U.S. patent application number 13/838438 was filed with the patent office on 2014-03-06 for multivalent and monovalent multispecific complexes and their uses.
This patent application is currently assigned to Zyngenia, Inc.. The applicant listed for this patent is Zyngenia, Inc.. Invention is credited to David M. Hilbert, Peter Kiener, David Lafleur, Viktor ROSCHKE.
Application Number | 20140065142 13/838438 |
Document ID | / |
Family ID | 46229931 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065142 |
Kind Code |
A1 |
ROSCHKE; Viktor ; et
al. |
March 6, 2014 |
Multivalent and Monovalent Multispecific Complexes and Their
Uses
Abstract
Compositions containing multivalent and monovalent multispecific
complexes having scaffolds such as antibodies that support such
binding functionalities are described. The use of and methods of
compositions containing multivalent and monovalent multispecific
complexes having scaffolds, such as antibodies, that support such
binding functionalities are also described.
Inventors: |
ROSCHKE; Viktor; (Bethesda,
MD) ; Lafleur; David; (Washington, DC) ;
Hilbert; David M.; (Bethesda, MD) ; Kiener;
Peter; (Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zyngenia, Inc. |
Gaithersburg |
MD |
US |
|
|
Assignee: |
Zyngenia, Inc.
Gaithersburg
MD
|
Family ID: |
46229931 |
Appl. No.: |
13/838438 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US12/39469 |
May 24, 2012 |
|
|
|
13838438 |
|
|
|
|
61489249 |
May 24, 2011 |
|
|
|
61597714 |
Feb 10, 2012 |
|
|
|
61610831 |
Mar 14, 2012 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
435/375; 530/387.3; 536/23.4 |
Current CPC
Class: |
C07K 2319/70 20130101;
C07K 16/241 20130101; C07K 16/2839 20130101; C07K 16/40 20130101;
C07K 2319/01 20130101; C07K 2319/35 20130101; C07K 2317/732
20130101; C07K 2317/31 20130101; C07K 16/24 20130101; C07K 14/47
20130101; C07K 16/2809 20130101; C07K 2317/60 20130101; C07K
2319/30 20130101; C07K 2319/74 20130101; C07K 16/2863 20130101;
A61K 45/06 20130101; A61K 39/3955 20130101; C07K 16/22 20130101;
C07K 16/2803 20130101; C07K 16/2848 20130101; C07K 2317/90
20130101; A61K 2039/505 20130101; C07K 16/32 20130101; C07K 14/00
20130101; C07K 2319/33 20130101; C07K 2319/40 20130101; C07K
2317/76 20130101; C07K 2317/21 20130101; C07K 7/08 20130101; C07K
2317/24 20130101; C07K 16/468 20130101; C07K 2317/73 20130101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 435/375; 536/23.4 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; C07K 16/22 20060101
C07K016/22; A61K 45/06 20060101 A61K045/06; C07K 14/00 20060101
C07K014/00; C07K 16/24 20060101 C07K016/24 |
Claims
1. A complex comprising an antibody and at least one modular
recognition domain (MRD), wherein the antibody and the MRD bind to
different targets or epitopes on the same cell or molecule, wherein
the MRD binding agonizes or antagonizes the MRD target under
physiological conditions, and wherein said MRD does not bind to and
agonize or antagonize said MRD target under physiological
conditions in the absence of said antibody.
2. The complex of claim 1, wherein in the absence of said antibody,
the MRD binds the MRD target with an EC50 of greater than 0.01,
0.1, 0.5 or 0.7 nM under physiological conditions.
3. The complex of claim 1, wherein the antibody and the MRD bind to
different targets or epitopes on a heteromeric or homomeric
protein.
4. A method for inhibiting the growth of a cell comprising
contacting the cell with a multispecific and multivalent complex
comprising an antibody and at least one modular recognition domain
(MRD), and a protein kinase inhibitor.
5. A method for inhibiting angiogenesis in a patient comprising
administering to said patient a therapeutically effective amount of
a multispecific and multivalent complex comprising an antibody and
at least one modular recognition domain (MRD), and a protein kinase
inhibitor.
6. The method of claim 4, wherein the antibody binds a target
selected from: VEGF, VEGFR1, EGFR, ErbB2, cMET, FGFR1, and
FGFR2.
7. (canceled)
8. (canceled)
9. (canceled)
10. The method of claim 4, wherein the protein kinase inhibitor is
a member selected from: imatinib, gefitinib, vandetanib, erlotinib,
sunitinib, lapatinib, and sorafenib.
11. A method for treating a patient having a disease or disorder
comprising administering to said patient a therapeutically
effective amount of a multispecific and multivalent complex
comprising an antibody and at least one modular recognition domain
(MRD), and a protein kinase inhibitor.
12. The method of claim 11, wherein the disease or disorder is
cancer.
13. The method of claim 11, wherein the disease or disorder is a
disease or disorder of the immune system.
14. (canceled)
15. (canceled)
16. The method of claim 11, wherein the antibody binds to a target
selected from: TNF, VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1,
FGFR2, and CD20.
17. (canceled)
18. (canceled)
19. (canceled)
20. The method of claim 11, wherein the protein kinase inhibitor is
a member selected from: imatinib, gefitinib, vandetanib, erlotinib,
sunitinib, lapatinib, and sorafenib.
21. The method of claim 11, wherein the protein kinase inhibitor is
a member selected from: lestaurtinib, tofacitinib, ruxolitinib,
SB1518, CYT387, LY3009104, TG101348, fostamatinib, BAY 61-3606, and
sunitinib.
22. A multivalent and multispecific complex comprising an antibody
and at least one modular recognition domain (MRD), wherein the
complex has a single binding site for a cell surface target.
23. (canceled)
24. The complex of claim 22, wherein the complex has 2, 3, 4, 5 or
more single binding sites for different targets.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. The complex of claim 22, wherein the complex has multiple
binding sites for a target on a diseased cell.
31. (canceled)
32. The complex of claim 22, wherein the complex has multiple
binding sites for a target on a tumor cell.
33. (canceled)
34. The complex of claim 22, wherein the complex has multiple
binding sites for a target on an immune cell.
35. (canceled)
36. The complex of claim 22, wherein the complex binds a target on
a leukocyte and a target on a tumor cell.
37. (canceled)
38. (canceled)
39. (canceled)
40. The complex of claim 22, wherein the complex has a binding site
for a target associated with an endogenous blood brain barrier
(BBB) receptor mediated transport system.
41. The complex of claim 40, wherein the complex has multiple
binding sites for a target associated with an endogenous BBB
receptor mediated transport system.
42. (canceled)
43. The complex of claim 22, wherein the single binding site is an
MRD.
44. The complex of claim 22, wherein the single binding site is an
antigen binding domain.
45. (canceled)
46. A polynucleotide encoding a heavy chain or light chain of the
MRD containing antibody in the complex of claim 22.
47. (canceled)
48. (canceled)
49. A pharmaceutical composition comprising the complex of claim
22.
50. A method for inhibiting the growth of a cell comprising
contacting the cell with the complex of claim 22.
51. A method for inhibiting angiogenesis in a patient comprising
administering to said patient a therapeutically effective amount of
the complex of claim 22.
52. A method for treating a patient having cancer comprising
administering to said patient a therapeutically effective amount of
the complex of claim 22.
53. A method for treating a patient having a disease or disorder of
the immune system comprising administering to said patient a
therapeutically effective amount of the complex of claim 22.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] Related applications U.S. 61/489,249, filed May 24, 2011,
U.S. 61/597,714, filed Feb. 10, 2012, U.S. 61/610,831, filed Mar.
