a result of these varying mechanisms for
cleavage, mechanisms of linking the drug to the antibody also vary
widely and any suitable linker can be used.
[0134] Suitable linkers may include any cleavable linker. In some
aspects, suitable linkers include a valine-citrulline (val-cit)
linker, a phenylalanine-lysine (phe-lys) linker, or a
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc)
linker, or contain a dipeptide attached to additional immolation
elements, such as
N.about.2.about.-acetyl-L-lysyl-L-valyl-L-citruline-p-aminobenzyloxyca-
rbonyl-N,N'-dimethylaminoethyl-CO-(AcLys-vc) linker, suitable for
transglutaminase-based conjugation technology. In another aspect,
suitable linkers include disulfide linkers, such as sulfanyl
pyridine (diS) linker and 2-(pyridin-2-yldisulfanyl)ethyl carbamoyl
(diS-C.sub.2OCO) linker. In another aspect, the linker may be a
non-cleavable linker, such as maleimidocaproyl (mc),
maleimido-heptanoyl (me) and maleimido-Peg6C2 (MalPeg6C2). In other
aspects, suitable linkers include linkers hydrolyzable at a
specific pH or a pH range, such as a hydrazone linker.
[0135] The linker may be covalently bound to the antibody through a
thioester linkage, for instance by reaction of a maleimide or
haloacetamide, present on the linker with a native or engineered
cysteine residue present on the antibody. In another aspect, the
linker may be covalently bound to the antibody through amide
linkages to lysine residues present on the antibody, for instance
by reaction of an N-hydroxy-succinimide activated carboxylic acid
present on the linker with a free amine of a lysine residue. In
another aspect, the linker may be covalently bound to the antibody
through amide linkages to the side chains of glutamine residues
present or engineered into the antibody, for instance by enzymatic
reaction catalyzed by a transglutaminase enzyme that creates a new
amide linkage from a primary amine present on the linker with a
side chain amide of a glutamine residue.
[0136] In some aspects, linkers of the present invention
include:
[0137] "mc-vc-PABC" or "vc-PABC" or "vc" linker having the
structure:
##STR00026##
[0138] "AcLys-vc-PABC-DMAE-CO" or "AcLys-vc" linker having the
structure:
##STR00027##
[0139] diS linker having the structure:
##STR00028##
and
[0140] diS-C.sub.2OCO linker having the structure:
##STR00029##
Methods of Preparing EDB ADCs
[0141] Provided herein are methods for preparing EDB ADCs of the
present invention. The present invention further provides for a
process for producing or generating conventionally and
site-specific conjugated EDB ADCs as disclosed herein and may
include (a) linking the linker to the drug; (b) conjugating the
linker-drug moiety to the antibody; and (c) purifying the antibody
drug conjugate. See Examples 3 and 4.
[0142] In some aspects, EDB ADCs may be generated using
conventional, non-specific conjugation of linker-payload moieties
through one or more cysteine residues of an anti-EDB antibody, or
an antigen binding fragment thereof.
[0143] In another aspect, EDB ADCs may be generated using
site-specific conjugation of linker-payload moieties though one or
more reactive cysteine residues engineered into an anti-EDB
antibody constant domain. Methods of preparing antibodies for
site-specific conjugation via engineered cysteine residues are
described in PCT International Publication No. WO2013/093809, which
is incorporated herein by reference in its entirety.
[0144] One or more amino acid residues of an anti-EDB antibody
heavy chain may be substituted to another amino acid, such as a
cysteine residue, for the purpose of conjugation to a drug or
payload. In one aspect, the invention provides an anti-EDB
antibody, or antigen binding fragment thereof, comprising an
antibody heavy chain constant region comprising an engineered
cysteine residue at position: 118 (114 according to Kabat), 246,
249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302,
303, 314, 315, 318, 320, 327, 332, 333, 334, 336, 345, 347, 354,
355, 358, 360, 362, 370, 373, 375, 376, 378, 380, 382, 386, 388,
390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428,
431, 432, 437, 438, 439, 443 or 444, or any combination thereof,
according to the numbering of the EU index of Kabat). In
particular, positions 118 (114 according to Kabat), 290, 334, 347,
373, 375, 380, 388, 392, 421, 443, or any combination thereof may
be used. Additional cysteine substitutions may be introduced.
[0145] In another aspect, the invention provides an anti-EDB
antibody, or antigen binding fragment thereof, comprising a heavy
chain constant domain comprising an engineered cysteine residue at
position 290 (K290C), according to the numbering of the EU index of
Kabat.
[0146] One or more amino acid residues of an anti-EDB antibody
light chain constant domain may be substituted to another amino
acid, such as a cysteine residue, for the purpose of conjugation to
a drug or payload. In one aspect, the invention provides an
anti-EDB antibody, or antigen binding fragment thereof, comprising
an antibody light chain constant region comprising (i) an
engineered cysteine residue at position 110, 111, 125, 149, 155,
158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208 or 210, or
any combination thereof, according to the numbering of Kabat); (ii)
an engineered cysteine residue at a position corresponding to
residue 4, 42, 81, 100, 103, or any combination thereof, of SEQ ID
NO: 37, when the constant domain is aligned with SEQ ID NO: 37
(kappa light chain); or (iii) an engineered cysteine residue at a
position corresponding to residue 4, 5, 19, 43, 49, 52, 55, 78, 81,
82, 84, 90, 96, 97, 98, 99, 101, or any combination thereof, of SEQ
ID NO: 38, when the constant domain is aligned with SEQ ID NO: 38
(lambda light chain). Additional cysteine substitutions may be
introduced.
[0147] In another aspect, the invention provides an anti-EDB
antibody or antigen binding fragment thereof comprising an antibody
kappa light chain constant region comprising (i) an engineered
cysteine residue at position 111, 149, 188, 207, 210, or any
combination thereof (preferably 111 or 210), according to the
numbering of Kabat; or (ii) an engineered cysteine residue at a
position corresponding to residue 4, 42, 81, 100, 103, or any
combination thereof, of SEQ ID NO: 37 (preferably residue 4 or
103), when the constant domain is aligned with SEQ ID NO: 37.
[0148] In another aspect, the invention provides an anti-EDB
antibody or antigen binding fragment thereof comprising an antibody
lambda light chain constant region comprising (i) an engineered
cysteine residue at position 110, 111, 125, 149, 155, 158, 161,
185, 188, 189, 191, 197, 205, 206, 207, 208, 210, or any
combination thereof (preferably 110, 111, 125, 149, or 155),
according to the numbering of Kabat; or (ii) an engineered cysteine
residue at a position corresponding to residue 4, 5, 19, 43, 49,
52, 55, 78, 81, 82, 84, 90, 96, 97, 98, 99, 101, or any combination
thereof of SEQ ID NO: 38 (preferably residue 4, 5, 19, 43, or 49),
when the constant domain is aligned with SEQ ID NO:38.
[0149] In another aspect, the invention provides an anti-EDB
antibody, or antigen binding fragment thereof, comprising a light
chain constant domain comprising (i) an engineered cysteine residue
at position 183 (.kappa.K183C), according to the numbering of
Kabat; or (ii) an engineered cysteine residue at a position
corresponding to residue 76 of SEQ ID NO: 37, when said constant
domain is aligned with SEQ ID NO: 37.
TABLE-US-00004 (C.kappa. constant domain) SEQ ID NO: 37 RTVAAPSVFI
FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS
TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (C.lamda. constant domain)
SEQ ID NO 38 GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK
AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS
[0150] In another aspect, EDB ADCs may be generated using
site-specific conjugation technology though one or more engineered
acyl donor glutamine-containing tags or endogenous glutamine
residues made reactive in an anti-EDB antibody constant region.
Methods of preparing antibodies for site-specific conjugation via
acyl donor glutamine-containing tags or glutamine residues are
described in PCT International Publication No. WO2012/059882, which
is incorporated herein by reference in its entirety.
[0151] In some aspects, the acyl donor glutamine-containing tag
comprises at least one glutamine (Q) and may be attached to
different position of the heavy and/or light chain (i.e., at the
N-terminus, C-terminus or internally). In another aspect, the acyl
donor glutamine-containing tag may comprise an amino acid sequence
selected from: LLQGG (SEQ ID NO: 39), LLQG (SEQ ID NO: 40), LSLSQG
(SEQ ID NO: 41), GGGLLQGG (SEQ ID NO: 42), GLLQG (SEQ ID NO: 43),
LLQ, GSPLAQSHGG (SEQ ID NO: 44), GLLQGGG (SEQ ID NO: 45), GLLQGG
(SEQ ID NO: 46), GLLQ (SEQ ID NO: 47), LLQLLQGA (SEQ ID NO: 48),
LLQGA (SEQ ID NO: 49), LLQYQGA (SEQ ID NO: 50), LLQGSG (SEQ ID NO:
51), LLQYQG (SEQ ID NO: 52), LLQLLQG (SEQ ID NO: 53), SLLQG (SEQ ID
NO: 54), LLQLQ (SEQ ID NO: 55), LLQLLQ (SEQ ID NO: 56), and LLQGR
(SEQ ID NO: 57). In some aspects, an acyl donor
glutamine-containing tag replaces wild type amino acid positions in
a heavy chain constant domain. In some aspects, an anti-EDB
antibody may comprise an acyl glutamine-containing tag having the
amino acid sequence LLQG (SEQ ID NO: 40) that replaces the amino
acids at positions E294-N297 (according to the EU index of Kabat)
of the heavy chain.
[0152] Optimal reaction conditions for the generation of ADCs may
be empirically determined by a variation of reaction variables such
as temperature, pH, linker-payload moiety input, and additive
concentration. Conditions suitable for conjugation of other drugs
may be determined by those skilled in the art without undue
experimentation. Representative methods for conjugating and
characterizing EDB ADCs are described in Examples 3 and 4.
[0153] Following conjugation, the conjugates may be separated,
purified from unconjugated reactants and/or aggregated forms of the
conjugates, and characterized by conventional methods. This
includes processes such as, but not limited to, mass spectrometry,
size exclusion chromatography (SEC), ultrafiltration/diafiltration,
ion exchange chromatography (IEC), chromatofocusing (CF),
site-directed mutagenesis, fluorescence-labeling, X-ray
crystallography, high performance liquid chromatography (HPLC),
fast protein liquid chromatography (FPLC), Sephacryl S-200
chromatography or hydrophobic interaction chromatography (HIC).
Suitable HIC media includes, but is not limited to, Phenyl
Sepharose 6 Fast Flow chromatographic medium, Butyl Sepharose 4
Fast Flow chromatographic medium, Octyl Sepharose 4 Fast Flow
chromatographic medium, Toyopearl Ether-650M chromatographic
medium, Macro-Prep methyl HIC medium or Macro-Prep t-Butyl HIC
medium.
[0154] Table 13 provides EDB ADCs produced according to the
conjugation and purification methods described herein and used to
generate data provided in the Examples.
[0155] In some aspects of the invention, EDB ADCs of the present
invention comprise (a) an antibody, or antigen binding fragment
thereof, that binds to EDB; (b) a linker and (c) a drug.
[0156] In another aspect of the invention, EDB ADCs of the present
invention comprise (a) an antibody, or antigen binding fragment
thereof, that binds to EDB; (b) a linker and (c) a drug, wherein
the linker is a cleavable or non-cleavable linker. In some aspects,
the linker is vc, diS, diS-C.sub.2OCO or AcLys-vc.
[0157] In another aspect of the invention, EDB ADCs of the present
invention comprise (a) an antibody, or antigen binding fragment
thereof, that binds to EDB; (b) a linker and (c) a drug, wherein
the drug is cytotoxic agent. In some aspects, the drug is an
auristatin. In some aspects, the drug is a CPI or CBI dimer. In
some aspects, the auristatin is 0101, 1569, 9411 or 4574. In some
aspects, the CPI dimer is CPI-8314 or CPI-0326.
[0158] In some aspects of the invention, EDB ADCs of the present
invention comprise (a) an antibody, or antigen binding fragment
thereof, comprising: a heavy chain comprising SEQ ID NO: 8 and a
light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ
ID NO: 8 and a light chain comprising SEQ ID NO: 31; a heavy chain
comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO:
15; a heavy chain comprising SEQ ID NO:17 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO:19 and
a light chain comprising SEQ ID NO: 15; a heavy chain comprising
SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 31; a heavy
chain comprising SEQ ID NO: 23 and a light chain comprising SEQ ID
NO: 15; a heavy chain comprising SEQ ID NO: 23 and a light chain
comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 25
and a light chain comprising SEQ ID NO: 15; a heavy chain
comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO:
31; a heavy chain comprising SEQ ID NO: 27 and a light chain
comprising SEQ ID NO: 15; heavy chain comprising SEQ ID NO: 27 and
a light chain comprising SEQ ID NO: 31; a heavy chain comprising
SEQ ID NO: 29 and a light chain comprising SEQ ID NO: 15; or a
heavy chain comprising SEQ ID NO: 29 and a light chain comprising
SEQ ID NO: 31; (b) a linker and (c) a drug. In some aspects, the
linker is a cleavable or non-cleavable linker. In some aspects, the
linker is vc, diS, diS-C.sub.2OCO or AcLys-vc. In some aspects, the
drug is cytotoxic agent. In some aspects, the drug is an
auristatin. In some aspects, the drug is a CPI or CBI dimer. In
some aspects, the auristatin is 0101, 1569, 9411 or 4574. In some
aspects, the CPI dimer is CPI-8314 or CPI-0326.
Uses of EDB ADCs
[0159] The anti-EDB antibodies and EDB ADCs of the present
invention are useful in various applications including, but are not
limited to, therapeutic treatment methods and diagnostic treatment
methods.
[0160] The present invention provides a method for treating EDB+
FN-expressing disorders or diseases, such as non-cancers or cancers
associated with EDB+ FN expression and/or EDB+ FN-expressing
cancers, in a subject. The invention also provides an EDB ADC, or a
pharmaceutical composition, as described herein, for use in a
method for treating an EDB+ FN-expressing disorder, such as
non-cancers or cancers associated with EDB+ FN expression and/or
EDB+ FN-expressing cancers, in a subject. The invention further
provides the use of an EDB ADC, or a pharmaceutical composition, as
described herein, in the manufacture of a medicament for treating
an EDB+ FN-expressing disorder, such as non-cancers or cancers
associated with EDB+ FN expression and/or EDB+ FN-expressing
cancers, in a subject.
[0161] In some aspects, the invention provides a method of
inhibiting tumor growth or progression in a subject who has an
EDB-expressing disorder, such as non-cancers or cancers associated
with EDB+ FN expression and/or EDB-expressing cancers, including
administering to the subject in need thereof an effective amount of
a composition (i.e., a pharmaceutical composition) having one or
more EDB ADCs described herein. In other aspects of the invention,
provided is a method of inhibiting metastasis of cancer cells
associated with EDB+ FN expression and/or EDB+ FN-expressing
cancers in a subject, including administering to the subject in
need thereof an effective amount of a composition (i.e., a
pharmaceutical composition) having one or more EDB ADCs described
herein. In other aspects of the invention, provided is a method of
inducing regression of a tumor associated with EDB+ FN expression
and/or EDB+ FN-expressing cancers in a subject, including
administering to the subject in need thereof an effective amount of
a composition (i.e., a pharmaceutical composition) having one or
more EDB ADCs described herein.
[0162] In some aspects, the EDB+ FN expression may be detected in
the extracellular matrix (ECM) adjacent to tumor cells. EDB+ FN may
be expressed by cells other than fibroblasts in the tumor
microenvironment, including tumor cells. The secreted EDB+ FN may
be then deposited in the matrix adjacent to tumor cells, or on the
plasma membrane of tumor cells. In other aspects, the invention
provides a pharmaceutical composition comprising one or more EDB
ADCs described herein for use in a method as described above. In
other aspects, the invention provides the use of one or more EDB
ADCs as described herein or a pharmaceutical composition comprising
the EDB ADCs as described herein in the manufacture of a medicament
for use in the methods described above.
[0163] Cancers associated with EDB+ FN expression and/or EDB+
FN-expressing cancers may generally include any cancer associated
with tissue remolding. Further, cancers associated with EDB+ FN
expression and/or EDB+ FN-expressing cancers may include, but are
not limited to, solid tumors and blood cancers. In some aspects,
solid tumors include, but are not limited to, thyroid cancer,
sarcoma, breast cancer, pancreatic cancer, glioblastoma,
gallbladder cancer, kidney cancer, skin cancer, uterine cancer,
mesothelioma, colorectal cancer, head and neck cancer, ovarian
cancer, bladder cancer, testicular cancer, prostate cancer, liver
cancer, endocrine cancer, thymus cancer, brain cancer, adrenal
cancer, eye cancer cervical cancer and lung cancer. In another
aspect, blood cancers include, but are not limited to, leukemia,
lymphoma and myeloma.