14, 2102, and International Application No. PCT/US2012/039469,
filed May 24, 2012 are herein incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to compositions containing
multivalent multispecific complexes and to compositions containing
multivalent and monovalent multispecific complexes having
scaffolds, such as antibodies, that support such binding
functionalities. The invention also generally relates to methods of
making these multispecific compositions and the diagnostic and
therapeutic uses of these compositions.
[0004] 2. Background
[0005] In recent years, drag discovery efforts have primarily
focused on identifying agents that modulate preselected individual
targets. However, agents directed to individual targets frequently
show limited efficacies and poor safety and resistance profiles, as
a result of the robustness, redundancy, crosstalk, compensatory
signaling networks and anti- or counter-signaling network
activities associated with the therapeutic target. Consequently,
drug discovery efforts have increasingly been directed toward the
discovery of new multicomponent based therapies.
[0006] The development of bispecific or multi-specific molecules
that target two or more targets simultaneously offers a novel and
promising solution for discovering new systems-oriented
multitargeted agents demonstrating improved efficacy and
pharmacological properties over conventional monotherapies.
Numerous attempts to develop multispecific molecules have been
based on immunoglobulin-like domains or subdomains. For example,
traditionally, bispecific antibodies have been prepared by
chemically linking two different monoclonal antibodies or by fusing
two hybridoma cell lines to produce a hybrid-hybridoma. Other
immunoglobulin-like domain-based technologies that have created
multispecific, and/or multivalent molecules include dAbs,
diabodies, TandAbs, nanobodies, BiTEs, SMIPs, DNLs, Affibodies,
Fynorners, Kunitz Domains, Albu-dabs, DARTs, DVD-IG, Covx-bodies,
peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knobs-in-Holes,
DuoBodies.TM. and triomAbs. Although each of these molecules may
bind one or more targets, they each present challenges with respect
to retention of typical Ig function (e.g., half-life, effector
function), production (e.g., yield, purity), valency, simultaneous
target recognition, and bioavailability.
[0007] Other attempts to generate multispecific and multivalent
molecules have relied on alternative scaffolds, based VASP
polypeptides, Avian pancreatic polypeptide (aPP), Tetranectin
(based on CTLD3), Affilin (based on .gamma.B-crystallin/ubiquitin)
knottins, SH3 domains, PDZ domains, Tendamistat, Transferrin, an
ankyrin consensus repeat domain (e.g., DARPins), lipocalin protein
folds (e.g., Duocalins), fibronectin (see for example, US
Application Publ. Nos. 2003/0170753 and 20090155275 which are
herein incorporated by reference), a domain of protein A (e.g.,
Affibodies) thioredoxin. Other attempts have relied on alternative
scaffolds fuse of associate polypeptides of interest with albumin
(e.g., ALBUdAb (Domantis/GSK) and ALB-Kunitz (Dyax)), unstructured
repeat sequences of 3 or 6 amino acids (e.g., PASylation.RTM.
technology and XTEN.RTM. technology), and sequences containing
elastin-like repeat domains (see for example, U.S. Pat. Appl. No.
61/442,106, which is herein incorporated by reference). To date,
these technologies have demonstrated limited clinical potential as
robust platforms for developing diverse multispecific and
multivalent therapeutic compositions.
[0008] The genetic complexity of most human malignancies and other
disorders strongly suggest that interfering with a single target or
pathway associated with these disorders is unlikely to produce
optimal or sustained therapeutic benefit. There is, therefore, a
great need for developing multispecific and multivalent
therapeutics such as multispecific antibodies that are capable of
interfering with the activity of multiple targets and/or signaling
mechanisms in or to optimize the therapeutic benefits of treatments
directed towards these disorders.
BRIEF SUMMARY OF THE INVENTION
[0009] The invention relates to compositions containing multivalent
as well as multivalent and monovalent, multispecific complexes
having scaffolds, such as, antibodies, that support such binding
functionalities. The invention is based in part on the surprising
discovery that multispecific and multivalent binding compositions,
such as those generated using the ZYBODY.TM. platform (Zyngenia,
Inc.; see, e.g., Intl. Pub. No. WO 2009/088805 which is herein
incorporated by reference) demonstrate dramatic synergistic
biological activity compared to conventional monotherapy
combinations. This synergistic activity is expected to extend to
novel therapies, for treating or preventing cancer, diseases or
disorders of the immune system (e.g., autoimmune diseases such as,
rheumatoid arthritis, and IBD), skeletal system (e.g.,
osteoporosis), cardiovascular system (e.g., stroke, heart disease),
nervous system (e.g., Alzheimer's), infectious disease (e.g., HIV),
and other diseases or disorders described herein or otherwise known
in the art.
[0010] In one embodiment the invention is directed to treating a
disease or disorder by administering a therapeutically effective
amount of a multivalent and monovalent multispecific composition to
a patient in need thereof. In a further embodiment, the invention
is directed to treating a disease or disorder by administering a
therapeutically effective amount of a multivalent and multispecific
MRD-containing antibody to a patient in need thereof.
[0011] In one embodiment, the multivalent and monovalent
multispecific composition contains 2 binding sites for three or
more targets. In an additional embodiment, the multivalent and
monovalent multispecific composition contains 2 binding sites for
four or more targets. In another embodiment, the multivalent and
monovalent multispecific composition contains 2 binding sites for
five or more targets. According to some embodiments, at least 1, 2,
3, 4 or more of the targets are located on a cell surface.
According to some embodiments, at least 1, 2, 3, 4 or more of the
targets are soluble targets (e.g., chemokines, cytokines, and
growth factors). In additional embodiments, the multivalent and
monovalent multispecific composition binds 1, 2, 3, 4 or more of
the targets described herein.
[0012] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition are associated
with cancer. In a further embodiment the targets bound by the
multivalent and monovalent multispecific composition are associated
with 1, 2, 3, 4 or more different signaling pathways or modes of
action associated with cancer.
[0013] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition are associated
with a disease or disorder of the immune system. In a further
embodiment the targets bound by the multivalent and monovalent
multispecific composition are associated with 1, 2, 3, 4 or more
different signaling, pathways or modes of action associated with a
disease or disorder of the immune system.
[0014] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition are associated
with a disease or disorder of the skeletal system (e.g.,
osteoporosis), cardiovascular system, nervous system, or an
infectious disease. In a further embodiment the targets bound by
the multivalent and monovalent multispecific composition are
associated with 1, 2, 3, 4, 5 or more different signaling pathways
or modes of action associated with a disease or disorder of the
skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease. In a further embodiment,
the multivalent and monovalent multispecific composition binds at
least 1, 2, 3, 4, 5 or more of the targets described herein.
[0015] In one embodiment, the multivalent and monovalent
multispecific composition contains 2 binding sites for three or
more targets. In an additional embodiment, the multivalent and
monovalent multispecific composition contains 2 binding sites for
four or more targets. In an additional embodiment, the multivalent
and monovalent multispecific composition contains 2 binding sites
for five or more targets.
[0016] In one embodiment, the multivalent and monovalent
multispecific composition contains 2 binding sites for three or
more targets. In an additional embodiment, the multivalent and
monovalent multispecific composition contains 2 binding sites for
four or more targets. In another embodiment, the multivalent and
monovalent multispecific composition contains 2 binding sites for
five or more targets. According to some embodiments, at least 1, 2,
3, 4, or more of the targets are associated with the cell membrane.
According to some embodiments, at least 1, 2, 3, 4, or more of the
targets are soluble targets (e.g., chemokines, cytokines, and
growth factors). In additional embodiments, the multivalent and
monovalent multispecific composition binds 1, 2, 3, 4, or more of
the targets described herein.
[0017] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition are associated
with cancer. In a further embodiment the targets bound by the
multivalent and monovalent multispecific composition are associated
with 1, 2, 3, 4, or more different signaling pathways or modes of
action associated with cancer.
[0018] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition are associated
with a disease or disorder of the immune system. In, a farther
embodiment the targets bound by the-multivalent and monovalent
multispecific composition are associated with 1, 2, 3, 4, or more
different signaling pathways or modes of action associated with a
disease or disorder of the immune system.
[0019] In additional embodiments, the multivalent and monovalent
multispecific composition binds (1) a target on a cell or tissue of
interest (e.g., a tumor associated antigen on a tumor cell, an
immune cell, a diseased cell or an infectious agent) and (2) a
target on an effector cell. According to one embodiment, the
binding of one or more targets by the multivalent and monovalent
multispecific composition directs an immune response to a cell,
tissue, infectious agent, or other location of interest in a
patient. In some embodiments the effector cell is a leukocyte, such
as a T cell or natural killer cell. In other embodiments, the
effector cell is an accessory cell, such as a myeloid cell or a
dendritic cell.
[0020] In additional embodiments, the multivalent and monovalent
multispecific composition binds (1) a target on a cell or tissue of
interest (e.g., a tumor associated antigen on a tumor cell, an
immune cell, a diseased cell or an infectious agent) and (2) a
target on a leukocyte, such as a T-cell receptor molecule.
According to one embodiment, the binding of one or more targets by
the multivalent and monovalent multispecific composition directs an
immune response to an infectious agent, cell, tissue, or other
location of interest in a patient. For example, in some embodiments
the multivalent and monovalent multispecific composition binds a
target on the surface of a T cell. In particular embodiments, the
composition binds a CD3 target selected from CD3 delta, CD3
epsilon, CD3 gamma, CD3 zeta, TCR alpha, TCR beta, and multimers of
proteins in the CD3 (TCR) complex. In specific embodiments the
multivalent and monovalent multispecific composition binds CD3. In
other embodiments, the multivalent and monovalent multispecific
composition binds CD2. In additional embodiments, the multivalent
and monovalent multispecific composition binds a target expressed
on a natural killer cell. Thus, in some embodiments, the
multivalent and monovalent multispecific composition binds a target
selected from: CD2, CD56, and CD161.
[0021] In additional embodiments, the multivalent and monovalent
multispecific composition hinds a target expressed on an accessory
(e.g., myeloid) cell. In some embodiments, the multivalent and
monovalent multispecific composition binds a target selected from:
CD64 (i.e., Fc gamma RI), an MHC class 2 and its invariant chain,
TLR1, TLR2, TLR4, TLR5, and TLR6.
[0022] In further embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD containing antibody) has a
single binding site (i.e., is monovalent) for a target. In some
embodiments, the multivalent and monovalent multispecific
composition has a single binding site for a target on a leukocyte,
such as a T-cell (e.g., CD3), and multiple binding sites (i.e., is
multivalent) for a target on, a cell or tissue of interest (e.g., a
tumor associated antigen on a tumor cell, such as a target
disclosed herein). In further embodiments, the multispecific
composition contains single binding sites for 2 different targets
(i.e., monovalently binds more than one different target). In
particular embodiments, the cell or tissue of interested is a
cancer cell, immune cell, diseased cell, or an infectious
agent.
[0023] In some embodiments, a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) has a
single binding site for CD3. In further embodiments, the
multivalent and monovalent multispecific composition has a single
binding site for CD3 and multiple binding sites for 1, 2, 3, 4, 5
or more different targets (e.g., a tumor antigen or other target
disclosed herein). In additional embodiments, the multispecific
composition has a single binding site for CD3 and a single binding
site for a different target (i.e., monovalently binds CD3 and a
different target). In other embodiments, a multivalent and
monovalent multispecific composition has a single binding site for
CD3 epsilon. In farther embodiments, the multivalent and monovalent
multispecific composition has a single binding site for CD3 epsilon
and multiple binding sites for 1, 2, 3, 4, 5 or more different
targets (e.g., a tumor antigen or other target disclosed herein).
In further embodiments, the multispecific composition has a single
binding site for CD3 epsilon and a single binding site for a
different target (i.e., monovalently binds CD3 epsilon and a
different target). In some embodiments, the multivalent and
monovalent multispecific composition has multiple binding sites for
a target on a cancer cell selected from breast cancer, colorectal
cancer, endometrial cancer, kidney (renal cell) cancer, lung
cancer, melanoma, Non-Hodgkin Lymphoma, leukemia, prostate cancer,
bladder cancer, pancreatic cancer, and thyroid cancer.
[0024] In further embodiments, the invention is directed to
treating a disease or disorder by administering a therapeutically
effective amount of a multivalent and monovalent multispecific
composition that has a single binding site for a target (i.e., that
monovalently binds a target) to a patient in need thereof. In some
embodiments, the administered multivalent and monovalent
multispecific composition has a single binding site for a target on
a leukocyte such as a T-cell (e.g., CD3). In further embodiments,
the administered multivalent and monovalent multispecific
composition has a single binding site for a target on a leukocyte
such as a T-cell (e.g., CD3) and multiple binding sites for (i.e.,
is capable of multivalently binding) a target located on a cell or
tissue of interest (e.g., a tumor antigen on a tumor cell). In
further embodiments, the multispecific composition has a single
binding site for a target on a leukocyte (e.g., CD3) and a single
binding site for a different target. In some embodiments, the cell
of interest is a tumor cell from a cancer selected from breast
cancer, colorectal cancer, endometrial cancer, kidney (renal cell)
cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia,
prostate cancer, bladder cancer, pancreatic cancer, and thyroid
cancer. In additional embodiments, the multivalent and monovalent
multispecific composition has multiple binding sites for a target
on a neurological tumor. In particular embodiments, the
neurological tumor is a glioma (e.g., a glioblastoma, glioblastoma
multiforme (GBM), and astrocytoma), ependymoma, oligodendroglioma,
neurofibroma, sarcoma, medulloblastoma, primitive neuroectodermal
tumor, pituitary adenoma, neuroblastoma or cancer of the meninges
(e.g., meningioma, meningiosarcoma and gliomatosis).
[0025] In further embodiments, the invention is directed to
treating a disease or disorder by administering to a patient in
need thereof, a therapeutically effective amount of a multivalent
and monovalent multispecific composition (e.g., an MRD-containing
antibody) that has a single binding site for a target (i.e., that
monovalently binds a target) and multiple binding sites for 1, 2,
3, 4, 5 or more different targets. In further embodiments, the
multivalent and monovalent multispecific composition has single
binding sites for 2 different targets. In some embodiments, the
multivalent and monovalent multispecific composition has multiple
binding sites for a target on a cancer cell selected from breast
cancer, colorectal cancer, endometrial cancer, kidney (renal cell)
cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma, leukemia,
prostate cancer, bladder cancer, pancreatic cancer, and thyroid
cancer.