[0164] The EDB ADCs of the present invention are useful in treating
EDB+ FN-expressing disorders, such as cancers associated with EDB+
FN expression and/or EDB+ FN-expressing cancers. EDB ADCs of the
invention may be used to treat cancers that express high levels of
EDB+ FN, moderate levels of EDB+ FN or low levels of EDB+ FN.
[0165] Thus, patients to be treated with EDB ADCs of the invention
may be selected based on biomarker expression, including but not
limited to mRNA (qPCR) of bulk tumor samples and elevated
expression of EDB+ FN protein which results in a patient population
selected for enriched target expression rather than tumor origin or
histology. Target expression can be measured as a function of the
number of cells staining combined with the intensity of the cells
staining.
[0166] Cancer growth or abnormal proliferation refers to any one of
a number of indices that suggest change within cells to a more
developed cancer form or disorder state. Inhibition of growth of
cancer cells or cells of a non-neoplastic proliferative disorder
may be assayed by methods known in the art, such as delayed tumor
growth and inhibition of metastasis. Other indices for measuring
inhibition of cancer growth include a decrease in cancer cell
survival, a decrease in tumor volume or morphology (for example, as
determined using computed tomographic (CT), sonography, or other
imaging method), destruction of tumor vasculature, improved
performance in delayed hypersensitivity skin test, an increase in
the activity of cytolytic T-lymphocytes, and a decrease in levels
of tumor-specific antigens.
[0167] Desired outcomes of the disclosed therapeutic methods are
generally quantifiable measures as compared to a control or
baseline measurement. As used herein, relative terms such as
"improve," "increase," or "reduce" indicate values relative to a
control or comparative molecule, such as a measurement in the same
individual prior to initiation of treatment described herein, or a
measurement in a control individual (or multiple control
individuals) in the absence of the treatment described herein. A
representative control individual is an individual afflicted with
the same form of cancer as the individual being treated, who is
about the same age as the individual being treated (to ensure that
the stages of the disorder in the treated individual and the
control individual are comparable.
[0168] Changes or improvements in response to therapy are generally
statistically significant. As used herein, the term "significance"
or "significant" relates to a statistical analysis of the
probability that there is a non-random association between two or
more entities. To determine whether or not a relationship is
"significant" or has "significance," statistical manipulations of
the data can be "p-value." Those p-values that fall below a
user-defined cut-off point are regarded as significant. A p-value
less than or equal to 0.1, less than 0.05, less than 0.01, less
than 0.005, or less than 0.001 may be regarded as significant.
In Vivo Detection and Diagnosis
[0169] In another aspect, provided is a method of detecting,
diagnosing, and/or monitoring an EDB+ FN-expressing disorder, such
as cancers associated with EDB+FN expression and/or EDB+
FN-expressing cancers. For example, the anti-EDB antibodies as
described herein can be labeled with a detectable moiety such as an
imaging agent and an enzyme-substrate label. The antibodies as
described herein can also be used for in vivo diagnostic assays,
such as in vivo imaging (e.g., PET or SPECT), or a staining
reagent.
[0170] Following administration of an EDB ADC to a subject, wherein
the drug is a detectable label, and after a time sufficient for
binding, the biodistribution of EDB+FN protein bound by the
antibody may be visualized. The disclosed diagnostic methods may be
used in combination with treatment methods. In addition, EDB ADCs
of the invention may be administered for the dual purpose of
detection and therapy.
[0171] Representative non-invasive detection methods include
scintigraphy (e.g., SPECT (Single Photon Emission Computed
Tomography), PET (Positron Emission Tomography), gamma camera
imaging, and rectilinear scanning), magnetic resonance imaging
(e.g., convention magnetic resonance imaging, magnetization
transfer imaging (MTI), proton magnetic resonance spectroscopy
(MRS), diffusion-weighted imaging (DWI) and functional MR imaging
(fMRI)), and ultrasound.
Formulations
[0172] The present invention further provides pharmaceutical
compositions including any of the EDB ADCs disclosed herein and a
pharmaceutically acceptable carrier. Further, the compositions may
include more than one EDB ADC disclosed herein.
[0173] The composition used in the present invention may further
include pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 21st
Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
"Pharmaceutically acceptable salt" as used herein refers to
pharmaceutically acceptable organic or inorganic salts of a
molecule or macromolecule. Pharmaceutically acceptable excipients
are further described herein.
[0174] Various formulations of the EDB ADCs may be used for
administration, including but not limited to, formulations
comprising a pharmaceutically acceptable excipient.
Pharmaceutically acceptable excipients are known in the art, and
are relatively inert substances that facilitate administration of a
pharmacologically effective substance. For example, an excipient
can give form or consistency, or act as a diluent. Suitable
excipients include but are not limited to stabilizing agents,
wetting and emulsifying agents, salts for varying osmolarity,
encapsulating agents, buffers, and skin penetration enhancers.
Excipients as well as formulations for parenteral and nonparenteral
drug delivery are set forth in Remington, The Science and Practice
of Pharmacy 20th Ed. Mack Publishing, 2000.
[0175] In some aspects of the invention, these agents may be
formulated for administration by injection (e.g.,
intraperitoneally, intravenously, subcutaneously, intramuscularly,
etc.). Accordingly, these agents can be combined with
pharmaceutically acceptable vehicles such as saline, Ringer's
solution, dextrose solution, and the like. The particular dosage
regimen, i.e., dose, timing and repetition, will depend on the
particular individual and that individual's medical history.
[0176] Therapeutic formulations of EDB ADCs used in accordance with
the present invention may be prepared for storage by mixing an
antibody having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington, The Science and Practice of Pharmacy 21st Ed. Mack
Publishing, 2005), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and may include buffers such as phosphate, citrate, and
other organic acids; salts such as sodium chloride; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens, such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0177] Therapeutic EDB ADC compositions are generally placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle. The compositions according to the present
invention may be in unit dosage forms such as tablets, pills,
capsules, powders, granules, solutions or suspensions, or
suppositories, for oral, parenteral or rectal administration, or
administration by inhalation or insufflation.
[0178] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently include between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0179] Suitable emulsions may be prepared using commercially
available fat emulsions, such as INTRALIPID.TM., LIPOSYN.TM.,
INFONUTROL.TM., LIPOFUNDIN.TM. and LIPIPHYSAN.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can include
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0. The emulsion
compositions can be those prepared by mixing an EDB ADC with
INTRALIPID.TM. or the components thereof (soybean oil, egg
phospholipids, glycerol and water).
[0180] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
including an EDB antibody or an EDB ADC as described herein and
instructions for use in accordance with any of the methods of the
invention described herein. Generally, these instructions include a
description of administration of the EDB antibody or EDB ADC for
the above described diagnostic or therapeutic treatments.
[0181] The instructions relating to the use of an EBD antibody or
an EDB ADC as described herein generally include information as to
dosage, dosing schedule, and route of administration for the
intended treatment. The containers may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the invention are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
[0182] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an EDB antibody or EDB ADC. The
container may further include a second pharmaceutically active
agent.
[0183] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit includes a
container and a label or package insert(s) on or associated with
the container.
Dose and Administration
[0184] The present invention provides for EDB ADCs administered in
an effective dosage. The phrase "effective dosage" or "effective
amount" as used herein refers to an amount of an ADC, drug,
payload, compound or pharmaceutical composition necessary to
achieve any one or more beneficial or desired therapeutic results.
For prophylactic use, beneficial or desired results include
eliminating or reducing the risk, lessening the severity, or
delaying the outset of the disorder, including biochemical,
histological and/or behavioral symptoms of the disorder, its
complications and intermediate pathological phenotypes presenting
during development of the disorder. For therapeutic use, beneficial
or desired results include clinical results such as reducing
incidence or amelioration of one or more symptoms of various EDB+
FN-expressing disorders, such as cancer, decreasing the dose of
other medications required to treat the disorder, enhancing the
effect of another medication, and/or delaying the progression of
the EDB+ FN-expressing disorders of patients.
[0185] An effective dosage can be administered in one or more
administrations. An effective dosage of an ADC, drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0186] For example, when administered to a cancer-bearing subject,
an effective amount includes an amount sufficient to elicit
anti-cancer activity, including cancer cell cytolysis, inhibition
of cancer cell proliferation, induction of cancer cell apoptosis,
reduction of cancer cell antigens, delayed tumor growth, and/or
inhibition of metastasis. Tumor shrinkage is well accepted as a
clinical surrogate marker for efficacy. Another well accepted
marker for efficacy is progression-free survival.
[0187] The EDB ADCs of the present invention can be administered to
an individual via any suitable route. It should be understood by
persons skilled in the art that the examples described herein are
not intended to be limiting but to be illustrative of the
techniques available. Accordingly, in some aspects of the
invention, the EDB ADC is administered to an individual in accord
with known methods, such as intravenous administration, e.g., as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerebrospinal, intracranial,
transdermal, subcutaneous, intra-articular, sublingually,
intrasynovial, via insufflation, intrathecal, oral, inhalation or
topical routes. Administration can be systemic, e.g., intravenous
administration, or localized. Commercially available nebulizers for
liquid formulations, including jet nebulizers and ultrasonic
nebulizers are useful for administration. Liquid formulations can
be directly nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, the EDB ADC may be aerosolized using
a fluorocarbon formulation and a metered dose inhaler, or inhaled
as a lyophilized and milled powder.
[0188] In some aspects of the invention, the EDB ADCs are
administered via site-specific or targeted local delivery
techniques. Examples of site-specific or targeted local delivery
techniques include various implantable depot sources of an EDC ADC
or local delivery catheters, such as infusion catheters, indwelling
catheters, or needle catheters, synthetic grafts, adventitial
wraps, shunts and stents or other implantable devices, site
specific carriers, direct injection, or direct application.
[0189] For the purpose of the present invention, the appropriate
dosage of an EDB ADC may depend on the particular EDB ADC (or
compositions thereof) employed, the type and severity of symptoms
to be treated, whether the agent is administered for therapeutic
purposes, previous therapy, the patient's clinical history and
response to the agent, the patient's clearance rate for the
administered agent, and the discretion of the attending physician.
The clinician may administer an EDB ADC until a dosage is reached
that achieves the desired result and beyond. Dose and/or frequency
can vary over course of treatment, but may stay constant as well.
Empirical considerations, such as the half-life, generally will
contribute to the determination of the dosage. For example,
antibodies that are compatible with the human immune system, such
as humanized antibodies or fully human antibodies, may be used to
prolong half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of symptoms, e.g.,
tumor growth inhibition or delay, etc. Alternatively, sustained
continuous release formulations of EDB ADCs may be appropriate.
Various formulations and devices for achieving sustained release
are known in the art.
[0190] For the purpose of the present invention, a typical daily
dosage might range from about any of 3 .mu.g/kg to 30 .mu.g/kg to
300 .mu.g/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more,
depending on the factors mentioned above. For example, dosage of
about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and
about 25 mg/kg may be used. For repeated administrations over
several days or longer, depending on the disorder, the treatment is
sustained until a desired suppression of symptoms occurs or until
sufficient therapeutic levels are achieved, for example, to inhibit
or delay tumor growth/progression or metatstasis of cancer cells.
Exemplary dosing regimens may include administering increasing
doses (e.g., initial dose of 1 mg/kg and gradual increase to one or
more higher doses every week or longer time period). Other dosage
regimens may also be useful, depending on the pattern of
pharmacokinetic decay that the practitioner wishes to achieve. For
example, in some aspects of the invention, dosing from one to four
times a week is contemplated. In other aspects, dosing once a month
or once every other month or every three months is contemplated, as
well as weekly, bi-weekly and every three weeks. The progress of
this therapy may be easily monitored by conventional techniques and
assays. The dosing regimen (including the EDB ADC used) can vary
over time.
[0191] In some aspects of the invention, dosages for an EDB ADC may
be determined empirically in individuals who have been given one or
more administration(s) of an EDB ADC. Individuals may be given
incremental dosages of an EDB ADC. To assess efficacy, an indicator
of the disorder can be followed.
[0192] Administration of an EDB ADC in accordance with the method
in the present invention can be continuous or intermittent,
depending, for example, upon the recipient's physiological
disorder, whether the purpose of the administration is therapeutic
or prophylactic, and other factors known to skilled practitioners.
The administration of an EDB ADC may be essentially continuous over
a preselected period of time or may be in a series of spaced
doses.
Combination Therapies
[0193] In some aspects of the invention, the methods described
herein further include a step of treating a subject with an
additional form of therapy. In some aspects, the additional form of
therapy is an additional anti-cancer therapy including, but not
limited to, be used in chemotherapy, radiation, surgery, hormone
therapy, and/or additional immunotherapy.
[0194] The disclosed EDB ADCs may be administered as an initial
treatment, or for treatment of cancers that are unresponsive to
conventional therapies. In addition, the EDB ADCs may combination
with other therapies (e.g., surgical excision, radiation,
additional anti-cancer drugs, etc.) to thereby elicit additive or
potentiated therapeutic effects and/or reduce cytotoxicity of some
anti-cancer agents. EDB ADCs of the invention may be
co-administered or co-formulated with additional agents, or
formulated for consecutive administration with additional agents in
any order.
[0195] EDB ADCs of the invention may be used in combination with
other therapeutic agents including, but not limited to, therapeutic
antibodies, ADCs, immunomodulating agents, cytotoxic agents, and
cytostatic agents. Representative agents useful for combination
therapy also include any of the drugs described herein above as
useful for preparation of an EDB ADC under the subheading
"Drugs."
[0196] Therapeutic agents include, but are not limited to, the
administration of a chemotherapeutic agent, a vaccine, a CAR-T
cell-based therapy, radiotherapy, a cytokine therapy, a vaccine, a
bispecific antibody, an ADC, an inhibitor of other
immunosuppressive pathways, an inhibitors of angiogenesis, a T cell
activator, an inhibitor of a metabolic pathway, an mTOR inhibitor,
an inhibitor of an adenosine pathway, a tyrosine kinase inhibitor
including but not limited to inlyta, ALK inhibitors and sunitinib,
a BRAF inhibitor, an epigenetic modifier, an inhibitors or depletor
of Treg cells and/or of myeloid-derived suppressor cells, a JAK
inhibitor, a STAT inhibitor, a cyclin-dependent kinase inhibitor, a
biotherapeutic agent (including but not limited to antibodies to
VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD20,
CD40, CD-40L, CTLA-4, OX-40, 4-1BB, and ICOS), an immunogenic agent
(for example, attenuated cancerous cells, tumor antigens, antigen
presenting cells such as dendritic cells pulsed with tumor derived
antigen or nucleic adds, immune stimulating cytokines (for example,
IL-2, IFNa2, GM-CSF), and cells transfected with genes encoding
immune stimulating cytokines such as but not limited to
GM-CSF).
[0197] Further representative antibodies, which may be used alone
or as an ADC, include, but are not limited to, anti-5T4 antibodies
(e.g., A1, A2, and A3), anti-CD19 antibodies, anti-CD20 antibodies
(e.g., RITUXAN.RTM., ZEVALIN.RTM., BEXXAR.RTM.), anti-CD22
antibodies, anti-antibodies (e.g., MYLOTARG.RTM.), anti CD33
antibody-drug conjugates, anti-Lewis Y antibodies (e.g., Hu3S193,
Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g.,
HERCEPTIN.RTM. (trastuzumab), MDX-210, OMNITARG.RTM. (pertuzumab,
rhuMAb 2C4)), anti-CD52 antibodies (e.g., CAMPATH.RTM.), anti-EGFR
antibodies (e.g., ERBITUX.RTM. (cetuximab), ABX-EGF (panitumumab)),
anti-VEGF antibodies (e.g., AVASTIN.RTM. (bevacizumab)),
anti-DNA/histone complex antibodies (e.g., ch-TNT-1/b), anti-CEA
antibodies (e.g., CEA-Cide, YMB-1003) hLM609, anti-CD47 antibodies
(e.g., 6H9), anti-VEGFR2 (or kinase insert domain-containing
receptor, KDR) antibodies (e.g., IMC-1C11), anti-Ep-CAM antibodies
(e.g., ING-1), anti-FAP antibodies (e.g., sibrotuzumab), anti-DR4
antibodies (e.g., TRAIL-R), anti-progesterone receptor antibodies
(e.g., 2C5), anti-CA19.9 antibodies (e.g., GIVAREX.RTM.) and
anti-fibrin antibodies (e.g., MH-1).