[0026] In additional embodiments, the invention is directed to
treating a disease or disorder by administering to a patient in
need thereof, a therapeutically effective amount of a multivalent
and monovalent multispecific composition (e.g., an MRD-containing
antibody) that has a single binding site for CD3 (e.g., CD3
epsilon) that monovalently binds CD3 and multiple binding sites for
1, 2, 3, 4, 5 or more different targets located on a cell or tissue
of interest (e.g., a tumor antigen on a tumor cell). In some
embodiments, the administered multivalent and monovalent
multispecific composition has a single binding site for CD3 (e.g.,
CD3 epsilon) and a single binding site for a different target and
also has multiple binding sites for a target located on a cell or
tissue of interest (e.g., a tumor antigen on a tumor cell). In some
embodiments, the multivalent and monovalent multispecific
composition has multiple binding sites for a target on a cancer
cell selected from breast cancer, colorectal cancer, endometrial
cancer, kidney (renal cell) cancer, lung cancer, melanoma,
Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer,
pancreatic cancer, and thyroid cancer.
[0027] In further embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) has a
single binding site for (i.e., monovalently binds) a cell surface
target that requires multimerization for signaling. In some
embodiments, the multivalent and monovalent multispecific
composition has a single binding site for a growth factor receptor.
In other embodiments, the multivalent and monovalent multispecific
composition has a single binding sire for a TNF receptor
superfamily member. In additional embodiments, the multispecific
composition additionally has a single binding site for a different
target i.e., monovalently binds more than one different
target).
[0028] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) binds a
target associated with an endogenous blood brain barrier (BBB)
receptor mediated transport system and is capable of crossing to
the brain (cerebrospinal fluid) side of the BBB. In some
embodiments, the multivalent and monovalent multispecific
composition has two or more binding sites for a target antigen
associated with an endogenous BBB receptor mediated transport
system. In additional embodiments, the multivalent and monovalent
multispecific composition has a single binding site for a tai get
associated with an endogenous BBB receptor mediated transport
system (e.g., the insulin receptor, transferrin receptor, leptin
receptor, lipoprotein receptor, and the IGF receptor mediated
transport systems). In further embodiments, the multivalent and
monovalent multispecific composition additionally binds 1, 2, 3, 4,
5, or more targets located on, the brain side of the BBB. In
particular embodiments, the MRD-containing antibody binds 1, 2, 3,
4, 5, or more targets associated with a neurological disease or
disorder. In another embodiment, the multivalent and monovalent
multispecific composition is administered to a patient to treat a
brain cancer, metastatic cancer of the brain, or primary cancer of
the brain. In a further embodiment, the multivalent and monovalent
multispecific composition is administered to a patient to treat
brain injury, stroke, spinal cord injury, or to manage pain.
[0029] In additional embodiments, targets bound by the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) are associated with a disease or disorder of the skeletal
system (e.g., osteoporosis), cardiovascular system, nervous system,
or an infectious disease. In a further embodiment a targets bound
by the multivalent and monovalent multispecific composition are
associated with 1, 2, 3, 4, 5 or more different signaling pathways
or modes of action associated with one or more of the above
diseases or disorders. In a further embodiment, the multivalent and
monovalent multispecific composition binds 1, 2, 3, 4, 5 or more of
the targets described herein.
[0030] In one embodiment, the multivalent and monovalent
multispecific composition is a ZYBODY.TM. (referred to herein as an
"MRD-containing antibody," or the like). In a further embodiment,
the MRD-containing antibody contains binding sites for three or
more targets. In an additional embodiment, the MRD-containing
antibody contains 2 binding sites for four or more targets. In an
additional embodiment, the MRD-containing antibody contains 2
binding sites for five or more targets.
[0031] In one embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
2 binding sites for three or more targets. In an additional
embodiment, the multispecific composition (e.g., MRD-containing
antibody) contains 2 binding sites for four or more targets. In
another embodiment, the multispecific composition (e.g.,
MRD-containing antibody) contains 2 binding sites for five or more
targets. According to some embodiments, at least 1, 2, 3, 4 or more
of the targets are located on a cell surface. According to some
embodiments, at least 1, 2, 3, 4 or more of the targets are soluble
targets (e.g., chemokines, cytokines, and growth factors). In
additional embodiments, the MRD-containing antibody binds at least
1, 2, 3, 4, 5 or more of the targets described herein.
[0032] In additional embodiments, the targets bound by the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) are associated with cancer. In a further
embodiment the targets bound by MRD-containing antibody are
associated with 1, 2, 3, 4 or more different signaling pathways or
modes of action associated with cancer.
[0033] In additional embodiments, a target bound by the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) is associated with a disease or disorder of the immune
system. In a further embodiment the targets bound by the
MRD-containing antibody are associated with 1, 2, 3, 4, 5 or more
different signaling pathways or modes of action associated with a
disease or disorder of the immune system.
[0034] In additional embodiments, a target bound by the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) is associated with a disease or disorder of the skeletal
system, cardiovascular system, nervous system, or an infectious
disease. In a further embodiment a target bound by the
MRD-containing antibody is associated with 1, 2, 3, 4 or more
different signaling pathways or modes of action associated with one
or more of the above diseases or disorders. In another embodiment,
the MRD-containing antibody binds 1, 2, 3, 4 or more of the targets
described herein.
[0035] The multivalent and multispecific compositions of the
invention (e.g., MRD-containing antibodies) provide the ability to
selectively target multiple targets (e.g., receptors and
microenvironment associated targets) having for example, different,
overlapping, or redundant mechanisms of action associated with the
etiology or pathophysiology of a disease or disorder.
[0036] In additional embodiments, the invention encompasses a
multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) that is covalently or otherwise associated
with a cytotoxic agent. According to some embodiments, the cytoxic
agent is covalently attached to an MRD-containing antibody by a
linker. According to some embodiments, the cytotoxic agent is a
chemotherapeutic agent, growth inhibitory agent, toxin (e.g., an
enzymatically active toxin of bacterial, fangal, plant, or animal
origin, or fragments thereof), radioactive isotope (i.e., a
radioconjugate), or prodrug. The compositions of the invention are
optionally linked to the cytotoxic agent by a linker. In particular
embodiments, a linker attaching the multivalent and monovalent
multispecific composition and the cytotoxic agent is cleavable by a
protease. In particular embodiments, a linker attaching the
multivalent and monovalent multispecific composition and the
cytotoxic agent is cleavable under low pH or reducing conditions.
Methods of using composition-cytoxic agent compositions of the
invention (e.g., MRD-containing antibody drug conjugates) are also
encompassed by the invention.
[0037] In additional embodiments, the multivalent and multispecific
compositions is covalently or otherwise associated with a cytotoxic
agent selected from, for example, a toxin, a chemotherapeutic
agent, a drug moiety (e.g., a chemotherapeutic agent or prodrug),
an antibiotic, a radioactive isotope, a chelating ligand DOTA,
DOTP, DOTMA, DTPA and TETA), and a nucleolytic enzyme. In
particular embodiments, the cytotoxic agent is selected from
auristatin and dolostantin, MMAE, MMAF, and a maytansinoid
derivative (e.g., the DM1 (N(2')-deacetyl-N
(2')-(3-mercapto-1-oxopropyl)-maytansine), DM3
(N(2')-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine), and DM4
(N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).
[0038] In further embodiments, a multivalent and monovalent
multispecific composition of the invention (e.g., an MRD-containing
antibody) is administered in combination with a multitargeting
therapeutic. In one embodiment, a multivalent and monovalent
muitispecific composition is administered in combination with a
multitargeting protein kinase inhibitor. In another embodiment, a
multivalent and monovalent multispecific composition is
administered in combination with an NFKB inhibitor. In an
additional embodiment, a multivalent and monovalent multispecific
composition is administered in combination with an HDAC inhibitor.
In a further embodiment, a multivalent and monovalent multispecific
composition is administered in combination with an HSP70 or HSP90
inhibitor. In a further embodiment, a multivalent and monovalent
multispecific composition is administered in combination with
chemotherapy.
[0039] In some embodiments, a multivalent and monovalent
multispecific composition of the invention (e.g., an MRD-containing
antibody) is administered in combination with a monospecific
therapeutic (e.g., a monoclonal antibody).
[0040] In some embodiments, a multivalent and monovalent
multispecific composition of the invention is a full-length
antibody comprising at least one modular recognition domain (MRD).
In some embodiments, the full-length antibody comprises multiple
MRDs. In additional embodiments, the full-length antibody comprises
more than one type of MRD (i.e., multiple MRDs having the same or
different specificities). Also embodied in the present invention
are variants and derivatives of such antibody complexes.
[0041] The MRDs of the MRD containing antibodies can be operably
attached to the antibodies at any location on the antibody (e.g.,
the amino terminus of the heavy chain or light chain or the
carboxyl terminus of the heavy chain or light chain), can be linked
at the same or different termini, and are optionally operably
linked to one another or to the antibody by a linker.
[0042] The antibodies of the MRD containing antibodies can be any
immunoglobulin molecule that binds to an antigen and can be of any
type, class, or subclass. In some embodiments, the antibody is
humanized or human. In other embodiments, the antibodies also
include modifications that do not interfere with their ability to
bind antigen. In particular embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
include modifications that increase ADCC, decrease ADCC, increase
CDC, or decrease CDC, that increase antibody half-life, or decrease
antibody half-life compared to the antibody without the
modification.
[0043] The antibodies of the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) of the invention can
be any antibody that binds to a target of therapeutic or diagnostic
value. In preferred embodiments, the antibody of the MRD-containing
antibody binds to a validated target. In some embodiments, the
antibodies corresponding to the MRD containing antibodies are in
clinical trials for regulatory approval. In some embodiments, the
antibodies corresponding to the MRD containing antibodies are
marketed.
[0044] In one embodiment, the antibody binds to a cell surface
antigen. In another embodiment, the antibody binds to an angiogenic
factor. In a further embodiment, the antibody binds to an
angiogenic receptor.
[0045] In some embodiments, the antibody of the MRD-containing
antibody binds to a target selected from: EGFR, ErbB2, ErbB3,
ErbB4, CD20, insulin-like growth factor-I receptor, VEGF, VEGF-R
and prostate specific membrane antigen. In additional embodiments
the antibody of the MRD-containing antibody binds to VEGF, VEGFR1,
EGFR, ErbB2, IGF-IR, cMET, FGFR1, FGFR2, and CD20.
[0046] In one embodiment, the antibody of the MRD-containing
antibody binds to EGFR. In another specific embodiment, the
antibody is Erbitux.RTM., nimotuzumab, or zalutumumab (e.g.,
Genmab). In another embodiment, the antibody binds to the same
epitope as Erbitux.RTM. antibody or competitively inhibits binding
of the Erbitux.RTM. antibody to EGFR. In a further specific
embodiment, the antibody is the Erbitux.RTM. antibody. In one
specific embodiment, the antibody binds to the same epitope as
Erbitux.RTM., nimotuzumab, zalutumumab (e.g., Genmab) antibody. In
another specific embodiment, the antibody component, MRD component,
and/or MRD-containing antibody competitively inhibits binding of
Erbitux.RTM., nimotuzumab, zalutumumab antibody to EGFR.
[0047] In one embodiment, an MRD-containing antibody binds EGFR and
a target selected from: HGF, CD64, CDCP1, RON, cMET, ErbB2, ErbB3,
IGF1R, PLGF, RGMa, PDGFRa, PDGFRh, VEGFR1, VEGFR2, TNFRSF10A (DR4),
TNFRSF10B (DR5), IGF1,2, IGF2, CD3, CD4, NKG2D and tetanus toxoid.
In some embodiments, the multivalent and monovalent multispecific
composition (e.g. MRD-containing antibodies) binds at least 1, 2,
3, 4, 5 or more of these targets. In specific embodiments, the
antibody component of the MRD-containing antibody binds EGFR. In
further embodiments, the antibody component of the MRD-containing
antibody is nimotuzumab, zalutumumab. In specific embodiments, the
antibody component of the MRD-containing antibody is
Erbitux.RTM..
[0048] In a specific embodiment, the antibody of the MRD-containing
antibody binds to ErbB2. In one embodiment, the antibody is
HERCEPTIN.RTM. (trastuzumab) antibody or competitively inhibits
HERCEPTIN.RTM. (trastuzumab) antibody binding to ErbB2.
[0049] In another specific embodiment, the antibody binds to VEGF.
In another specific embodiment, the antibody binds to the same
epitope as AVASTIN.RTM. (bevacizumab) antibody or competitively
inhibits AVASTIN.RTM. antibody. In a further specific embodiment,
the antibody is the AVASTIN.RTM. antibody.
[0050] In some embodiments, the antibody binds to a target that is
associated with a disease or disorder of the immune system. In one
embodiment, the antibody binds to TNF. In another specific
embodiment, the antibody binds to the same epitope as HUMIRA.RTM.
(adalimumab) antibody or competitively inhibits HUMIRA.RTM.
antibody. In a further specific embodiment, the antibody is the
HUMIRA.RTM. antibody. In one embodiment, the antibody binds to TNF.
In another specific embodiment, the antibody binds to the same
epitope as SIMPONI.TM. (golimumab) antibody or competitively
inhibits SIMPONI.TM. antibody. In a further specific embodiment,
the antibody is the SIMPONI.TM. antibody.
[0051] In some embodiments, the antibody component of the MRD
containing antibody binds to a target that is associated with a
disease or disorder of the metabolic, cardiovascular,
musculoskeletal, neurological, or skeletal system. In other
embodiments, the antibody component of the MRD containing antibody
binds to a target that is associated with yeast, fungal, viral or
bacterial infection or disease.
[0052] In one embodiment, the MRD is about 2 to 150 amino acids. In
another embodiment, the MRD is about 2 to 60 amino acids. MRDs can
be linked to an antibody or other MRDs directly or through a
linker. The MRDs can be any target binding peptide. In some
embodiments, the MRD target is a soluble factor. In other
embodiments, the MRD target is a transmembrane protein such as a
cell surface receptor. In another embodiment, the target of the MRD
is a cellular antigen. In a specific embodiment, the target of the
MRD is CD20.
[0053] In another embodiment, the target of the MRD is an integrin.
In one aspect, the peptide sequence of the integrin targeting MRD
is YCRGDCT (SEQ ID NO:3). In another aspect, the peptide sequence
of the integrin targeting MRD, is PCRGDCL (SEQ ID NO:4). In yet
another aspect, the peptide sequence of the integrin targeting MRD
is TCRGDCY (SEQ ID NO:5). In another aspect, the peptide sequence
of the integrin targeting MRD is LCRGDCF (SEQ ID NO:5).
[0054] In an additional embodiment, the target of the MRD is an
angiogenic cytokine. In one aspect, the peptide sequence of the
angiogenic cytokine targeting (i.e., binding) MRD is
MGAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:7).
[0055] In one embodiment, the target of the MRD is ErbB2. In
another embodiment, the target to which the MRD binds is ErbB3. In
an additional embodiment, the target to which the MRD binds is
tumor-associated surface antigen or an epithelial cell adhesion
molecule (Ep-CAM).
[0056] In one embodiment, the target to which the MRD binds is
VEGF. In one aspect, the peptide sequence of the VEGF targeting MRD
is VEPNCDIHVMWEWECFERL (SEQ ID NO:13).