[0198] Examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CBI-TMI), eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
the enediyne antibiotics (e.g. calicheamicin, especially
calicheamicin gamma) I and calicheamicin phiM, see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromomophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin, and deoxydoxorubicin), pegylated liposomal
doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin; anti-metabolites such as methotrexate and
5-fluorouracil (5-FU); folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2, 2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"),
cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel;
chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; vinorelbine; novantrone; teniposide; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0199] Also included are anti-hormonal agents that act to regulate
or inhibit hormone action on tumors such as anti-estrogens and
selective estrogen receptor modulators (SERMs), including, for
example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY1 17018, onapristone, and toremifene
(Fareston); aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate,
exemestane, formestane, fadrozole, vorozole, letrozole, and
anastrozole; and anti-androgens such as flutamide, nilutamide,
bicalutamide, leuprolide, and goserelin; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0200] In some aspects, EDB ADCs may be used in combination
crizotinib, palbociclib, gemcitabine, cyclophosphamide,
fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib,
sunitinib malate, tofacitinib, bevacizumab, rituximab, and
traztuzumab.
[0201] In one aspect, after treatment with EDB ADCs an increase in
tumor infiltrating lymphocytes, an increase in CD8/CD4 ratios, an
increase in F4/80+ macrophages, and/or an increase in
immunomodulatory proteins such as PDL1 and 41BB, or any combination
thereof, may occur. Thus, the combination of an EDB ADC and an
immune checkpoint inhibitor or IO agent, such as an anti-41BB
agonist and/or anti-PDL1 antagonist monoclonal antibody may be
effective. (See Example 12). Further, EDB ADCs of the invention
alone may have immunodulatory, and immune-oncology (IO) agent
enabling mechanisms, that maybe increased with combination
therapy.
[0202] In some aspects, an EDB ADC may be used in combination with
one or more other therapeutic agents targeting an immune checkpoint
modulator, including but not limited to, an agent (such as an
antibody) targeting PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC
(PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L,
TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD1 12,
LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1), OX40,
OX40L, GITR, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR3, TL1A, CD40,
CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2
(CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84), SLAMF6
(NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28,
CEACAM1 (CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7,
CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-1 1, CEACAM1-4C1,
CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosine pathway
(A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1,
or an innate immune response modulator.
[0203] For combination therapies, an EDB ADC and/or one or more
additional therapeutic agents are administered within any time
frame suitable for performance of the intended therapy. Thus, the
single agents may be administered substantially simultaneously
(i.e., as a single formulation or within minutes or hours) or
consecutively in any order. For example, single agent treatments
may be administered within about 1 year of each other, such as
within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or 1
week(s), or within about 5, 4, 3, 2 or 1 day(s).
[0204] The disclosed combination therapies may elicit a synergistic
therapeutic effect, i.e., an effect greater than the sum of their
individual effects or therapeutic outcomes. For example, a
synergistic therapeutic effect may be an effect of at least about
two-fold greater than the therapeutic effect elicited by a single
agent, or the sum of the therapeutic effects elicited by the single
agents of a given combination, or at least about five-fold greater,
or at least about ten-fold greater, or at least about twenty-fold
greater, or at least about fifty-fold greater, or at least about
one hundred-fold greater. A synergistic therapeutic effect may also
be observed as an increase in therapeutic effect of at least 10%
compared to the therapeutic effect elicited by a single agent, or
the sum of the therapeutic effects elicited by the single agents of
a given combination, or at least 20%, or at least 30%, or at least
40%, or at least 50%, or at least 60%, or at least 70%, or at least
80%, or at least 90%, or at least 100%, or more. A synergistic
effect is also an effect that permits reduced dosing of therapeutic
agents when they are used in combination.
EXAMPLES
[0205] The following examples of specific aspects for carrying out
the present invention are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way. Indeed, various modifications of the invention in addition
to those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
Example 1
Generation of Anti-EDB Antibodies and Preparation for
Conjugation
Generation of Anti-EDB Antibodies
[0206] The cDNA encoding various fully human antibodies that bind
to EDB were constructed using standard molecular biology
methodology and derived from the L19 human monoclonal antibody
which specifically binds to EDB (herein after "anti-EDB-L19" or
"EDB-L19" antibody). The EDB-L19 antibody comprises a human IgG1
constant region with G1m(a) allotype having aspartic acid (D) at
position 356 and leucine (L) at position 358 (according to the EU
index of Kabat) and a human Kappa light chain constant region. The
EDB-L19 antibody heavy and light chain variable regions are set
forth in SEQ ID NOS. 1 and 10, respectively, and the heavy and
light chains are set forth in SEQ ID NOS. 8 and 15,
respectively.
[0207] To produce a non-immunogenic antibody, a non-G1m(a) allotype
having glutamic acid (E) at position 356 and methionine (M) at
position 358 (according to the EU index of Kabat) was introduced
into the EDB-L19 heavy chain. To generate the heavy chain, the
nucleotide sequence encoding the EDB-L19 heavy chain variable
region was fused to the human IgG1 constant region cDNA with the
Glm.sup.z, non-(a), non-(x) allotype. In some aspects, the
antibodies were further altered to decrease the charge variant of
an antibody and increase homogeneity by eliminating the C-terminal
lysine (K) of the EDB-L19 antibody IgG1 constant region generating
EDB-PFE HC (SEQ ID NO: 17). The EDB-PFE antibody heavy and light
chains are set forth in SEQ ID NOS. 17 and 15, respectively.
[0208] As shown in Table 4, imaged capillary electrophoresis (iCE)
was performed using an iCE3 with Prince Autosampler to determine
the percent of charge variants for the antibody preparations. The
EDB-L19 antibody had a substantial increase in basic species and a
decrease of the main peak of antibody as a result of incomplete
C-terminal lysine processing during cell culture compared to the
EDB-PFE antibody.
TABLE-US-00005 TABLE 4 Percent (%) of charge variants for EDB
antibodies. EDB Antibody % Acidic % Main % Basic EDB-L19 Ab 19.60
49.44 30.96 (HC with C-terminal Lys) EDB-PFE Ab 23.16 71.92 4.92
(HC without C-terminal Lys)
Antibodies for Site-Specific Conjugation Via Engineered Cysteine
Residues
[0209] Methods for preparing anti-EDB antibodies for site-specific
conjugation to various linker-payloads through reactive engineered
cysteine residues were generally performed as described in PCT
International Publication No. WO2013/093809, which is incorporated
herein by reference in its entirety. One or more residues on either
the heavy chain, such as position K290 (according to the EU index
of Kabat, or the light chain, such as K183 (according to Kabat)
were altered to a cysteine (C) residue by site directed
mutagenesis.
[0210] In some aspects, position K290 (according to the EU index of
Kabat) in the human IgG1 heavy chain constant region of the EDB-PFE
antibody was substituted with a reactive cysteine (C) to enable
site-specific conjugation generating EDB-(K290C) HC (SEQ ID NO:
19). In other aspects, residue K183 (according to Kabat) in the
human Kappa light chain constant region was substituted to a
reactive cysteine (C) to enable site-specific conjugation
generating EDB-(.kappa.K1830) LC (SEQ ID NO: 31).
Antibodies for Site Specific Conjugation Via Engineered Glutamine
Residues
[0211] Anti-EDB antibodies were expressed having human IgG1
subtypes engineered with reactive glutamine residues, such as
glutamine-containing ("Q") tags, at various amino acid positions
for conjugation to various linker-payloads. Methods for preparing
anti-EDB antibodies for site-specific conjugation through reactive
glutamine residues were generally performed as described in PCT
International Publication WO2012/059882, which is incorporated
herein by reference in its entirety.
[0212] In some aspects, a H16-glutamine tag LLQG (SEQ ID NO: 40)
was engineered within the human IgG1-Fc region of the EDB-PFE
antibody to enable a DAR 2 transglutaminase mediated site-specific
conjugation. For example, in the EDB-PFE antibody heavy chain the
amino acids at positions E294-N297 (according to the EU index of
Kabat) were replaced with the H16-glutamine-containing tag LLQG
(SEQ ID NO: 40). In other aspects, the antibodies were further
altered to increase specificity of conjugation to the engineered
H16-glutamine-containing tag. The lysine (K) amino acid at position
222 (according to the EU index of Kabat) on the heavy chain was
substituted with an arginine (R) generating EDB-(H16-K222R) HC (SEQ
ID NO. 27). The K222R substitution provided an increase in
homogenous ADCs, improved intermolecular crosslinking between the
antibody and linker-payload, and/or significant decrease in
interchain crosslinking with the H16-glutamine-containing tag on
the C-terminus of the antibody light chain.
Potential Chemical Liabilities
[0213] Potential chemical liabilities, especially within CDRs, may
impact molecular heterogeneity and result in antigen binding a
putative protein glycation sites. Protein glycation is a
non-enzymatic glycosylation that can occur in recombinant
antibodies during cell culture and glycated proteins can undergo
further reactions to generate poorly characterized heterogeneous
products, collectively termed advanced glycation end products. To
mitigate potential glycation liability, position K94 (numbering of
Kabat) adjacent to CDR3 in the EDB-L19 heavy chain variable region
was mutated to an arginine (R) to generate EDB-(K94R) VH (SEQ ID
NO: 21) and was then fused to a human IgG1 constant region to
generate EDB-(K94R) HC (SEQ ID NO: 23). The K94R glycation mutation
was also introduced within the EDB-(K290C) and EDB-(H16-K222R)
heavy chains engineered for site-specific conjugation to generate
EDB-(K94R-K290C) HC (SEQ ID NO: 25) and EDB-(K94R-H16-K222R) HC
(SEQ ID NO: 29), respectively.
Example 2
Characterization of EDB Antibody Variant Binding Properties
Binding Affinity Analysis
[0214] Surface plasmon resonance (SPR) was used to characterize
binding kinetics of the anti-EDB antibody variants to recombinant
human, cynomolgus monkey and rat 7-EDB-89 (SEQ ID NO: 34, SEQ ID
NO: 35 and SEQ ID NO: 36, respectively) and to confirm that binding
properties of the anti-EDB antibodies having the K94R glycation
mutation were fully retained. Binding is detected by surface SPR of
laser light refracting from the surface. Analysis of the signal
kinetics on-rate (ka) and off-rate (kd), allows the discrimination
between non-specific and specific interactions.
[0215] An anti-human IgG antibody (GE Healthcare) was covalently
amine coupled onto all 4 flow cells of a CM5 carboxymethylated
dextran coated sensor chip to a density of about 10,000 resonance
units (RUs) following the manufacturer's protocol and then each
anti-EDB antibody variant was captured to a level of approximately
60-90 RUs. The running and sample buffer used was HBS-EP+ buffer
(0.01M HEPES, 0.15M NaCl, 3 mM EDTA, and 0.05% v/v surfactant P20
pH7.4). A 3-fold serial dilution series of 7-EDB-89 ranging in
concentration from 600 nM to 11.1 nM was injected over the surface
at a flow rate of 50 .mu.L/minute for a 60 second association and
120 second dissociation. The surface was then regenerated with a 30
second pulse of 3M MgCl.sub.2, a 30 second pulse of an ionic
regeneration buffer (0.46M KSCN, 1.83 M MgCl2, 0.92 M urea, and
1.83 M guanidine-HCl pH7.4) and then equilibrated with a 30 second
pulse of HBS-EP+ running buffer. All SPR assays were performed at
25.degree. C. with a data collection rate of 1 Hz using a
BIAcore.RTM. T200 instrument (GE Healthcare). The resulting
sensorgrams were double referenced (Myszka, D. G., J. Mol.
Recognit., 12:279-284, 1999) using both a control surface and
buffer injections. The rate constants were determined by fitting
the data to a 1:1 Langmuir model with BIAcore.RTM. T200 evaluation
software v2.0 and the equation K.sub.D=k.sub.d/k.sub.a. Each
experiment was run in duplicate and the average K.sub.D was
determined. As shown in Table 5, the EDB-L19 and EDB-(K94R)
antibodies exhibit comparable binding to human 7-EDB-89.
t1/2=half-life, Rmax=maximum response, RU=resonance units.
TABLE-US-00006 TABLE 5 Binding properties of EDB-L19 and EDB-(K94R)
antibodies. 7-EDB-89 Antibody ka (1/Ms) kd(1/s) t1/2 (s) Rmax (RU)
Chi2/Rmax K.sub.D (nM) Human (1) EDB-L19 5.03E+05 1.16E-01 5.97
61.7 0.24% 230 Human (2) EDB-L19 4.86E+05 1.13E-01 6.13 61.5 0.20%
232 231 .+-. 1.4 Avg .+-. SD Human (1) EDB-(K94R) 5.39E+05 1.24E-01
5.59 36.4 0.08% 230 Human (2) EDB-(K94R) 5.02E+05 1.13E-01 6.13
35.1 0.17% 226 228 .+-. 2.8 Avg .+-. SD
[0216] Further, the binding affinities of EDB-L19 and
EDB-(.kappa.K183C-K94R-K290C) antibodies to human, cynomolgus
monkey and rat 7-EDB-89 were determined. As show in Table 6, the
binding affinities of the EDB-L19 and EDB-(.kappa.K183C-K94R-K2900)
were antibodies were similar. As show in Table 7, the binding
affinities of EDB-(.kappa.K183C-K94R-K2900) antibody to human,
cynomolgus monkey and rat 7-EDB-89 were comparable confirming
cross-species reactivity was retained after engineering EDB-L19
antibody to enable site-specific conjugation and removal of
putative glycation site.
TABLE-US-00007 TABLE 6 Binding properties of anti-EDB antibodies
7-EDB-89. EDB-L19 antibody EDB-(.kappa.K183C-K94R-K290C) ka kd KD
ka kd KD 7-EDB-89 (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) Human
6.15E+05 9.75E-02 159 1.40E+06 3.12E-01 223 Monkey 5.60E+05
1.05E-01 188 ND ND ND Rat 5.08E+05 1.07E-01 210 ND ND ND
TABLE-US-00008 TABLE 7 Binding properties of
EDB-(.kappa.K183C-K94R-K290C) antibody to 7-EDB-89. 7-EDB-89
Antibody ka (1/Ms) kd(1/s) t1/2 (s) Rmax (RU) Chi2/Rmax K.sub.D
(nM) Human EDB- 3.42E+05 1.16E-01 6.0 67.0 0.20% 340.0
(.kappa.K183C- K94R-K290C) Cyno EDB- 3.30E+05 1.19E-01 5.8 62.6
0.29% 361.5 monkey (.kappa.K183C- K94R-K290C) Rat EDB- 2.98E+05
1.23E-01 5.7 62.1 0.31% 412.5 (.kappa.K183C- K94R-K290C)
Competitive Binding by ELISA
[0217] Binding properties of EDB-(K94R) and
EDB-(.kappa.K183C-K94R-K2900) antibodies were further evaluated
using a competition ELISA with biotinylated EDB-L19 to confirm
binding to EDB was fully maintained. Human 7-EDB-89 (SEQ ID NO: 34)
was immobilized (100 ng/well) onto a 96-well ELISA plate and 20
ng/mL biotinylated EDB-L19 antibody was added to compete with
varying concentrations of the modified anti-EDB antibody samples
and binding was detected using an anti-Streptavidin-HRP antibody
(Southern Biotech, Birmingham, Ala.).
[0218] As shown in FIG. 1A and Table 8, the EDB-L19 and EDB-(K94R)
antibodies had similar half maximal inhibition concentration
values. FIG. 1B and Table 9 show that the EDB-(K94R) and
EDB-(.kappa.K183C-K94R-K290C) antibodies also had similar half
maximal inhibition concentration values. This indicates that the
EDB-(K94R) and EDB-(.kappa.K183C-K94R-K290C modified antibodies
retained EDB binding properties and that the (K94R) modification of
the heavy chain and/or the introduction of reactive engineered
cysteines for site-specific conjugation did not alter binding to
EDB.
TABLE-US-00009 TABLE 8 Competition with bioEDB-L19 for binding to
human 7-EDB-89. Antibody IC50 [nM] EDB-L19 12.7 EDB-(K94R) 13.1
TABLE-US-00010 TABLE 9 Competition with bioEDB-L19 for binding to
human 7-EDB-89. Antibody IC.sub.50 [nM] EDB-(K94R) 19.7
EDB-(.kappa.K183C-K94R-K290C) 19.0
Avidity Analysis
[0219] The affinity for EDB-L19 antibody binding EDB was determined
to be a low binding interaction at .about.230 nM. Therefore, SPR
was used to investigate whether avidity impacted binding to
differential target levels within the tumor microenvironment.
Varying densities of human 7-EDB-89 (SEQ ID NO: 34) were covalently
amine coupled onto individual flow cells of a CM5 carboxymethylated
dextran coated sensor chip. The running and sample buffer was as
described above for the binding affinity analysis. A 3-fold serial
dilution series of EDB-L19 antibody ranging in concentration from 6
nM to 0.074 nM was injected at a flow rate of 50 .mu.L/minute for a
110 second association and 900 second dissociation. The surface was
then regenerated with two 30 second pulse of an ionic regeneration
buffer (0.46M KSCN, 1.83 M MgCl2, 0.92 M urea, and 1.83 M
guanidine-HCl pH7.4) and then equilibrated with a 30 second pulse
of HBS-EP+ running buffer. Each experiment was run in duplicate and
the average ka, kd and K.sub.D was determined.