[0057] In one embodiment, the target to which the MRD binds is an
insulin-like growth factor-I receptor (IGF1R). An illustrative
IGF1R targeting MRD includes, for example, a peptide sequence
having the formula: NFYQCIDLLMAYPAEKSRGQWQECRTGG (SEQ ID
NO:37);
[0058] In one embodiment, the target of the MRD is a tumor antigen.
The "tumor antigen" as used herein may be understood as both those
antigens (including mutations) exclusively expressed on tumor cells
(i.e., tumor-specific antigens) and those antigens expressed on
tumor cells and normal cells (e.g., antigens overexpressed on tumor
cells).
[0059] In one embodiment, the target of the MRD is an epidermal
growth factor receptor (EGFR). In another embodiment of the present
invention, the target of the MRD is an angiogenic factor. In an
additional embodiment, the target of the MRD is an angiogenic
receptor.
[0060] In another embodiment, the MRD is a vascular homing
peptide.
[0061] In one embodiment, the target of the MRD is a nerve growth
factor.
[0062] In another embodiment, the antibody and/or MRD binds to
EGFR, ErbB2, ErbB3, ErbB4, CD20, insulin-like growth factor-I
receptor, or prostate specific membrane antigen.
[0063] The present invention also relates to an isolated
polynucleotide comprising a nucleotide sequence encoding an
MRD-containing antibody. In one aspect, a vector comprises a
polynucleotide sequence encoding an MRD-containing antibody. In
another aspect, the polynucleotide sequence encoding an
MRD-containing antibody is operatively linked with a regulatory
sequence that controls expression on the polynucleotide. In an
additional aspect, a host cell comprises the polynucleotide
sequence encoding an MRD-containing antibody.
[0064] Methods of making multivalent and multispecific compositions
(e.g., MRD-containing antibodies) are also provided, as are the use
of these MRD-antibody fusions in diagnostic and therapeutic
applications. The present invention also relates to methods of
designing and making multivalent and multispecific compositions
(e.g., MRD-containing antibodies) having a full-length antibody
comprising a MRD. In one aspect, the MRD is derived from a phage
display library. In another aspect, the MRD is derived from natural
ligands. In another aspect, the MRD is derived from yeast display
of RNA display technology.
[0065] The present invention also relates to a method of treating
or preventing a disease or disorder in a subject (patient) in need
thereof, comprising administering an antibody comprising an MRD to
the subject (patient). In one aspect, the disease is cancer. In
another aspect, undesired angiogenesis in inhibited. In another
aspect, angiogenesis is modulated. In yet another aspect, tumor
growth is inhibited.
[0066] Certain embodiments provide for methods of treating or
preventing a disease, disorder, or injury comprising administering
to a patient in need thereof, a therapeutically effective amount of
a multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) to a patient in need thereof. In some
embodiments, the disease, disorder or injury is cancer. In other
embodiments, the disease, disorder or injury is a disorder of the
immune system. In one embodiment, the disorder of the immune system
is inflammation. In another embodiment, the disorder of the immune
system is an autoimmune disease. In an additional embodiment, the
disorder of the immune system is selected from the group consisting
of: rheumatoid arthritis, Crohn's disease, systemic lupus
erythematous, inflammatory bowel disease, psoriasis, diabetes
ulcerative colitis, and multiple sclerosis. In one embodiment, the
disease, disorder or injury is a metabolic disease. In another
embodiment, the disease, disorder, or injury is an infectious
disease. In specific embodiments, the infectious disease is human
immunodeficiency virus (HIV) infection or AIDS, botulism, anthrax,
or clostridium difficile. In other embodiments, the disease,
disorder, or injury is neurological. In a specific embodiment, the
neurological disease, disorder or injury is pain. In a more
specific embodiment, the pain is, acute pain or chronic pain.
[0067] In another embodiment, a method of treatment or prevention
comprising administering an additional therapeutic agent along with
an antibody comprising an MRD is provided. In other embodiments,
the methods of treatment or prevention comprise administering an
antibody comprising more than one type of MRD.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0068] FIG. 1 shows the schematic representation of different
designs of multi-specific and multivalent molecules. MRDs are
depicted as triangles, circles, diamonds, and squares.
[0069] FIG. 2A shows a typical peptibody as a C-terminal fusion
with the heavy chain of Fc.
[0070] FIG. 2B shows an MRD containing antibody with a C-terminal
MRD fusion with the light chain of the antibody.
[0071] FIG. 2C shows an MRD containing antibody with an N-terminal
MRD fusion with the light chain of the antibody.
[0072] FIG. 2D shows an MRD containing antibody with unique MRD
peptides fused to each terminus of the antibody.
[0073] FIG. 3 depicts the results of an enzyme linked immunosorbent
assay (ELISA) in which integrin and Ang2 were bound by an
anti-integrin antibody (JC7U) fused to an Ang2 targeting MRD
(2xCon4).
[0074] FIG. 4 depicts the results of an ELISA in which, integrin
and Ang2 were bound by an anti-integrin antibody (JC7U) fused to an
Ang2 targeting MRD (2xCon4).
[0075] FIG. 5 depicts the results of an ELISA in which an
anti-Erb132 antibody was fused to an MRD which targets Ang2.
[0076] FIG. 6 depicts the results of an ELISA in which an Ang2
targeting MRD was fused to a hepatocyte, growth factor receptor
(cMET) binding antibody.
[0077] FIG. 7 depicts the results of an ELISA in which an integrin
targeting MRD was fused to an ErbB2-binding antibody.
[0078] FIG. 8 depicts the results of an ELISA in which an integrin
targeting MRD was fused to a hepatocyte growth factor receptor
binding antibody.
[0079] FIG. 9 depicts the results of an ELISA in which an
insulin-like growth factor-I receptor targeting MRD was fused to an
ErbB2-binding antibody.
[0080] FIG. 10 depicts the results of an ELISA in which a
VEGF-targeting MRD was fused to an ErbB2-binding antibody.
[0081] FIG. 11 depicts the results of an ELISA in which an integrin
targeting MRD was fused to a catalytic antibody.
[0082] FIG. 12 depicts the results of an ELISA in which an
Ang2-targeting MRD was fused to a catalytic antibody.
[0083] FIG. 13 depicts the results of an ELISA in which an integrin
targeting MRD and an Ang2 targeting MRD were fused to an
ErbB2-binding antibody.
[0084] FIG. 14 depicts the results of an ELISA in which an integrin
targeting MRD was fused to an ErbB2-binding antibody.
[0085] FIG. 15 depicts the results of an ELISA in which an
integrin, Ang2, or insulin-like growth factor-I receptor-targeting
MRD was fused to an ErbB2 or hepatocyte growth factor
receptor-binding antibody with a short linker peptide.
[0086] FIG. 16 depicts the results of an ELISA in which an
integrin, Ang2, or insulin-like growth factor-I receptor-targeting
MRD was fused to an ErbB2 or hepatocyte growth factor
receptor-binding antibody with a long linker peptide.
[0087] FIG. 17A depicts the dose response curves of MRD-maltose
binding protein (MBP) fusions assayed for direct binding to
Ang2.
[0088] FIG. 17B indicates MRD-MBP fasion proteins tested, the amino
acid sequence of the MRD, and the EC50 values (calculated using a 4
parameter fit). The MXD sequence motif in the MRD components of the
MRD-MBP fusions is underlined and mutated residues are in bold and
italics.