[0220] As shown in Table 10, the results showed that as the level
of immobilized human 7-EDB-89 increased, the off-rates (kd) slowed
and subsequent affinities were increased. The apparent K.sub.D
values were proportional to the immobilization levels of human
7-EDB-89 and confirmed that EDB-L19 antibody binds EDB with a large
avidity component.
TABLE-US-00011 TABLE 10 Apparent K.sub.D Values of EDB Antibody
Binding EDB. Analyte Ligand ka (1/Ms) kd(1/s) t1/2 (min) Rmax (RU)
Chi2/Rmax KD (pM) EDB-L19 7-EDB-89 4.34E+06 2.06E-04 3364.1 194.8
4.77% 47.4 High: 650 RU EDB-L19 7-EDB-89 4.42E+06 1.72E-04 4029.1
187.7 0.73% 38.8 High: 650 RU AVG .+-. STD 4.38E+06 1.89E-04 43.1
.+-. 6.08 EDB-L19 7-EDB-89 4.46E+06 8.83E-04 784.8 38.9 2.20% 198
Med: 90 RU EDB-L19 7-EDB-89 2.71E+06 4.53E-04 1529.8 40.6 2.54% 167
Med: 90 RU AVG .+-. STD 3.59E+06 6.68E-04 182.5 .+-. 21.9 EDB-L19
7-EDB-89 2.02E+06 1.13E-03 613.3 27.5 9.49% 557 Low: 50 RU EDB-L19
7-EDB-89 2.81E+06 7.65E-04 905.9 23.6 2.06% 272 Low: 50 RU AVG .+-.
STD 2.42E+06 9.48E-04 414.5 .+-. 201.5
Polyreactivity of Anti-EDB Antibodies
[0221] Polyreactivity has been associated with rapid clearance in
vivo (Hotzel et al. mAbs 4(6):753-760, 2012) and undesirable
protein-protein interactions (Xu et al. Protein Eng Des Sel 26(10):
663-670 (2013). A DNA and Insulin direct binding ELISA has been
shown to correlate with known pharmacokinetics (PK) of clinically
validated antibodies. Serial dilutions of antibodies starting at 10
.mu.g/mL in quadruplicate were assessed in a low stringency assay
for binding to either DNA or Insulin that was directly coated onto
an ELISA plate.
[0222] As shown in Table 11, both the EDB-(K94R) and the
EDB-(.kappa.K183C-K94R-K290C) antibodies have very low
polyreactivity scores that are comparable or better than the
negative control which has optimal PK properties. Further, the
polyreactivity scores were significantly lower than the positive
control antibody having poor PK and resulting in rapid
clearance.
TABLE-US-00012 TABLE 11 Polyreactivity Scores of anti-EDB
antibodies. Polyreactivity Score Antibody DNA Insulin Negative
Control 4.805 5.027 Positive Control 15.741 12.171 EDB-(K94R) 0.429
2.725 EDB-(.kappa.K183C-K94R-K290C) 0.412 4.267
FcRn Chomatography
[0223] FcRn chromatography was utilized to investigate potential
charge-mediated influence of the introduction of reactive
engineered cysteines into a wild type IgG1 constant region on
FcRn-dependent pharmacokinetics. Evaluation of antibodies using
FcRn column methodology has demonstrated that the elution time
exhibited a positive correlation with human and non-human primate
clearance (Schoch A. et al. PNAS, 2015, Vol. 112). FcRn affinity
columns were prepared according to Schlothauer et al., MAbs 5(4):
576-586, 2013. Next, 50 .mu.g of EDB-(.kappa.K183C-K94R-K2900)
antibody or EDB-(.kappa.K183C-K94R-K2900)-vc-0101 ADC was injected
and then eluted by a linear pH gradient (30 CV) from pH 5.5-8.8
within 60 minutes using 20 mM MES, 150 mM NaCl, pH5.5 and 20 mM
Tris, 150 mM NaCl, pH 8.8 as eluents.
[0224] As shown in Table 12, the FcRn column relative elution time
of the EDB-(.kappa.K183C-K94R-K290C) antibody and
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 ADC were consistent with
acceptable PK parameters. These data demonstrate that the
incorporation of reactive engineered cysteine residue K290 into the
IgG1 constant region does not impact FcRn binding.
TABLE-US-00013 TABLE 12 FcRn column relative elution time. FcRn
Relative Elution time Peak Width at Antibody or ADC (min) 50%
Height EDB-(.kappa.K183C-K94R-K290C) 0.62 1.19
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 2.00 1.57
Example 3
Bioconjugation of EDB ADCs
[0225] Anti-EDB antibodies of the present invention were conjugated
to drugs/payloads via linkers to generate EDB ADCs. The conjugation
method used was either conventional conjugation (i.e. via random
cysteine residues) or site-specific conjugation (i.e., via
engineered cysteine residues or engineered glutamine residues).
Table 13 shows the conjugation methods used for various EDB
ADCs.
Method A: Conventional Conjugation Via Cysteine Residues
[0226] Anti-EDB antibody at 27 mg/ml in PBS, pH7.2 was reduced with
2.3 to 2.6 times (m/m) of TCEP at 37.degree. C. for 2 hours and
then conjugated. Molar ratio was generally at 2.5 times but
optimized depending on the amount of antibody conjugate to achieve
an optimal final average DAR of about 4.0. The partially reduced
antibody conjugated with 6 to 7 times (m/m) of linker-payload in
PBS with 10% DMA at 25.degree. C. for 1 hour. Excess linker-payload
was quenched with L-cysteine at 25.degree. C. for 15 minutes. The
crude ADC was dialyzed overnight in PBS at 4-6.degree. C.
[0227] The crude ADC was purified by size exclusion chromatography
(SEC) on Superdex 200 in PBS and collected monomer peak was either
stored at 4-6.degree. C. or dialyzed in 20 mM histidine, 8.5%
sucrose, pH 5.8; sterile filtered and frozen at -70.degree. C.
Negative control huNeg-8.8 antibody was conjugated by the same
method.
Method B: Site-Specific Conjugation Via Engineered Cysteine
Residues
[0228] Two grams anti-EDB antibody, generated with reactive
engineered cysteine residues, at 27.2 mg/ml in PBS, pH7.2 was
reduced with 15 times (m/m) of TCEP at 37.degree. C. for 7 hours
and desalted on Sephadex.RTM. G-25 in PBS to remove excess TCEP.
The inter-chain cysteines were oxidized with 30 times DHA (m/m) at
4-6.degree. C. overnight. DHA was removed by desalting on
Sephadex.RTM. G-25 in PBS. For ADC having a higher degree of
glutathione capping, instead of the preferred cysteine capping of
the site-specific cysteine, 100 times TCEP (m/m) was used for
reduction.
[0229] The reduced and oxidized antibody was conjugated with 9
times (m/m) of linker-payload in PBS with 10% DMA at 25.degree. C.
for 2 hours. Excess linker-payload was quenched with 9 times (m/m)
of L-cysteine at 25.degree. C. for 15 minutes. The crude ADC was
dialyzed overnight in PBS at 4-6.degree. C.
[0230] The crude ADC was purified by SEC on Superdex 200 in PBS and
collected monomer peak was dialyzed in 20 mM histidine, 8.5%
sucrose, pH 5.8; sterile filtered and frozen at -70.degree. C.
Negative control huNeg-8.8 antibody was conjugated by the same
method.
Method C: Site-Specific Conjugation Via Engineered Glutamine
Residues
[0231] Anti-EDB antibody, generated with reactive engineered
glutamine residues, was dialyzed in the reaction buffer; 100 mM
phosphate, 200 mM NaCl, pH 7.0. 20 mg/ml of antibody was conjugated
to linker-payload (10 times m/m) at room temperature for 15 hours,
using 1 unit of commercial purified transglutaminase (TG) per mg of
antibody, with mixing, in 100 mM potassium phosphate, 200 mM NaCl,
10% DMSO. The crude ADC was centrifuged and the supernatant was
purified by SEC.
[0232] The crude ADC was purified by SEC on Superdex 200 in PBS,
collected monomer peak was and dialyzed in 20 mM histidine, 8.5%
sucrose, pH 5.8; sterile filtered and frozen at -70.degree. C.
Negative control huNeg-8.8 antibody was conjugated by the same
method.
Method D: Conventional Conjugation Via Cysteine Residues Using
Disulfide Linkers
[0233] Anti-EDB antibody at 27 mg/ml in PBS, pH7.2 was partially
reduced using 5 times (m/m) of TCEP at 37.degree. C. for 2 hours
and desalted using a Sephadex.RTM. G-25 SEC.
[0234] The partially reduced antibody was conjugated with 12-15
times (m/m) of reduced linker-payload in 67 mM HEPES, pH7.0 with
0.7 mM DTPA and 7% DMA at 25.degree. C. for 15 minutes. Excess
linker-payload was quenched with 20 times NEM (m/m) at 25.degree.
C. for 15 minutes.
[0235] The crude ADC was purified by SEC on Superdex 200 in PBS
with 50 mM DHA and 50 mM DTPA, and collected monomer peak which was
stored at 4-6.degree. C. A negative control was conjugate by the
same method.
TABLE-US-00014 TABLE 13 Structures of various EDB ADCs (X
represents an antibody). ADC# ADC Structure Method ADC1 EDB-
L19-vc- 0101 ##STR00030## A ADC2 EDB- (.kappa.K183C- K290C)-
vc-0101 ##STR00031## B ADC3 EDB- (K94R)- vc-0101 ##STR00032## A
ADC4 EDB- (.kappa.K183C- K94R- K290C)- vc-0101 ##STR00033## B ADC5
EDB- L19-diS- DM1 ##STR00034## D ADC6 EDB- L19-diS- C.sub.2OCO-
1569 ##STR00035## D ADC7 EDB L19-vc- 9411 ##STR00036## A ADC8
EDB-diS- L19-4574 ##STR00037## D ADC9 EDB- (H16- K222R)- AcLys-
vc-CPI- 8314 ##STR00038## C ADC10 EDB- L19-vc- 1569 ##STR00039##
A
Example 4
Characterization of EDB ADCs
[0236] The EDB ADCs of the present invention were characterized
using a combination of size-exclusion chromatography (SEC), LC-MS
and hydrophobic interaction chromatography (HIC). The average
drug:antibody ratio (DAR) was determined by a mass spectrometry
(MS). Table 14 provides analytic characteristics of various EDB
ADCs.
[0237] LC-MS:
[0238] Column=Waters BEH300-C4, 2.1.times.100 mm (P/N=186004496);
Instrument=Acquity UPLC with an SQD2 mass spec detector; Flow
rate=0.7 mL/min; Temperature=80.degree. C.; Buffer A=water+0.1%
formic acid; Buffer B=acetonitrile+0.1% formic acid. The gradient
runs from 3% B to 95% B over 2 minutes, holds at 95% B for 0.75
min, and then re-equilibrates at 3% B. The sample is reduced with
TCEP or DTT immediately prior to injection. The eluate is monitored
by LCMS (400-2000 daltons) and the protein peak is deconvoluted
using MaxEnt1. DAR is reported as a weight average loading as has
been previously described.
[0239] SEC:
[0240] Column: Superdex200 (5/150 GL); Mobile phase: Phosphate
buffered saline containing 2% acetonitrile, pH 7.4; Flow rate=0.25
mL/min; Temperature=ambient; Instrument: Agilent 1100 HPLC.
[0241] HIC:
[0242] Column: TSKGel Butyl NPR, 4.6 mm.times.3.5 cm
(P/N=S0557-835); Buffer A=1.5 M ammonium sulfate containing 10 mM
phosphate, pH 7; Buffer B=10 mM phosphate, pH 7+20% isopropyl
alcohol; Flow rate=0.8 mL/min; Temperature=ambient; Gradient=0% B
to 100% B over 12 minutes, hold at 100% B for 2 minutes, then
re-equilibrate at 100% A; Instrument: Agilent 1100 HPLC.
TABLE-US-00015 TABLE 14 Analytical characteristics of EDB ADCs.
Isolated HPLC-HIC Observed DAR DAR yield retention .DELTA. mass for
(LC/MS (HIC ADC# ADC (%) time HC Method) Method) ADC1
EDB-L19-vc-0101 64 8.7 1342 3.4 3.4 ADC2 EDB-(.kappa.K183C- 63 8.9
1341 3.8 4.0 K290C)-vc-0101 ADC3 EDB-(K94R)-vc- 65 8.6 1342 3.7 4.1
0101 ADC4 EDB-(.kappa.K183C- 71 8.9 1341 3.8 3.9 K94R-K290C)-vc-
0101 ADC5 EDB-L19-diS-DM1 80 8.9 738 4.4 4.9 ADC6 EDB-L19-diS- 84
6.4 889.6 4.4 4.7 C.sub.2OCO-1569 ADC7 EDB-L19-vc-9411 80 8.3 1397
5.3 4.9 ADC8 EDB-L19-diS-4574 75 6.5 816 5.2 5.2 ADC9
EDB-(H16-K222R)- 78 5.2 1343 2.0 1.7 AcLys-vc-CPI-8314 ADC10
EDB-L19-vc-1569 82 7.4 1385 4.3 4.2
Example 5
EDB+ FN Expression
[0243] To conduct a broad investigation of cancer indications for
EDB ADC based therapy, EDB+ FN expression was analyzed at the
protein and mRNA level in human tumors and PDX models.
RNA-Seq Analysis of EDB+ FN Expression
[0244] RNA-Seq data was analyzed from 10660 individual tumor
samples collected as part of The Cancer Genome Atlas (TOGA) project
(National Cancer Institute at HIH, Bethesda, Md.) expanding 31
tumor types. The isoform level expression data were obtained from
OmicSoft software (Cary, N.C.). EDB+ FN expression was calculated
as the summation of expression levels of the isoforms of
fibronectin (FN1) which harbor EDB. The expression levels were
measured by fragment per kilobase of transcript per million reads
(FPKM) and the summary statistics of EDB+ FN expression levels for
each tumor type is shown in Table 15. Generally, the gene is
considered expressed if the FPKM is about 1 or higher.
[0245] Table 15 shows the RNA-Seq analysis of EDB+ FN in human
tumors. EDB+FN expression is demonstrated in a broad range of human
tumor indications, including but not limited to, thyroid carcinoma,
sarcoma, breast carcinoma, pancreatic adenocarcinoma, glioblastoma,
cholangiocarcinoma, lung adenocarcinoma, renal carcinoma, melanoma,
uterine carcinosarcoma, mesothelioma, lung squamous cell carcinoma,
rectum and colon adenocarcinoma, liver hepatocellular carcinoma,
colon carcinoma, ovariam serous cystadenocarcinoma, and bladder
carcinoma.
TABLE-US-00016 TABLE 15 RNA-Seq analysis of EDB+ FN in TCGA
samples. Medium Value Upper Lower Maximum Tumor type/disease (FPKM)
Quantile Quantile Value Thyroid carcinoma 216.69 628.03 14.20
3541.43 Sarcoma 96.86 226.41 22.20 1450.21 Breast invasive
carcinoma 36.92 77.75 14.77 1062.56 Pancreatic adenocarcinoma 35.08
67.03 14.02 549.68 Glioblastoma multiforme 28.74 56.74 11.76
1171.19 Cholangiocarcinoma 27.77 55.99 10.34 458.05 Lung
adenocarcinoma 23.31 49.16 10.85 1105.41 Kidney renal clear cell
22.91 39.34 11.49 346.83 carcinoma Skin Cutaneous Melanoma 22.13
54.48 7.89 2131.95 Uterine Carcinosarcoma 21.08 56.70 7.28 185.43
Mesothelioma 20.13 52.36 3.64 205.08 Lung squamous cell 19.13 42.86
8.75 1004.73 carcinoma Rectum adenocarcinoma 15.69 33.20 6.02
221.89 Liver hepatocellular 13.29 37.22 4.32 472.23 carcinoma Colon
adenocarcinoma 12.24 27.77 3.87 275.90 Head and Neck squamous 11.37
32.52 3.47 1111.18 cell carcinoma Ovarian serous 11.25 27.51 5.00
425.09 cystadenocarcinoma Bladder Urothelial 10.03 29.64 2.28
467.12 Carcinoma Testicular Germ Cell 8.68 72.28 2.94 1395.34
Tumors Prostate adenocarcinoma 5.92 11.03 2.74 648.96 Kidney
Chromophobe 5.21 8.07 1.71 1788.92 Pheochromocytoma and 4.95 12.24
2.06 118.84 Paraganglioma Thymoma 3.58 27.15 0.30 1173.35 Brain
Lower Grade Glioma 3.19 6.94 1.53 163.17 Adrenocortical carcinoma
2.40 4.87 0.52 112.69 Uterine Corpus Endometrial 2.20 7.36 0.49
172.08 Carcinoma Uveal Melanoma 1.81 3.72 0.89 16.59 Cervical
squamous cell 1.79 6.34 0.56 258.49 carcinoma & endocervical
adenocarcinoma Kidney renal papillary cell 1.76 6.80 0.64 2291.27
carcinoma Lymphoid Neoplasm 0.34 1.11 0.12 51.63 Diffuse Large
B-cell Lymphoma Acute Myeloid Leukemia 0.00 0.13 0.00 5.84
[0246] Gene expression quantification was performed on the RNA-Seq
data of 160 Pfizer internal patient derived xenograft (PDX) models
from breast cancer, ovarian cancer, head & neck cancer,
colorectal cancer, melanoma, pancreatic, non-small cell lung cancer
(NSCLC) and small cell lung cancer using RSEM program. See Li et
al., BMC Bioinformatics, 12:323, 2011. EDB+ FN expression was
calculated as the summation of expression levels of the isoforms of
fibronectin (FN1) which harbor EDB. As shown in FIG. 2, EDB+ FN was
expressed at varying levels (all samples had levels>1) across
all tumor types analyzed. Data represented as fragment per kilobase
of transcript per million reads (FPKM).