[0089] FIG. 18A depicts the results of an assay for direct binding
of a HERCEPTIN.RTM. based zybody (i.e. an MRD containing
HERCEPTIN.RTM. antibody sequences) antibody-MRDs and a
HERCEPTIN.RTM. antibody to Her2 (ErbB2) Fc in the presence of
biotinylated Ang2. Binding was detected with HRP-conjugated
anti-human kappa chain mAb.
[0090] FIG. 18B depicts the results of an assay for direct binding
of a HERCEPTIN.RTM. based zybody (i.e., an MRD containing
HERCEPTIN.RTM. antibody sequences) and a HERCEPTIN.RTM. antibody to
Her2 Fc in the presence of biotinylated Ang2. Binding was detected
with horseradish peroxidase (HRP)-conjugated streptavidin.
[0091] FIG. 19A depicts the results of an assay for direct binding
of anti body-MRDs and an AVASTIN.RTM. antibody to VEGF in the
presence of biotinylated Ang2. Binding was detected with
HRP-conjugated anti-human kappa chain mAb.
[0092] FIG. 19B depicts the results of an assay for direct binding
of antibody-MRDs and an AVASTIN.RTM. antibody to VEGF in the
presence of biotinylated Ang2. Binding was detected with
HRP-conjugated streptavidin.
[0093] FIG. 20A depicts the results of a flow cytometry assay which
demonstrates that antibody-MRDs simultaneously bind Her2 and Ang2
on BT-474 breast cancer cells.
[0094] FIG. 20B depicts binding of antibody-MRDs to HER2 on BT-474
breast cancer cells.
[0095] FIG. 21 depicts the results of an ELISA assay that
demonstrates the inhibitory effect of antibody-MRDs on TIE-2
binding to plate immobilized Ang2.
[0096] FIG. 22 depicts the results of a competitive binding assay
that demonstrates the inhibition of binding of biotinylated
antibody by antibody-MRD and unlabeled antibody.
[0097] FIG. 23 depicts the results of a competitive binding assay
that illustrates the inhibition of labeled antibody binding to
BT-474 cells by antibody-MRDs and unlabeled antibody.
[0098] FIG. 24A depicts the fitted dose curves illustrating the
inhibition of BT-474 cell proliferation by HERCEPTIN.RTM. with the
lm32 MRD (SEQ ID NO:8) fused to the heavy chain and
HERCEPTIN.RTM..
[0099] FIG. 24B depicts the fitted dose curves illustrating the
inhibition of BT-474 cell proliferation by HERCEPTIN.RTM. with the
lm32 MRD fused to the light chain and HERCEPTIN.RTM..
[0100] FIG. 24C depicts the fitted dose curves illustrating the
inhibition of BT-474 cell proliferation by HERCEPTIN.RTM. with the
2xcon4 MRD fused to the heavy chain and HERCEPTIN.RTM..
[0101] FIG. 25A depicts the results of a cytotoxicity assay
illustrating ADCC-mediated killing of BT-474 cells by
HERCEPTIN.RTM. with the Lm32 MRD fused to the heavy chain,
HERCEPTIN.RTM. with the lm32 MRD fused to the light chain, and
HERCEPTIN.RTM..
[0102] FIG. 25B depicts the results of a cytotoxicity assay
illustrating ADCC-mediated killing of BT-474 cells by
HERCEPTIN.RTM. with the 2xcon4 MRD fused to the heavy chain, and
HERCEPTIN.RTM..
[0103] FIG. 26A depicts the inhibition of HUVEC proliferation by
AVASTIN.RTM. with the lm32 MRD fused to the heavy chain and
AVASTIN.RTM. using HUVECs obtained from GlycoTech (Gaithersburg,
Md.).
[0104] FIG. 26B depicts the inhibition of HUVEC proliferation by
AVASTIN.RTM. with the lm32 MRD fused to the heavy chain and
AVASTIN.RTM. using HUVECs obtained from Lonza.
[0105] FIG. 27 depicts the effect of RITUXIMAB.RTM.,
HERCEPTIN.RTM., and an MRD-containing antibody on tumor volume in
vivo.
[0106] FIG. 28 depicts the increased effect of an
antibody-containing MRD on receptor phosphorylation and AKT
activation compared to the effect of an antibody in combination
with the MRD.
[0107] FIG. 29A depicts the increased effect of a bispecific
MRD-containing antibody on cell proliferation compared to the
effect of the antibody or the antibody in combination with the
MRD.
[0108] FIG. 29B depicts the increased effect of a pentaspecific
MRD-containing antibody on cell proliferation compared to the
effect of the antibody or the antibody in combination with the
MRD.
[0109] FIG. 30 depicts the increased efficacy of a HUMIRA antibody
containing an Ang2-binding MRD in an arthritis model compared to
HUMIRA.
[0110] FIG. 31 shows inhibition of EGF-induced signaling in SK-BR3
cells by zybodies.
[0111] FIG. 32 shows inhibition of Heregulin-induced signaling in
SK-BR3 zybodies.
[0112] FIG. 33 shows inhibition of EGF and Heregulin-induced
signaling in SK-BR3 cells by zybodies.
[0113] FIG. 34 shows a bar-graph (A) and flow-cytometry results (B)
depicting the down-regulation of EGFR expression on SK-BR3 cells by
zybodies.
[0114] FIG. 35 shows down-regulation of EGFR in SKBR3 cells by
zybodies.
DETAILED DESCRIPTION OF THE INVENTION
[0115] The following provides a description of antibodies
containing at least one modular recognition domain (MRD). The
linkage of one or more MRDs to an antibody results in a multi
specific molecule of the invention that retains structural and
functional properties of traditional antibodies or Fc optimized
antibodies and can readily be synthesized using conventional
antibody expression systems and techniques. The antibody can be any
suitable antigen-binding immunoglobulin, and the MRDs can be, any
suitable target-binding peptide. The MRDs can be operably linked to
any location on the antibody, and the attachment can be direct or
indirect (e.g., through a chemical or polypeptide linker).
Compositions of antibodies comprising an MRD, methods of
manufacturing antibodies comprising an MRD, and methods of using
antibodies comprising MRDs are also described in the sections
below.
[0116] The section headings used herein are for organizational
purposes only and are not to be construed as in any way limiting
the subject matter described.
[0117] Standard techniques may be used for recombinant DNA
molecule, protein, and antibody production, as well as for tissue
culture and cell transformation. Enzymatic reactions and
purification techniques are typically performed according to the
manufacturer's specifications or as commonly accomplished in the
art using conventional procedures such as those set forth in Harlow
et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988) and Sambrook et al., (Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)) (both herein incorporated by
reference), or as described herein. Unless specific definitions are
provided, the nomenclature utilized in connection with, and the
laboratory procedures and techniques of analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein, are those known and used in the art.
Standard techniques may be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, delivery, and
treatment of patients.
I. DEFINITIONS
[0118] The terms "multivalent and monovalent multispecific
complexes", "multivalent and multispecific complexes",
"MRD-containing antibodies," "antibody-MRD molecules,"
"MRD-antibody molecules," "antibodies comprising an MRD" and
"Zybodies" are used interchangeably herein and do not encompass a
peptibody. Each of these terms may also be used herein to refer to
a "complex" of the invention. Multivalent and monovalent
multispecific complexes can contain MRDs, antibodies, cytoxic
agents, and binding motifs in addition to MRDs that bind to one or
more targets. For example, a multivalent and monovalent
multispecific complex (e.g., an MRD-containing antibody) can
contain a portion of, or a derivative of, a binding sequence
contained in, antibody (e.g., a single binding domain, a ScFv, a
CDR region) and/or can also include a cytotoxic agent (e.g., a
therapeutic agent). Such molecules are also described in U.S.