Immunohistochemistry (IHC) Detection of EDB+ FN Expression
[0247] EDB+ FN protein expression in human cancer was validated by
IHC using EDB-L19 antibody in frozen sections. Eight micron fresh
frozen tissue sections that were embedded in Tissue-Tek O.C.T.
Compound (Sakura Finetek) were fixed for 4 minutes in a 3:1 mixture
of acetone to 100% ethanol and then dipped in 10% neutral buffered
formalin for 20 seconds. Slides were rinsed in TBS. Endogenous
peroxidase activity was inactivated with Peroxidazed 1 (Biocare
Medical) for 10 minutes. Non-specific protein interactions were
blocked for 10 minutes with Background Punisher (Biocare Medical).
EDB-L19 antibody or isotype negative control huNeg-8.8 antibody was
pre-complexed with rabbit anti-human IgG (Jackson ImmunoResearch)
at a final concentration of 3 .mu.g/ml and 0.5 .mu.g/ml
respectively, for 1 hour at room temperature. The pre-complexed
mixture was incubated with excess whole human IgG (Jackson
ImmunoResearch) for 15 minutes at room temperature and was added to
the slides for 1 hour. Sections were washed in TBS and incubated
with SignalStain Boost Rabbit HRP (CellSignaling Technologies) for
30 minutes. Chromogenic signal was developed with DAB+ (Dako) for 5
minutes, and subsequently quenched with distilled H.sub.20. Slides
were briefly counterstained with CAT Hematoxylin (Biocare Medical),
washed in water, dehydrated in graded alcohols, cleared in xylene,
and coverslipped with Permount Mounting Medium (FisherChemicals).
Analysis of expression was performed and confirmed.
[0248] As shown in Table 16, EDB+ FN protein was expressed at
moderate to high levels across the all human cancer indications
profiled, including head and neck carcinoma (data not shown),
pancreatic carcinoma, non-small cell lung carcinoma (NSCLC),
ovarian carcinoma and breast carcinoma. Expression in all tumors
was dominantly stromal (including fibroblastic and that associated
with the vasculature), though some staining of tumor cells was also
observed.
TABLE-US-00017 TABLE 16 EDB+ FN protein expression in human cancer
assessed by IHC assay. % samples with EDB+ FN Tumor # patient
stromal positivity type samples Negative/Low Moderate/High
Pancreatic 20 30 70 Lung 15 0 100 Breast 12 8 92 Ovarian 10 0
100
Example 6
In Vitro Binding of EDB ADCs
[0249] To assess the relative binding of anti-EDB antibodies and
EDB ADCs to EDB, MaxiSorp 96-well plates were coated with 0.5 or 1
.mu.g/ml of human 7-EDB-89 (SEQ ID NO: 34) in PBS and incubated
overnight at 4.degree. C. with gentle shaking. Plates were then
emptied, washed with 200 .mu.l PBS and blocked with 100 .mu.l of
Blocking Buffer (ThermoScientific) for 3 hours at room temperature.
Blocking buffer was removed, wells were washed with PBS and
incubated with 100 .mu.l of anti-EDB antibodies or EDB ADCs which
were serially diluted (4-fold) in ELISA Assay Buffer (EAB, 0.5%
BSA/0.02% Tween-20/PBS). The first column of the plate was left
empty and the last column of the plate was filled with EAB as blank
controls. The plate was incubated at room temperature for 3 hours.
Reagents were removed and plate washed with 200 .mu.l of 0.03%
Tween-20 in PBS (PBST). Anti-human IgG-Fc-HRP (Thermo/Pierce)
diluted 1:5000 in EAB was added as 100 .mu.l to the wells and
incubated for 15 minutes at room temperature. The plate was washed
with 200 .mu.l of PBST, then 100 .mu.l of BioFX TMB (Fisher) was
added and the color allowed to develop for 4 minutes at room
temperature. The reaction was stopped with 100 .mu.l of 0.2 N
sulfuric acid and absorbance at 450 nm was read on a Victor plate
reader (Perkin Elmer, Waltham, Mass.).
[0250] Table 17 provides the relative binding of anti-EDB
antibodies and EDB ADCs to human 7-EDB-89 protein fragment bound to
a 96-well plate in ELISA format. All antibodies and ADCs targeting
EDB bound to the target protein with similar affinity in the range
of 19 pM to 58 pM. In contrast, non-EDB targeting antibodies and
ADCs have high EC.sub.50 values>10,000 pM. Representative ELISA
binding curves are illustrated in FIGS. 3A and 3B.
TABLE-US-00018 TABLE 17 Anti-EDB antibody and ADC binding to human
EDB. ADC or Antibody Avg EC.sub.50 # ADC or Antibody Name (pM) SD
Ab1 EDB-L19 27.0 -- ADC1 EDB-L19-vc-0101 37.8 12.8 ADC11
Neg-vc-0101 >10,000 -- Ab2 EDB-(.kappa.K183C-K290C) 30.2 1.6
ADC2 EDB-(.kappa.K183C-K290C)-vc-0101 58.4 17.0 ADC12
Neg-(.kappa.K183C-K290C)-vc-0101 >10,000 ND Ab3 EDB-(K94R) 15.0
-- ADC3 EDB-(K94R)-vc-0101 37.1 14.6 Ab4
EDB-(.kappa.K183C-K94R-K290C) 44.8 8.7 ADC4
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 56.7 13.5 ADC5
EDB-L19-diS-DM1 21.3 -- ADC6 EDB-L19-diS-C.sub.2OCO-1569 30.7 --
ADC7 EDB-L19-vc-9411 37.5 -- ADC15 Neg-vc-9411 >10,000 -- ADC8
EDB-L19-diS-4574 31.9 -- Ab5 EDB-(H16-K222R) 19.3 -- ADC9
EDB-(H16-K222R)-AcLys-vc-CPI-8314 39.4 2.5 ADC17
Neg-(H16-K222R)-AcLys-vc-CPI-8314 >10,000 -- Mean EC.sub.50 .+-.
standard deviation and number (n) of determinations. ND = not
determined.
Example 7
In Vitro Cytotoxicity of EDB ADCs
Cell Culture
[0251] WI38-VA13 are SV40-transformed human lung fibroblasts
obtained from ATCC and maintained in MEM Eagles media (Cell-Gro),
supplemented with 10% FBS, 1% MEM non-essential amino acids, 1%
sodium pyruvate, 100 units/ml penicillin-streptomycin, and 2 mM
GlutaMax. HT29 are derived from human colorectal carcinoma (ATCC)
and maintained in DMEM media supplemented with 10% FBS and 1%
glutamine.
EDB+ FN Transcript Detection
[0252] For gene expression and transcript analysis of EDB+ FN,
adherent proliferating WI38-VA13 and HT29 cells were dissociated
from cell-culture flasks with TrypLE Express (Gibco). The RNeasy
Mini Kit (Qiagen) was used to purify total RNA from the collected
cell pellets. The residual DNA was removed by RNase-Free DNase Set
(Qiagen) during RNA purification. High Capacity RNA-to-cDNA Kit
(Applied Biosystems) was used for reverse transcription of total
RNA to cDNA. The cDNA was analyzed by quantitative real-time PCR
using TaqMan Universal Master Mix II, with UNG (Applied
Biosystems). EDB+ FN signal was detected by TaqMan primer
Hs01565271_m1 and normalized with the average of both signals from
ACTB (TaqMan primer Hs99999903_m1) and GAPDH (TaqMan primer
Hs99999905_m1). All primers were from ThermoFisher Scientific. Data
from a representative experiment is shown.
EDB+ FN Protein Detection by Western Blotting
[0253] For detection of EDB+ FN by western blotting, adherent
proliferating WI38-VA13 and HT29 cells were harvested by cell
scraping. Cell lysates were prepared in Cell Lysis Buffer (Cell
Signaling Technology) with protease inhibitors and phosphatase
inhibitors. Tumor lysate was prepared in either RIPA Lysis Buffer
or 2.times. Cell Lysis Buffer (Cell Signaling Technology) with
protease inhibitors and phosphatase inhibitors. Protein lysates
were analyzed by SDS-PAGE and followed by western blotting.
Proteins were transferred to nitrocellulose membrane and then
blocked with 5% milk/TBS, followed by incubation with EDB-L19
antibody and anti-GAPDH antibody (Cell Signaling Technology)
overnight at 4.degree. C. After washing, the anti-EDB blot was
incubated with ECL HRP-linked anti-human IgG secondary antibody (GE
Healthcare) for 1 hour at room temperature. After washing, the
EDB+FN signal was developed by Pierce ECL 2 Western Blotting
Substrate (Thermo Scientific) and detected by X-ray films. The
anti-GAPDH blot was incubated with Alexa Fluor 680 conjugated
anti-rabbit IgG secondary antibody (Invitrogen) in blocking buffer
for 1 hour at room temperature. After washing, the GAPDH signal was
detected by LI-COR Odyssey Imaging System. Densitometric analysis
of EDB+FN western blots was conducted using the Bio-Rad GS-800
Calibrated Imaging Densitometer and quantified using Quantity One
version 4.6.9 software. Data from a representative experiment is
shown.
[0254] FIG. 4 shows EDB+ FN expression by western blot in WI38-VA13
and HT29 cells. EDB+ FN is expressed in the WI38-VA13 cell line and
the HT29 colon carcinoma cell line is negative when grown in
vitro.
EDB+ FN Protein Detection by Flow Cytometry
[0255] EDB-L19 antibody was used to measure the expression of EDB+
FN on the cell surface of WI38-VA13 or HT29 cells by flow
cytometry. Cells were dissociated by non-enzymatic cell
dissociation buffer (Gibco) and incubated with cold flow buffer
(FB, 3% BSA/PBS+Ca+Mg) on ice for blocking. Cells were then
incubated with primary antibodies on ice in FB. After the
incubation, cells were washed with cold PBS--Ca--Mg and then
incubated with viability stain (Biosciences) to discriminate live
and dead cells, according to the manufacture's procedure. The
signals were analyzed on a BD Fortessa flow cytometer and data were
analyzed using BD FACS DIVA software. Data from a representative
experiment is shown.
[0256] Table 18 summarizes the results from western blot, qRT-PCR
and flow cytometry. The data demonstrates that WI38-VA13 is EDB+ FN
positive and HT29 is EDB+ FN negative.
TABLE-US-00019 TABLE 18 Characterization of EDB+ FN expression in
WI38 VA13 and HT29 cells Western Flow cytometric binding qRT-PCR
(normalized (MFI-GeoMean Cell Line (2.sup.(-ddC(t)) density
(OD/mm2)) (EDB+ FN unstained)) WI38-VA13 0.224247 475.397 4480 HT29
0.000049 0.093 2
In Vitro Cytotoxicity Assays
[0257] Proliferating WI38-VA13 or HT29 cells were harvested from
culture flasks with non-enzymatic cell dissociation buffer and
cultured overnight in 96-well plates (Corning) at 1000 cells/well
in a humidified chamber (37.degree. C., 5% CO2). The next day,
cells were treated with EDB ADCs or isotype control non-EDB-binding
ADCs by adding 50 .mu.l of 3.times. stocks in duplicate at 10
concentrations. In some experiments, cells were plated at 1500
cells/well and treated the same day. Cells were then incubated with
EDB ADCs or isotype control non-EDB-binding ADCs for four days. On
harvest day, 50 .mu.l of Cell Titer Glo (Promega) was added to the
cells and incubated 0.5 hours at room temperature. Luminescence was
measured on a Victor plate reader (Perkin Elmer, Waltham, Mass.).
Relative cell viability was determined as a percentage of untreated
control wells. IC.sub.50 values were calculated using
four-parameter logistic model #203 with XLfit v4.2 (IDBS).
[0258] Table 19 shows the IC.sub.50 (ng/ml of antibody) of the EDB
ADC treatments in cytotoxicity assays performed on WI38-VA13 (EDB+
FN positive tumor cell line) and HT29 colon carcinoma cells (EDB+
FN negative tumor cell line). The EDB ADCs induced cell death in
the EDB+ FN expressing cell line. The IC.sub.50 values were similar
for all EDB ADCs having vc-0101 linker-payload, in the range of
approximately 184 ng/ml to 216 ng/ml (EDB-L19-vc-0101,
EDB-(.kappa.K183C-K290C)-vc-0101, EDB-(K94R)-vc-0101,
EDB-(.kappa.K183C-K94R-K290C)-vc-0101). The negative control
vc-0101 ADCs were substantially less potent, with IC.sub.50 values
approximately 70- to 200-fold higher than EDB-vc-0101 ADCs. All
vc-0101 ADCs had 46- to 83-fold higher IC.sub.50 values in the EDB+
FN negative tumor cell line, HT29. Therefore, EDB ADCs were
dependent on EDB+ FN expression for their in vitro
cytotoxicity.
[0259] Other auristatin-based EDB ADCs with "vc" protease-cleavable
linkers, EDB-L19-vc-9411and EDB-L19-vc-1569, also showed potent
cytotoxicity in WA38-VA13 cells with high selectivity of about 50-
to 180-fold compared with the corresponding negative control ADCs
and selectivity of about 25- to 140-fold compared with the
non-expressing cell line. The EDB-L19-diS-DM1 ADC had similar
potency as the vc-0101 ADCs, however much lower selectivity
compared with the negative control ADC (about 3-fold) and with HT29
cells (about 0.9-fold).
TABLE-US-00020 TABLE 19 In vitro cytotoxicity of EDB ADCs and
control non-EDB-binding ADCs. WI38-VA13 HT29 ADC # ADC Name Avg
IC.sub.50 SD n Avg IC.sub.50 SD n ADC1 EDB-L19-vc-0101 184 143 23
15,346 4448 5 ADC11 Neg-vc-0101 19,585 6762 16 10,731 8193 24 ADC2
EDB-(.kappa.K183C-K290C)-vc-0101 198 176 6 9,276 83 2 ADC12
Neg-(.kappa.K183C-K290C)-vc-0101 >40,000 ND 4 21,913 2635 2 ADC3
EDB-(K94R)-vc-0101 184 138 7 10,577 2065 2 ADC4
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 216 94 6 15,584 58 3 ADC5
EDB-L19-diS-DM1 268 150 8 237 180 2 ADC13 Neg-diS-DM1 879 82 5 ND
ND ND ADC6 EDB-L19-diS-C2OCO-1569 21 8 6 5 3 2 ADC14
Neg-diS-C2OCO-1569 36 6 3 ND ND ND ADC7 EDB-L19-vc-9411 46 22 3
1,153 -- 1 ADC15 Neg-vc-9411 2,514 260 3 1,243 -- 1 ADC8
EDB-L19-diS-4574 487 406 4 429 228 2 ADC16 Neg-diS-4574 1,279 -- 1
ND ND ND ADC9 EDB-(H16-K222R)-AcLys-vc-CPI-8314 34 30 5 3,449 -- 1
ADC17 Neg-AcLys-vc-CPI-8314 2,656 876 3 15,110 15,408 2 ADC10
EDB-L19-vc-1569 40 11 2 5,702 -- 1 ADC18 Neg-vc-1569 7283 -- 1 ND
ND ND Mean IC.sub.50 .+-. standard deviation and number (n) of
determinations. ND = not determined.
[0260] As shown in Table 20, the unconjugated payloads were highly
potent in both cell lines, independent of EDB+ FN expression,
indicating that these cells are sensitive to the cytotoxic agents
used as ADC payloads.
TABLE-US-00021 TABLE 20 In vitro cytotoxicity potency of various
unconjugated compounds. WI38-VA13 HT29 Avg IC.sub.50 Avg IC.sub.50
Payload Name (nM) SD n (nM) SD n Payload-1569 0.269 0.134 7 0.074
0.080 3 Payload-DM1 3.06 2.77 5 2.63 2.30 8 Payload-0101 0.392
0.326 12 0.090 0.043 14 Payload-0326 <0.001 ND 2 0.049 0.028 2
Payload-4574 3.54 1.14 2 3.65 1.89 2 Payload-9411 0.519 0.144 2
0.197 0.177 3 Payload-Cemadotin 24.2 2.14 2 43.8 -- 1 Mean
IC.sub.50 .+-. standard deviation and number (n) of determinations.
ND = not determined.
Example 8
In Vivo Efficacy of EDB ADCs
[0261] EDB ADCs were evaluated in cell line xenograft (CLX),
patient derived xenograft (PDX) and syngeneic tumor models.
Expression of EDB+ FN was detected using an immunohistochemical
(IHC) assay as previously described herein.
[0262] To generate CLX models, 8.times.10.sup.6 to
10.times.10.sup.6 cells of H-1975, HT29, or Ramos tumor lines were
implanted into female athymic nude mice subcutaneously. Ramos and
H-1975 cells for inoculation were suspended in 50% and 100%
Matrigel (BD Biosciences), respectively. For the Ramos model, the
animals received whole body irradiation (4 Gy) before cell
inoculation to facilitate the establishment of tumors. When the
average tumor volume reached approximately 160 to 320 mm.sup.3, the
animals were randomized into treatment groups, with 8-10 mice in
each group, ADCs or vehicle (PBS) were administered intravenously
on day 0 and then the animals were dosed once every 4 days for 4 to
8 doses. Tumors were measured once or twice weekly and tumor volume
was calculated as volume
(mm.sup.3)=(width.times.width.times.length)/2. The body weight of
animals was monitored for 4 to 9 weeks and no animal weight loss
was observed in any treatment groups.
[0263] To generate PDX models, tumors were collected from donor
animals and tumor fragments approximately 3.times.3 mm were
implanted subcutaneously into the flank of female athymic nude mice
(for PDX-NSX-11122 model) or NOD SCID mice (for PDX-PAX-13565 and
PDX-PAX-12534 models) by using a 10 gage trocar. When average tumor
volume reached approximately 160 to 260 mm.sup.3 the mice were
randomized into treatment groups, with 7-10 mice in each group.
ADCs or vehicle (PBS) dosing regime and administration route as
well as tumor measurement procedures are the same as described
above for CLX models. The body weight of animals was monitored for
5 to 14 weeks and no animal weight loss was observed in any
treatment groups. Tumor growth inhibition is plotted as an average
of tumor size.+-.SEM.
Expression of EDB+ FN
[0264] As shown in Table 21, expression of EDB+ FN in the H-1975,
HT29 and Ramos CLX models, PDX-NSX-11122, PDX-PAX-13565 and
PDX-PAX-12534 PDX models and EMT-6 syngeneic syngeneic tumor models
was measured by binding of EDB-L19 antibody and subsequent
detection in IHC assay. The CLX HT-29 was a moderate expressing CLX
however was negative when examined in vitro due to the predominance
of protein expression in the CLX being derived from the tumor
stroma.
TABLE-US-00022 TABLE 21 Expression of EDB+ FN EDB+ FN Overall
Efficacy Model Tumor Type Expression PDX-NSX-11122 NSCLC PDX High
EMT-6 Syngeneic mouse mammary High carcinoma (breast) PDX-PAX-13565
Pancreatic adenocarcinoma PDX Moderate/High H-1975 NSCLC CLX
Moderate/High HT29 Colorectal cancer CLX Moderate Ramos Burkitt's
lymphoma CLX Moderate PDX-PAX-12534 Pancreatic adenocarcinoma PDX
Low/Moderate
PDX-NSX-11122 NSCLC PDX
[0265] The effects of various ADCs were evaluated in PDX-NSX-11122,
a NSCLC PDX model of human cancer that expresses high levels of
EDB+ FN. FIG. 5A shows the anti-tumor activity for EDB-L19-vc-0101
at 0.3, 0.75, 1.5 and 3 mg/kg. The data demonstrates that
EDB-L19-vc-0101 showed tumor regression in a dose dependent manner
at 3 mg/kg and 1.5 mg/kg.
[0266] Anti-tumor efficacy of vc-linked ADCs was compared to
disulfide-linked ADCs. FIGS. 5B and 5C show the anti-tumor activity
of EDB-L19-vc-0101 at 3 mg/kg as compared to 10 mg/kg of disulfide
linked EDB-L19-diS-DM1 and EDB-L19-vc-0101 at 1 and 3 mg/kg as
compared to 5 mg/kg of disulfide linked
EDB-L19-diS-C.sub.2OCO-1569, respectively. As shown in FIGS. 5B and
5C, EDB-L19-vc-0101 demonstrated greater efficacy as compared to
isotype negative control ADCs and ADCs that were generated using a
disulfide linker, EDB-L19-diS-DM1 and EDB-L19-dis-C.sub.2OCO-1569.
Further, animals bearing tumors that were treated with
EDB-L19-vc-0101 had delayed tumor growth at 1 mg/kg and complete
regressions at 3 mg/kg. The data demonstrates that EDB-L19-vc-0101
(ADC1) inhibits growth of PDX-NSX-11122 NSCLC xenografts in a
dose-dependent manner.
[0267] The activity of site-specific and conventionally conjugated
ADCs was evaluated. FIG. 5D shows the anti-tumor efficacy of the
site-specific conjugated EDB-(.kappa.K183C+K290C)-vc-0101 compared
to the conventionally conjugated EDB-L19-vc-0101 at the doses of
0.3, 1 and 3 mg/kg and 1.5 mg/kg, respectively. The dose-level
based efficacy was comparable and the
EDB-(.kappa.K183C+K2900)-vc-0101 led to tumor regression in a dose
dependent manner.
[0268] The activity of vc-0101 EDB ADCs having various mutations
was assessed. FIG. 5E shows the anti-tumor efficacy of
site-specific conjugated EDB-(.kappa.K183C-K94R-K2900)-vc-0101 at
the doses of 0.3, 1 and 3 mg/kg.
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 induced tumor regression at 1
and 3 mg/kg. FIG. 5F shows the tumor growth inhibition curves for
the 10 individual tumor bearing mice in the
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 group dosed at 3 mg/kg of
FIG. 5E. The tumor regressions in the 3 mg/kg group were complete
and durable in 8 of 10 mice (80%) at the end of the study (95
days).
H-1975 NSCLC CLX
[0269] The effects of various vc-linked auristatin and CPI ADCs
were evaluated in H-1975, a moderate to high EDB+ FN expressing
NSCLC CLX model of human cancer. FIG. 6A shows EDB-L19-vc-0101
assessed for anti-tumor activity at 0.3, 0.75, 1.5 and 3 mg/mg. The
data demonstrates that EDB-L19-vc-0101 showed tumor regression in a
dose dependent manner at 3 mg/kg, and at as low as 1.5 mg/kg. FIG.
6B shows EDB-L19-vc-0101 and EDB-L19-vc-1569 were evaluated for
anti-tumor activity at 0.3, 1 and 3 mg/kg. The data demonstrates
that EDB-L19-vc-0101 and EDB-L19-vc-1569 showed tumor regression in
a dose dependent manner.
[0270] The anti-tumor activity of vc-linked auristatin ADCs were
compared to CPI ADCs. As shown in FIG. 6C, EDB-L19-vc-0101 and
EDB-(H16-K222R)-AcLys-vc-CPI-8314 were assessed at 0.5, 1.5 and 3
mg/kg and 0.1, 0.3 and 1 mg/kg, respectively. EDB-L19-vc-0101 and
EDB-(H16-K222R)-AcLys-vc-CPI-8314 both showed tumor regression at
the highest doses evaluated.
[0271] The activity of site-specific and conventionally conjugated
EDB ADCs was evaluated. FIG. 6D shows the anti-tumor efficacy of
the site-specific conjugated EDB-(.kappa.K183C+K2900)-vc-0101
compared to conventionally conjugated EDB-L19-vc-0101 at the doses
of 0.5, 1.5 and 3 mg/kg. The dose-level based efficacy was
comparable and the EDB-(.kappa.K183C+K290C)-vc-0101 led to tumor
regression in a dose dependent manner.
[0272] The activity of vc-0101 EDB ADCs having various mutations
was assessed. FIG. 6E shows the anti-tumor efficacy of
EDB-L19-vc-0101 and EDB-(K94R)-vc-0101 at 1 and 3 mg/kg. FIG. 6F
shows the anti-tumor efficacy of site-specific
EDB-(.kappa.K183C+K290C)-vc-0101 and
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 at 1 and 3 mg/kg. The 4 ADCs
demonstrated similar efficacy in the H-1975 model irrespective of
whether they contained the .kappa.K183C-K2900 and/or K94R
mutations. In addition, all ADCs tested resulted in robust
anti-tumor efficacy including tumor regressions at 3 mg/kg. These
data demonstrate that the introduction of the .kappa.K183C-K2900
and/or K94R mutations did not negatively impact the efficacy of the
ADCs.
HT29 Colon CLX
[0273] The effects of various vc-linked auristatin ADCs were
evaluated in HT29, a moderate EDB+ FN expressing colon CLX model of
human cancer. As shown in FIG. 7, EDB-L19-vc-0101 and
EDB-L19-vc-9411 were tested for anti-tumor activity at 3 mg/kg.
Both EDB-L19-vc-0101 and EDB-L19-vc-9411 showed tumor regression at
the 3 mg/kg dose over time.
PDX-PAX-13565 and PDX-PAX-12534 Pancreatic PDXs
[0274] The anti-tumor efficacy of EDB-L19-vc-0101 was evaluated in
human pancreatic PDX models. As shown in FIG. 8A, EDB-L19-vc-0101
was assessed at 0.3, 1 and 3 mg/kg in PDX-PAX-13565, a moderate to
high EDB+ FN expressing pancreatic PDX. As shown in FIG. 8B,
EDB-L19-vc-0101 was assessed at 0.3, 1 and 3 mg/kg in
PDX-PAX-12534, a low to moderate EDB+ FN expressing pancreatic PDX.
EDB-L19-vc-0101 demonstrated tumor regression in a dose dependent
manner in both pancreatic PDX models evaluated.
Ramos Lymphoma CLX
[0275] The anti-tumor efficacy of EDB-L19-vc-0101 was evaluated in
Ramos, a moderate EDB+ FN expressing lymphoma CLX model.
EDB-L19-vc-0101 was assessed for anti-tumor activity at 1 and 3
mg/kg. As shown in FIG. 9, EDB-L19-vc-0101 showed tumor regression
at the 3 mg/kg dose in a dose dependent manner.
EMT-6 Breast Syngeneic Model
[0276] The anti-tumor efficacy of
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 was evaluated in EMT-6, a
mouse syngeneic breast carcinoma model in an immuncompetent
background. As shown in FIG. 10A,
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 demonstrated tumor growth
inhibition at 4.5 mg/kg. The tumor growth inhibition was plotted as
an average of tumor size in eleven tumor bearing animals.+-.SEM.
FIG. 10B shows the tumor growth inhibition curves for the 11
individual tumor bearing mice in the
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 group dosed at 4.5 mg/kg. The
tumor regressions in the 4.5 mg/kg group were complete and durable
in 9 of 11 mice (82%) at the end of the study (34 days).
Ovarian
[0277] The activity of EDB-(.kappa.K183C-K94R-K2900)-vc-0101 was
examined in ovarian and breast carcinoma human PDX models which
express EDB+ FN. Activity has been observed at 3 mg/kg and 10 mg/kg
dose levels (data not shown).
Example 9
Pharmacokinetics (PK)
[0278] Exposure of conventionally conjugated EDB-L19-vc-0101 and
site-specific conjugated EDB-(.kappa.K183C-K94R-K2900)-vc-0101
conjugated antibody drug conjugates were determined after an
intravenous (IV) bolus dose administration of either 5 or 6 mg/kg
in cynomolgus monkeys, respectively. Concentrations of total
antibody (total Ab; measurement of both conjugated mAb and
unconjugated mAb), ADC (mAb that is conjugated to at least one drug
molecule) were measured using ligand binding assays (LBA) and
concentrations of the released payload 0101 were measured using
mass spectrometery. Quantitation of total Ab and ADC concentrations
was achieved by ligand binding assay (LBA) using the Gyrolab.RTM.
workstation with fluorescence detection. The Biotinylated capture
protein used was a sheep anti-hlgG and the detection antibody was
Alexa Fluor 647 goat anti-hlgG for total antibody or Alexa Fluor
647 anti-0101 mAb for ADC (data was processed by the Watson v 7.4
LIMS system). In vivo samples were prepared for unconjugated
payload analysis using protein precipitation and injected onto an
AB Sciex AP15500 (QTRAP) mass spectrometer using positive Turbo
IonSpray electrospray ionization (ESI) and multiple reaction
monitoring (MRM) mode. The transitions of 743.6.fwdarw.188.0 and
751.6.fwdarw.188.0 were used for the analyte and deuterated
internal standard, respectively. Data acquisition and processing
were carried out with Analyst software version 1.5.2 (Applied
Biosystems/MDS Sciex, Canada).
[0279] The pharmacokinetics of total Ab, ADC and released payload
from EDB-L19-vc-0101 ADC (at 5 mg/kg) and
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 ADC (6 mg/kg) dosed
cynomolgus monkeys are shown in Table 22. Exposure of the
site-specific conjugated EDB-(.kappa.K183C-K94R-K290C)-vc-0101 ADC
showed both increased exposure (.about.2.3.times. increase as
measured by dose normalized AUC) and increased conjugation
stability when compared to the conventional conjugate. Conjugation
stability was assessed by both the higher ADC/Ab ratio (84% versus
75%) and by the lower released payload exposure (dose normalized
AUC; 0.0058 versus 0.0082 .mu.g*h/mL) for the site-specific
conjugated EDB-(.kappa.K183C-K94R-K290C)-vc-0101 ADC compared to
the conventional EDB-L19-vc-0101 ADC, respectively. NA=not
applicable.
TABLE-US-00023 TABLE 22 Summary of pharmacokinetics in non-human
primates. Dose C.sub.max AUC.sub.0-504 Terminal T.sub.1/2 AUC/
ADC/Ab ADC (mg/kg) Analyte (.mu.g/mL) (.mu.g*hr/mL) (day) Dose (%)
EDB-L19- 5 Ab 114 .+-. 6907 .+-. 5.1 .+-. 1381 .+-. -- vc-0101 27
1997 2.2 399 (ADC1) ADC 110 .+-. 5190 .+-. 4.6 .+-. 1038 .+-. 75
.+-. 31 1453 1.0 291 2 Payload 0.00053 .+-. 0.0411 .+-. NA 0.0082
.+-. -- 0.00025 0.0160 0.0032 EDB- 6 Ab 164 .+-. 17600 .+-. 6.4
.+-. 2933 .+-. -- (.kappa.K183C- 36 3045 1.3 507 K94R- ADC 156 .+-.
14567 .+-. 5.9 .+-. 2428 .+-. 84 .+-. K290C)-vc- 30 2122 1.1 354 3
0101 Payload 0.00024 .+-. 0.0349 .+-. NA 0.0058 .+-. -- (ADC4)
0.00021 0.0030 0.0005
Example 10
Thermal Stability Assessment for EDB ADCs
[0280] Differential Scanning calorimetry (DCS) was used to
determine the thermal stability of the anti-EDB antibody variants
and corresponding conventional and site-specific conjugated EDB
ADCs. Samples formulated in PBS-CMF pH 7.2 were dispensed into the
sample tray of a MicroCal VP-Capillary DSC with Autosampler (GE
Healthcare Bio-Sciences, Piscataway, N.J.), equilibrated for 5
minutes at 10.degree. C. and then scanned up to 110.degree. C. at a
rate of 100.degree. C. per hour. A filtering period of 16 seconds
was selected. Raw data was baseline corrected and the protein
concentration was normalized. Origin Software 7.0 (OriginLab
Corporation, Northampton, Mass.) was used to fit the data to an
MN2-State Model with an appropriate number of transitions.
[0281] As shown in Table 23, various anti-EDB antibodies and EDB
ADCs, using both site-specific and conventional conjugation
technology, were evaluated and exhibited favorable thermal
stability as determined by the first melting transition
(Tm1)>65.degree. C. These results demonstrate that the
EDB-(.kappa.K183C-K94R-K290C antibody and
.kappa.K183C-K94R-K2900-vc-0101 ADC incorporating engineered
cysteine residues were thermally stable.
TABLE-US-00024 TABLE 23 Thermal Stability of EDB Antibody Variants
and EDB ADCs T.sub.m1 T.sub.m2 T.sub.m3 ADCs EDB-L19-vc-0101 66.00
.+-. 0.15 80.97 .+-. 0.25 84.11 .+-. 0.06 EDB-(K94R)-vc-0101 65.61
.+-. 0.14 80.24 .+-. 0.22 83.43 .+-. 0.05 EDB-(.kappa.K183C-K94R-
66.00 .+-. 0.10 80.24 .+-. 0.43 83.27 .+-. 0.10 K290C)-vc-0101
Antibodies EDB-(.kappa.K183C-K94R-K290C) 75.28 .+-. 0.12 81.56 .+-.
0.37 84.24 .+-. 0.12 EDB-L19 72* 82* 85* *Values determined in a
different experiment from others reported in table
Example 11
Toxicity Studies
[0282] The nonclinical safety profile of conventional conjugated
EDB-L19-vc-0101 and site-specific conjugated
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 was characterized in
exploratory repeat-dose (Q3W.times.3) studies in Wistar-Han rats
and cynomolgus monkeys. The rat and cynomolgus monkey were
considered pharmacologically relevant nonclinical species for
toxicity evaluation due to 100% protein sequence homology with
human EDB, as well as similar binding affinity of the antibodies
EDB-L19 and EDB-(.kappa.K183C-K94R-K2900) to rat, human and monkey
by Biacore assay, as demonstrated in Example 2.
[0283] EDB-L19-vc-0101 was evaluated in Wistar Han rats and
cynomolgus monkeys up to 10 and 5 mg/kg/dose, respectively, and
EDB-(.kappa.K183C-K94R-K290C)-vc-0101 was evaluated in cynomolgus
monkeys up to 12 mg/kg/dose. Rats or monkeys were dosed
intravenously once every 3 weeks (on Days 1, 22 and 43) and were
euthanized on Day 46 (3 days after the 3.sup.rd dose). Animals were
evaluated for clinical signs, changes in body weight, food
consumption, clinical pathology parameters, organ weights, and
macroscopic and microscopic observations. No mortality or
significant changes in clinical condition of animals were noted in
these studies.
[0284] There was no indication of target-dependent toxicity in EDB+
FN expressing tissues/organs in rats and monkeys. In both species,
the major toxicity was reversible myelosuppression with associated
hematological changes. In monkeys, marked transient neutropenia was
seen with conventionally conjugated EDB-L19-vc-0101 at 5 mg/kg/dose
while only minimal effects on neutrophil counts were seen with
site-specific conjugated EDB-(.kappa.K183C-K94R-K2900)-vc-0101 at 6
mg/kg/dose, as shown in Table 24 and FIG. 11. Points represent mean
and error bars represent .+-.1 standard deviation (SD) from the
mean.
[0285] The data demonstrates significant alleviation of
myelosuppression by site-specific conjugation. The toxicity profile
of EDB-L19-vc-0101 and EDB-(.kappa.K183C-K94R-K2900)-vc-0101 was
consistent with target-independent effects of these conjugates and
the highest non-severely toxic doses (HNSTD) for EDB-L19-vc-0101
and EDB-(.kappa.K183C-K94R-K2900)-vc-0101 were determined to be 5
mg/kg/dose and 12 mg/kg/dose, respectively.
TABLE-US-00025 TABLE 24 Absolute neutrophil counts in cynomolgus
monkeys over the study duration. EDB-(.kappa.K183C-K94R- 0 mg/kg
EDB-L19-vc-0101 K290C)-vc-0101 (vehicle) (5 mg/kg) (6 mg/kg) Animal
Animal Animal Animal Animal Animal Day # 1 # 2 # 1 # 2 # 1 # 2 -7
3.26 2.9 8.48 4.67 2.41 7.42 7 3.54 2.52 7.08 2.29 3.96 4.3 10 3.16
6.83 0.11 0.82 2.66 1.56 15 3.06 1.98 11.41 3.65 1.37 1.44 31 3.87
4.17 0.39 2.07 1.91 2.22 38 3.09 5.63 16.17 2.73 1.97 1.13 45 3.53
2.07 13.02 1.83 1.4 3.78
Example 12
IO Combinations
[0286] As cancer cells die, they release antigens that are taken up
and presented by dendritic cells (DCs). Because of the mutations in
these tumor cells some of these antigens include cancer neoepitopes
which have the potential to be presented by the mature DCs to T
cells, thereby activating them and inducing anti-tumor targeting.
However, negative regulatory mechanisms are upregulated in cancer
patients. For example, signaling through checkpoints such as the
PD-1/PD-L1 pathways may limit the recognition of the neoepitopes
and activation of T cells.
[0287] Payloads conjugated to antibodies in the ADC format may
participate to engage the dendritic cell maturation pathways
resulting in increased tumor antigen cross presentation, which
allows for T cell priming and increased tumor T cell targeting. The
EDB ADCs of the present invention, comprising various payloads such
as Payload-0101, were utilized to improve immune recognition of
tumor neoantigens by creating immunogenic tumor environments. These
environments become responsive to immune-oncology agents that block
the negative regulatory pathways, when the EDB ADC and
immune-oncology agent is given in combination.
[0288] Data from efficacy studies of EMT6 syngeneic tumors treated
with EDB-L19-vc-0101 suggests an effector response to Payload-0101
was induced. Increased infiltration of CD3+ T cells was observed in
EDB-L19-vc-0101 treated tumors vs. vehicle controls. Additionally,
increased expression of PDL1 in treated tumors was observed,
suggesting IFN.gamma. release due to the increased effector T cell
response.
[0289] Combining EDB ADCs with agents that target immunomodulatory
pathways, such as anti-PDL1 antagonist antibodies or anti-41BB
agonist antibodies, will likely improve anti-tumor efficacy and
provide more durable responses.
Example 13
Biomarker/Mechanism of Action
[0290] NSCLC PDX model PDX-NSX-11122 was developed in nude mice as
previously described. EDB-(K94R)-vc-0101,
EDB-(.kappa.K183C-K94R-K2900)-vc-0101, and Neg-vc-0101 ADC, were
administered by tail vein injection at 3 mg/kg (4 animals per
timepoint per group). At 96 hours after a single administration,
animals were anaesthetized and were perfused with saline. Following
saline perfusion, tumors were removed and prepared for measurement
of antibody and ADC via ligand binding assay (LBA), or were
prepared for immunohistochemistry (II-1C).
Ligand Binding Assay (LBA)
[0291] For LBA assays, 5.times. buffer was added to the tumor
samples. The tissue extraction reagent (Invitrogen) contained 1%
protease inhibitor (Sigma), (v/w) with a final dilution of 6.times.
(.mu.g/mL homogenate.fwdarw..mu.g/g tissue). Stainless steel beads
were added and the tissue was homogenize using a Mini-Beadbeater-96
(BioSpec). The homogenate (.about.100-300 .mu.L depending on sample
size) was transferred to an appropriate vial (Marsh tube) and
centrifuge at 14000 rpm for 10 minutes (4.degree. C.). The
centrifuged homogenate was diluted (MRD) with Super Block.TM. for
analysis according to analytical protocol.
[0292] As shown in Table 25, ligand binding assays were used to
determine mean total antibody and ADC plasma concentrations
(.mu.g/mL) tumor concentrations (.mu.g/g) following the single dose
administration of EDB ADCs. The data demonstrates that
EDB-(K94R)-vc-0101 and EDB-(.kappa.K183C-K94R-K2900)-vc-0101 were
detected at the site of the tumor at increased levels as measured
by total antibody and ADC as compared to the Neg-vc-0101.
Furthermore, there was a decreased plasma to tumor ratio for both
the ADC and the total antibody observed for EDB-(K94R)-vc-0101 and
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 compared to Neg-vc-0101
indicating the increased efficiency of tumor specific targeting of
EDB targeting ADCs.
TABLE-US-00026 TABLE X Mean total Ab and ADC plasma and tumor
concentrations in NSCLC PDX. Plasma at Tumor at Plasma/ 96 hours 96
hours Tumor ADC Modality (.mu.g/mL) (.mu.g/g) ratio
EDB-(K94R)-vc-0101 Total Ab 13.1 .+-. 3.7 10.2 .+-. 2.6 1.3 ADC
12.0 .+-. 4.0 8.93 .+-. 1.77 1.3 EDB-(.kappa.K183C- Total Ab 10.3
.+-. 2.4 7.17 .+-. 2.40 1.4 K94R-K290C)-vc- ADC 8.19 .+-. 2.03 6.01
.+-. 1.96 1.4 0101 Neg-vc-0101 Total Ab 24.2 .+-. 6.6 4.74 .+-.
0.84 5.1 ADC 20.2 .+-. 5.8 3.67 .+-. 0.80 5.5
Immunohistochemistry (IHC)
[0293] For immunohistochemical detection of ADC distribution and
downstream biomarkers of response, samples were fixed in 10%
neutral buffered formalin for 48 hours. After fixation, samples
were embedded in paraffin and sectioned at 5 .mu.M. Cut paraffin
section were deparaffinized in xylene substitute and rehydrated
with graded alcohols to distilled water. Antigens were retrieved
in: 10 mM Citrate buffer pH 6.0 (Invitrogen) for phospho-Histone H3
and cleaved caspase 3 detection or Borg Decloaker buffer pH 9.5
(Biocare Medical) for anti-human IgG detection and anti-0101
detection in a pressure cooker (Electron Microscopy Sciences) and
cooled to room temperature. Endogenous peroxidase was blocked with
3% hydrogen peroxide for 10 minutes. Non-specific protein
interactions were blocked with Protein block (DAKO) for 20 minutes.
Tissue sections were incubated with primary antibody for 1 hour at
room temperature. Primary antibodies were: 0.3 .mu.g/mL anti-human
Pan IgG antibody (Epitomics); 10 .mu.g/mL anti-0101 Ab; 0.13
.mu.g/mL anti-phospho Histone H3 (pHH3, Cell Signaling
Technologies); 1.3 .mu.g/mL anti-Cleaved Caspase 3 (Cell Signaling
Technologies). To avoid mouse on mouse detection, anti-0101 isotype
antibodies were labeled with AlexaFluor 488 using Alexa Fluor 488
protein labeling kit (Life Technologies). Unlabeled primary
antibodies were detected with Signalstain Boost reagent (Cell
Signaling Technologies) for 30 minutes at room temperature.
AlexaFluor 488 labeled primary antibodies were detected with 1
.mu.g/ml rabbit anti-AlexaFluor488 (Life Technologies) for 45
minutes at room temperature, followed by incubation with
Signalstain Boost reagent (Cell Signaling Technologies) for 30
minutes at room temperature. DAB+(3',3'-Diaminobenzidine; Dako) was
used to develop color for 5 minutes. Sections were briefly
counterstained in hematoxylin, washed in water, dehydrated in
graded alcohols, cleared in xylene substitute, and coverslipped
with Permount Mounting Medium.
[0294] At 96 hours after a single dose, both the conventional
EDB-(K94R)-vc-0101 and site-specific conjugated
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 were similarly detected by
anti-human IgG IHC in the PDX-NSX-11122 PDX model. An increase in
pHH3 positive cells, a marker of mitotic arrest, was observed in
tumors treated with both EDB-(K94R)-vc-0101 and
EDB-(.kappa.K183C-K94R-K2900)-vc-0101 compared to those treated
with the negative control ADC (Neg-vc-0101). The majority of the
cells harboring the pHH3 mitotic arrest marker were neoplastic
cells, suggesting the bystander effect. Cleaved caspase 3 stain
indicated increased apoptosis in those tumors treated with
EDB-(K94R)-vc-0101 (and EDB-(.kappa.K183C-K94R-K2900)-vc-0101
compared to the tumors treated with the negative control ADC
(Neg-vc-0101).
Sequence CWU 1
1
571116PRTArtificial SequenceSynthetic peptide sequence 1Glu Val Gln
Leu Leu 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 Phe 20 25 30Ser
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110Thr Val Ser Ser 1152348DNAArtificial
SequenceSynthetic nucleotide sequence 2gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agtttttcga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gttcgggtac cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gaaaccgttt 300ccgtattttg actactgggg ccagggaacc
ctggtcaccg tctcgagt 34835PRTArtificial SequenceSynthetic peptide
sequence 3Ser Phe Ser Met Ser1 547PRTArtificial SequenceSynthetic
peptide sequence 4Gly Phe Thr Phe Ser Ser Phe1 5517PRTArtificial
SequenceSynthetic peptide sequence 5Ser Ile Ser Gly Ser Ser Gly Thr
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly66PRTArtificial
SequenceSynthetic peptide sequence 6Ser Gly Ser Ser Gly Thr1
577PRTArtificial SequenceSynthetic peptide sequence 7Pro Phe Pro
Tyr Phe Asp Tyr1 58446PRTArtificial SequenceSynthetic peptide
sequence 8Glu Val Gln Leu Leu 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 Phe 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150
155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265
270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390
395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 435 440 44591338DNAArtificial SequenceSynthetic
nucleotide sequence 9gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agtttttcga
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcatct
attagtggta gttcgggtac cacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaagac acggccgtat attactgtgc gaaaccgttt
300ccgtattttg actactgggg ccagggaacc ctggtcaccg tctcgagtgc
gtcgaccaag 360ggcccatcgg tcttccccct ggcaccctcc tccaagagca
cctctggggg cacagcggcc 420ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 480gccctgacca gcggcgtgca
caccttcccg gctgtcctac agtcctcagg actctactcc 540ctcagcagcg
tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac
600gtgaatcaca agcccagcaa caccaaggtg gacaagaaag ttgagcccaa
atcttgtgac 660aaaactcaca catgcccacc gtgcccagca cctgaactcc
tggggggacc gtcagtcttc 720ctcttccccc caaaacccaa ggacaccctc
atgatctccc ggacccctga ggtcacatgc 780gtggtggtgg acgtgagcca
cgaagaccct gaggtcaagt tcaactggta cgtggacggc 840gtggaggtgc
ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt
900gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga
gtacaagtgc 960aaggtctcca acaaagccct cccagccccc atcgagaaaa
ccatctccaa agccaaaggg 1020cagccccgag aaccacaggt gtacaccctg
cccccatccc gggatgagct gaccaagaac 1080caggtcagcc tgacctgcct
ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1140gagagcaatg
ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac
1200ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca
gcaggggaac 1260gtcttctcat gctccgtgat gcatgaggct ctgcacaacc
actacacgca gaagagcctc 1320tccctgtctc cgggtaaa
133810108PRTArtificial SequenceSynthetic peptide sequence 10Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr
Gly Arg Ile Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10511324DNAArtificial SequenceSynthetic nucleotide sequence
11gaaattgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcagctttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat tatgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagacgggtc gtattccgcc gacgttcggc 300caagggacca aggtggaaat caaa
3241212PRTArtificial SequenceSynthetic peptide sequence 12Arg Ala
Ser Gln Ser Val Ser Ser Ser Phe Leu Ala1 5 10137PRTArtificial
SequenceSynthetic peptide sequence 13Tyr Ala Ser Ser Arg Ala Thr1
5149PRTArtificial SequenceSynthetic peptide sequence 14Gln Gln Thr
Gly Arg Ile Pro Pro Thr1 515215PRTArtificial SequenceSynthetic
peptide sequence 15Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Tyr Ala Ser Ser Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro 85 90 95Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 21516645DNAArtificial SequenceSynthetic nucleotide sequence
16gaaattgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcagctttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat tatgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagacgggtc gtattccgcc gacgttcggc 300caagggacca aggtggaaat
caaacgtacg gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc
gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagca aagcagacta cgagaaacac
aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac
aaagagcttc aacaggggag agtgt 64517445PRTArtificial SequenceSynthetic
peptide sequence 17Glu Val Gln Leu Leu 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 Phe 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135
140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445181335DNAArtificial
SequenceSynthetic nucleotide sequence 18gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agtttttcga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gttcgggtac cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gaaaccgttt 300ccgtattttg actactgggg ccagggaacc
ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg tcttccccct
ggcaccctcc tccaagagca cctctggggg cacagcggcc 420ctgggctgcc
tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc
480gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg
actctactcc 540ctcagcagcg tggtgaccgt gccctccagc agcttgggca
cccagaccta catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg
gacaagaaag ttgagcccaa atcttgtgac 660aaaactcaca catgcccacc
gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc
780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta
cgtggacggc 840gtggaggtgc ataatgccaa gacaaagccg cgggaggagc
agtacaacag cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag
gactggctga atggcaagga gtacaagtgc 960aaggtctcca acaaagccct
cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag
aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac
1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc
cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac aagaccacgc
ctcccgtgct ggactccgac 1200ggctccttct tcctctatag caagctcacc
gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat gctccgtgat
gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320tccctgtccc cgggt
133519445PRTArtificial SequenceSynthetic peptide sequence 19Glu Val
Gln Leu Leu 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 Phe 20 25
30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Cys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445201335DNAArtificial
SequenceSynthetic nucleotide sequence 20gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agtttttcga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gttcgggtac cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gaaaccgttt 300ccgtattttg actactgggg ccagggaacc
ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg tcttccccct
ggcaccctcc tccaagagca cctctggggg cacagcggcc 420ctgggctgcc
tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc
480gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg
actctactcc 540ctcagcagcg tggtgaccgt gccctccagc agcttgggca
cccagaccta catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg
gacaagaaag ttgagcccaa atcttgtgac 660aaaactcaca catgcccacc
gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc
780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta
cgtggacggc 840gtggaggtgc ataatgccaa gacatgcccg cgggaggagc
agtacaacag cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag
gactggctga atggcaagga gtacaagtgc 960aaggtctcca acaaagccct
cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag
aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac
1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc
cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac aagaccacgc
ctcccgtgct ggactccgac 1200ggctccttct tcctctatag caagctcacc
gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat gctccgtgat
gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320tccctgtccc cgggt
133521116PRTArtificial SequenceSynthetic peptide sequence 21Glu Val
Gln Leu Leu 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 Phe 20 25
30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Pro Phe Pro Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11522348DNAArtificial SequenceSynthetic nucleotide sequence
22gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc agtttttcga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatct attagtggta gttcgggtac
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaagac
acggccgtat attactgtgc gagaccgttt 300ccgtattttg actactgggg
ccagggaacc ctggtcaccg tctcgagt 34823445PRTArtificial
SequenceSynthetic peptide sequence 23Glu Val Gln Leu Leu 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 Phe 20 25 30Ser Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser
Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215
220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445241335DNAArtificial SequenceSynthetic nucleotide sequence
24gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc agtttttcga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatct attagtggta gttcgggtac
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaagac
acggccgtat attactgtgc gagaccgttt 300ccgtattttg actactgggg
ccagggaacc ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg
tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc
420ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg
gaactcaggc 480gccctgacca gcggcgtgca caccttcccg gctgtcctac
agtcctcagg actctactcc 540ctcagcagcg tggtgaccgt gccctccagc
agcttgggca cccagaccta catctgcaac 600gtgaatcaca agcccagcaa
caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660aaaactcaca
catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc
720ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga
ggtcacatgc 780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt
tcaactggta cgtggacggc 840gtggaggtgc ataatgccaa gacaaagccg
cgggaggagc agtacaacag cacgtaccgt 900gtggtcagcg tcctcaccgt
cctgcaccag gactggctga atggcaagga gtacaagtgc 960aaggtctcca
acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg
1020cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat
gaccaagaac 1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca
gcgacatcgc cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac
aagaccacgc ctcccgtgct ggactccgac 1200ggctccttct tcctctatag
caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc
1320tccctgtccc cgggt 133525445PRTArtificial SequenceSynthetic
peptide sequence 25Glu Val Gln Leu Leu 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 Phe 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Phe Pro
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135
140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Cys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445261335DNAArtificial
SequenceSynthetic nucleotide sequence 26gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agtttttcga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gttcgggtac cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gagaccgttt 300ccgtattttg actactgggg ccagggaacc
ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg tcttccccct
ggcaccctcc tccaagagca cctctggggg cacagcggcc 420ctgggctgcc
tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc
480gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg
actctactcc 540ctcagcagcg tggtgaccgt gccctccagc agcttgggca
cccagaccta catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg
gacaagaaag ttgagcccaa atcttgtgac 660aaaactcaca catgcccacc
gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc
780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta
cgtggacggc 840gtggaggtgc ataatgccaa gacatgcccg cgggaggagc
agtacaacag cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag
gactggctga atggcaagga gtacaagtgc 960aaggtctcca acaaagccct
cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag
aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac
1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc
cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac aagaccacgc
ctcccgtgct ggactccgac 1200ggctccttct tcctctatag caagctcacc
gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat gctccgtgat
gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320tccctgtccc ccgga
133527445PRTArtificial SequenceSynthetic peptide sequence 27Glu Val
Gln Leu Leu 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 Phe 20 25
30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170
175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Arg Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Leu Leu Gln Gly Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445281335DNAArtificial SequenceSynthetic nucleotide sequence
28gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag
cctctggatt cacctttagc agtttttcga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatct attagtggta gttcgggtac
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaagac
acggccgtat attactgtgc gaaaccgttt 300ccgtattttg actactgggg
ccagggaacc ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg
tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc
420ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg
gaactcaggc 480gccctgacca gcggcgtgca caccttcccg gctgtcctac
agtcctcagg actctactcc 540ctcagcagcg tagtgaccgt gccctccagc
agcttgggca cccagaccta catctgcaac 600gtgaatcaca agcccagcaa
caccaaggtg gacaagaaag ttgagcccaa atcttgtgac 660cgcactcaca
catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc
720ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga
ggtcacatgc 780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt
tcaactggta cgtggacggc 840gtggaggtgc ataatgccaa gacaaagccg
cgggagctgc tgcaggggag cacgtaccgt 900gtggtcagcg tcctcaccgt
cctgcaccag gactggctga atggcaagga gtacaagtgc 960aaggtctcca
acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg
1020cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat
gaccaagaac 1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca
gcgacatcgc cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac
aagaccacgc ctcccgtgct ggactccgac 1200ggctccttct tcctctatag
caagctcacc gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc
1320tccctgtctc cgggt 133529445PRTArtificial SequenceSynthetic
peptide sequence 29Glu Val Gln Leu Leu 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 Phe 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Ser Gly Thr
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Pro Phe Pro
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135
140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr 195 200 205Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Arg Thr His Thr 210 215 220Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Lys Pro Arg Glu Leu Leu Gln Gly Ser Thr Tyr Arg
Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445301335DNAArtificial
SequenceSynthetic nucleotide sequence 30gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agtttttcga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gttcgggtac cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gagaccgttt 300ccgtattttg actactgggg ccagggaacc
ctggtcaccg tctcgagtgc gtcgaccaag 360ggcccatcgg tcttccccct
ggcaccctcc tccaagagca cctctggggg cacagcggcc 420ctgggctgcc
tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc
480gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg
actctactcc 540ctcagcagcg tagtgaccgt gccctccagc agcttgggca
cccagaccta catctgcaac 600gtgaatcaca agcccagcaa caccaaggtg
gacaagaaag ttgagcccaa atcttgtgac 660cgcactcaca catgcccacc
gtgcccagca cctgaactcc tggggggacc gtcagtcttc 720ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc
780gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta
cgtggacggc 840gtggaggtgc ataatgccaa gacaaagccg cgggagctgc
tgcaggggag cacgtaccgt 900gtggtcagcg tcctcaccgt cctgcaccag
gactggctga atggcaagga gtacaagtgc 960aaggtctcca acaaagccct
cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1020cagccccgag
aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac
1080caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc
cgtggagtgg 1140gagagcaatg ggcagccgga gaacaactac aagaccacgc
ctcccgtgct ggactccgac 1200ggctccttct tcctctatag caagctcacc
gtggacaaga gcaggtggca gcaggggaac 1260gtcttctcat gctccgtgat
gcatgaggct ctgcacaacc actacacgca gaagagcctc 1320tccctgtccc ccgga
133531215PRTArtificial SequenceSynthetic peptide sequence 31Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr
Gly Arg Ile Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170
175Ser Ser Thr Leu Thr Leu Ser Cys Ala Asp Tyr Glu Lys His Lys Val
180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu Cys 210
21532645DNAArtificial SequenceSynthetic nucleotide sequence
32gaaattgtgt taacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agcagctttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat tatgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagacgggtc gtattccgcc gacgttcggc 300caagggacca aggtggaaat
caaacgaact gtggctgcac catctgtctt catcttcccg 360ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
420tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc
gggtaactcc 480caggagagtg tcacagagca ggacagcaag gacagcacct
acagcctcag cagcaccctg 540acgctgagct gcgcagacta cgagaaacac
aaagtctacg cctgcgaagt cacccatcag 600ggcctgagct cgcccgtcac
aaagagcttc aacaggggag agtgt 6453391PRTHomo sapiens 33Glu Val Pro
Gln Leu Thr Asp Leu Ser Phe Val Asp Ile Thr Asp Ser1 5 10 15Ser Ile
Gly Leu Arg Trp Thr Pro Leu Asn Ser Ser Thr Ile Ile Gly 20 25 30Tyr
Arg Ile Thr Val Val Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu 35 40
45Asp Phe Val Asp Ser Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu
50 55 60Pro Gly Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly
Gly65 70 75 80Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr 85
9034374PRTArtificial SequenceSynthetic peptide sequence 34Val Val
Thr Gln Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn1 5 10 15Pro
Asp Thr Gly Val Leu Ala Val Ser Trp Glu Arg Ser Thr Thr Pro 20 25
30Asp Ile Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln
35 40 45Gly Asn Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys
Thr 50 55 60Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val
Tyr Thr65 70 75 80Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp
Thr Ile Ile Pro 85 90 95Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val
Asp Ile Thr Asp Ser 100 105 110Ser Ile Gly Leu Arg Trp Thr Pro Leu
Asn Ser Ser Thr Ile Ile Gly 115 120 125Tyr Arg Ile Thr Val Val Ala
Ala Gly Glu Gly Ile Pro Ile Phe Glu 130 135 140Asp Phe Val Asp Ser
Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu145 150 155 160Pro Gly
Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly 165 170
175Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Val Pro Pro Pro
180 185 190Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg
Val Thr 195 200 205Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe
Leu Val Arg Tyr 210 215 220Ser Pro Val Lys Asn Glu Glu Asp Val Ala
Glu Leu Ser Ile Ser Pro225 230 235 240Ser Asp Asn Ala Val Val Leu
Thr Asn Leu Leu Pro Gly Thr Glu Tyr 245 250 255Val Val Ser Val Ser
Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu 260 265 270Arg Gly Arg
Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe 275 280 285Ser
Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 290 295
300Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Phe
Ser305 310 315 320Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg
Asn Ser Ile Thr 325 330 335Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr
Val Val Ser Ile Val Ala 340 345 350Leu Asn Gly Arg Glu Glu Ser Pro
Leu Leu Ile Gly Arg Ser Arg Ser 355 360 365His His His His His His
37035374PRTArtificial SequenceSynthetic peptide sequence 35Val Val
Thr Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Thr Asn1 5 10 15Pro
Asp Thr Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro 20 25
30Asp Ile Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln
35 40 45Gly Tyr Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys
Thr 50 55 60Phe Asp Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val
Tyr Thr65 70 75 80Val Lys Asp Asp Lys Glu Ser Val Pro Ile Ser Asp
Thr Ile Ile Pro 85 90 95Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val
Asp Ile Thr Asp Ser 100 105 110Ser Ile Gly Leu Arg Trp Thr Pro Leu
Asn Ser Ser Thr Ile Ile Gly 115 120 125Tyr Arg Ile Thr Val Val Ala
Ala Gly Glu Gly Ile Pro Ile Phe Glu 130 135 140Asp Phe Val Asp Ser
Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu145 150 155 160Pro Gly
Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly 165 170
175Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Val Pro Pro Pro
180 185 190Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg
Val Thr 195 200 205Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe
Leu Val Arg Tyr 210 215 220Ser Pro Val Lys Asn Glu Glu Asp Val Ala
Glu Leu Ser Ile Ser Pro225 230 235 240Ser Asp Asn Ala Val Val Leu
Thr Asn Leu Leu Pro Gly Thr Glu Tyr 245 250 255Val Val Ser Val Ser
Ser Val Tyr Glu Gln His Glu Ser Thr Pro Leu 260 265 270Arg Gly Arg
Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Ile Asp Phe 275 280 285Ser
Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile Ala Pro Arg 290 295
300Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu His Met
Ser305 310 315 320Gly Arg Pro Arg Glu Asp Arg Val Pro Pro Ser Arg
Asn Ser Ile Thr 325 330 335Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr
Val Val Ser Ile Val Ala 340 345 350Leu Asn Gly Arg Glu Glu Ser Pro
Leu Leu Ile Gly Arg Ser Arg Ser 355 360 365His His His His His His
37036374PRTArtificial SequenceSynthetic peptide sequence 36Val Val
Thr Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn1 5 10 15Pro
Asp Thr Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro 20 25
30Asp Ile Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln
35 40 45Gly Thr Ala Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys
Thr 50 55 60Phe Glu Asn Leu Asn Pro Gly Leu Glu Tyr Asn Val Ser Val
Tyr Thr65 70 75 80Val Lys Asp Asp Lys Glu Ser Ala Pro Ile Ser Asp
Thr Val Ile Pro 85 90 95Glu Val Pro Gln Leu Thr Asp Leu Ser Phe Val
Asp Ile Thr Asp Ser 100 105 110Ser Ile Gly Leu Arg Trp Thr Pro Leu
Asn Ser Ser Thr Ile Ile Gly 115 120 125Tyr Arg Ile Thr Val Val Ala
Ala Gly Glu Gly Ile Pro Ile Phe Glu 130 135 140Asp Phe Val Asp Ser
Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu145 150 155 160Pro Gly
Ile Asp Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly 165 170
175Glu Ser Ala Pro Thr Thr Leu Thr Gln Gln Thr Ala Val Pro Pro Pro
180 185 190Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met Arg
Val Thr 195 200 205Trp Ala Pro Pro Pro Ser Ile Glu Leu Thr Asn Leu
Leu Val Arg Tyr 210 215 220Ser Pro Val Lys Asn Glu Glu Asp Val Ala
Glu Leu Ser Ile Ser Pro225 230 235 240Ser Asp Asn Ala Val Val Leu
Thr Asn Leu Leu Pro Gly Thr Glu Tyr 245 250 255Leu Val Ser Val Ser
Ser Val Tyr Glu Gln His Glu Ser Ile Pro Leu 260 265 270Arg Gly Arg
Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly Phe Asp Ser 275 280 285Ser
Asp Val Thr Ala Asn Ser Phe Thr Val His Trp Val Ala Pro Arg 290 295
300Ala Pro Ile Thr Gly Tyr Ile Ile Arg His His Ala Glu His Ser
Ala305 310 315 320Gly Arg Pro Arg Gln Asp Arg Val Pro Pro Ser Arg
Asn Ser Ile Thr 325 330 335Leu Thr Asn Leu Asn Pro Gly Thr Glu Tyr
Ile Val Thr Ile Ile Ala 340 345 350Val Asn Gly Arg Glu Glu Ser Pro
Pro Leu Ile Gly Arg Ser Arg Ser 355 360 365His
His His His His His 37037107PRTArtificial SequenceSynthetic peptide
sequence 37Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 10538106PRTArtificial SequenceSynthetic peptide
sequence 38Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro
Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala
Asp Gly Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser
Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val 85 90 95Glu Lys Thr Val Ala Pro Thr
Glu Cys Ser 100 105395PRTArtificial SequenceSynthetic peptide
sequence 39Leu Leu Gln Gly Gly1 5404PRTArtificial SequenceSynthetic
peptide sequence 40Leu Leu Gln Gly1416PRTArtificial
SequenceSynthetic peptide sequence 41Leu Ser Leu Ser Gln Gly1
5428PRTArtificial SequenceSynthetic peptide sequence 42Gly Gly Gly
Leu Leu Gln Gly Gly1 5435PRTArtificial SequenceSynthetic peptide
sequence 43Gly Leu Leu Gln Gly1 54410PRTArtificial
SequenceSynthetic peptide sequence 44Gly Ser Pro Leu Ala Gln Ser
His Gly Gly1 5 10457PRTArtificial SequenceSynthetic peptide
sequence 45Gly Leu Leu Gln Gly Gly Gly1 5466PRTArtificial
SequenceSynthetic peptide sequence 46Gly Leu Leu Gln Gly Gly1
5474PRTArtificial SequenceSynthetic peptide sequence 47Gly Leu Leu
Gln1488PRTArtificial SequenceSynthetic peptide sequence 48Leu Leu
Gln Leu Leu Gln Gly Ala1 5495PRTArtificial SequenceSynthetic
peptide sequence 49Leu Leu Gln Gly Ala1 5507PRTArtificial
SequenceSynthetic peptide sequence 50Leu Leu Gln Tyr Gln Gly Ala1
5516PRTArtificial SequenceSynthetic peptide sequence 51Leu Leu Gln
Gly Ser Gly1 5526PRTArtificial SequenceSynthetic peptide sequence
52Leu Leu Gln Tyr Gln Gly1 5537PRTArtificial SequenceSynthetic
peptide sequence 53Leu Leu Gln Leu Leu Gln Gly1 5545PRTArtificial
SequenceSynthetic peptide sequence 54Ser Leu Leu Gln Gly1
5555PRTArtificial SequenceSynthetic peptide sequence 55Leu Leu Gln
Leu Gln1 5566PRTArtificial SequenceSynthetic peptide sequence 56Leu
Leu Gln Leu Leu Gln1 5575PRTArtificial SequenceSynthetic peptide
sequence 57Leu Leu Gln Gly Arg1 5
* * * * *