Provisional Application No. 61/481,063, which is herein
incorporated by reference in its entirety. The terms "multivalent
and monovalent multispecific complex(es)" and "multivalent and
monovalent multispecific complexes" as used herein therefore refer
to compositions that are able to bind 2 or more targets and that
contain one binding site and/or multiple binding sites for
different epitopes. Thus, this term is intended to include
complexes containing multiple binding sites for each different
epitope bound by the complex, or alternatively, complexes that
contain at least one single binding site for a different epitope.
The different epitopes can be on the same or different targets.
Multivalent and monovalent multispecific complexes can be
multivalent and, multispecific and can therefore bind two or more
targets and have two or more binding sites for each of the targets
bound by the complex. Multivalent and monovalent multispecific
complexes can also have one (or more) single binding sites for one
(or more) target(s) and multiple binding sites for other targets
and accordingly, these complexes are monovalent (with respect to
the single binding site(s)), multivalent and multispecific.
Moreover, multivalent and monovalent multispecific complexes can be
monovalent and multispecific and thus, only contain single binding
sites for two or more different targets.
[0119] The term "multivalent and monovalent multispecific
complex-drug complex" or "MRD-containing antibody-cytotoxic agent"
as used herein, refers to a multivalent and monovalent
multispecific complex containing one or more cytotoxic agents.
[0120] The term "cytotoxic agent" as used herein, includes any
agent that is detrimental to cells including for example, substance
that inhibits or prevents the function of cells and/or causes
destruction of cells. The term is intended to include a
chemotherapeutic agent, a drug moiety (e.g., a cytokine or
prodrug), an antibiotic, a radioactive isotope, a chelating ligand
(e.g., DOTA, DOTP, DOTMA, DTPA and TETA), a nucleolytic enzyme, a
toxins such as a small molecule toxin or enzymatically active toxin
of bacterial, fungal, plant or animal origin, including fragments
and/or variants of these toxins. In particular embodiments, the
cytotoxic agent is a member selected from auristatin, dolostantin,
MMAE, MMAF, a maytansinoid derivative (e.g., the DM1
(N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine), DM3
(N(2')-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine) and DM4
(N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).
[0121] The term "antibody" is used herein to refer to
immunoglobulin molecules that are able to bind antigens through an
antigen binding domain (i.e., antibody combining site). The term
"antibody" includes polyclonal, oligoclonal (mixtures of
antibodies), and monoclonal antibodies, chimeric, single chain, and
humanized antibodies. The term "antibody" also includes human
antibodies. In some embodiments, an antibody comprises at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as VH) and a heavy chain
constant region. The heavy chain constant region is comprised of
three domains; CH1, CH2, and CH3. Each light chain is comprised of
a light chain variable region (abbreviated herein as VL) and a
light chain constant region. The light chain constant region is
comprised of one domain, CL. The VH and VL regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each VH and VL is
composed of three CDRs and four FRs, arranged from amino-terminus
to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. In other embodiments, the antibody is a homomeric
heavy chain antibody (e.g., camelid antibodies) which lacks the
first constant region domain (CH1) but retains an otherwise intact
heavy chain and is able to bind antigens through an antigen binding
domain. The variable regions of the heavy and light chains in the
antibody-MRD fusions of the invention contain a functional binding
domain that interacts with an antigen.
[0122] The term "monoclonal antibody" typically refers to a
population of antibody molecules that contain only one species of
antibody combining site capable of immunoreacting with a particular
epitope. A monoclonal antibody thus typically displays a single
binding affinity for any epitope with which it immunoreacts. As
used herein, a "monoclonal antibody" may also contain an antibody
molecule having a plurality of antibody combining sites (i.e., a
plurality of variable domains), each immunospecific for a different
epitope, e.g., a bispecific monoclonal antibody. Thus, as used
herein, a "monoclonal antibody" refers to a homogeneous antibody
population involved in the highly specific recognition and binding
of one or two (in the case of a bispecific monoclonal antibody)
antigenic determinants, or epitopes. This is in contrast to
polyclonal antibodies that typically include different antibodies
directed against different antigenic determinants. The term
"monoclonal antibody" refers to such antibodies made in any number
of manners including but not limited to by hybridoma, phage
selection, recombinant expression, yeast, and transgenic
animals.
[0123] A "dual-specific antibody" is used herein to refer to an
immunoglobulin molecule that contains dual-variable-domain
immunoglobulins, where the dual-variable-domain can be engineered
from any two monoclonal antibodies.
[0124] The term "chimeric antibodies" refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g., mouse, rat,
rabbit, etc.) with the desired specificity and/or affinity while
the constant regions are homologous to the sequences in antibodies
derived from another species (usually human) to avoid eliciting an
immune response in that species.
[0125] The term "humanized antibody" refers to forms of non-human
(e.g., murine) antibodies that are specific immunoglobulin chains,
chimeric immunoglobulins, or fragments thereof that contain minimal
non-human (e.g., murine) sequences. Typically, humanized antibodies
are human immunoglobulins in which residues from the
complementarity determining region (CDR) are replaced by residues
from the CDR of a non-human species (e.g., mouse, rat, rabbit,
hamster) that have the desired specificity and/or affinity (Jones
et al., Nature, 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536
(1988)). In some instances, the Fv framework region (FR) residues
of a human immunoglobulin are replaced with the corresponding
residues in an antibody from a non-human species that has the
desired specificity and/or affinity. The humanized antibody can be
further modified by the substitution of additional residues either
in the Fv framework region and/or within the replaced non-human
residues to refine and optimize antibody specificity, affinity,
and/or capability. In general, the humanized antibody will comprise
substantially all of at least one, and typically two or three,
variable domains containing all or substantially all of the CDR
regions that correspond to the non-human immunoglobulin whereas all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody can also
comprise an immunoglobulin constant region or domain (Fc),
typically that of a human immunoglobulin. Examples of methods used
to generate humanized antibodies are described in U.S. Pat. No.
5,225,539, U.S. Pat. No. 4,816,567, Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,
Taniguchi et al., EP 171496; Morrison et al., EP 173494,
WO86/01533; WO8702671; Boulianne et al., Nature 312:643 (1984); and
Neuberger et al., Nature 314:268 (1985), each of which is herein
incorporated by reference in its entirety.
[0126] As used herein, "human" antibodies include antibodies having
the amino acid sequence of a human immunoglobulin or one or more
human germlines and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more
human immunoglobulins and that do not express endogenous
immunoglobulins, as described infra and, for example in, U.S. Pat.
No. 5,939,598 by Kucherlapati et al., A human antibody may still be
considered "human" even if amino acid substitutions are made in the
antibody. Examples of methods used to generate human antibodies are
described in: Int. Appl. Publ. Nos. WO98/24893, WO92/01047,
WO96/34096, and WO96/33735; European Pat. No. 0 598 877; U.S. Pat.
Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016,
5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598; and
Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), which are
herein incorporated by reference.
[0127] An "antibody combining site" is that structural portion of
an antibody molecule comprised of heavy and light chain variable
and hypervariable regions that specifically binds (immunoreacts
with) an antigen. The term "immunoreact" in its various forms means
specific binding between an antigenic determinant-containing
molecule and a molecule containing an antibody combining site such
as a whole antibody molecule or a portion thereof.
[0128] In naturally occurring antibodies, the six "complementarity
determining regions" or "CDRs" present in each antigen binding
domain are short, non-contiguous sequences of amino acids that are
specifically positioned to form the antigen binding domain as the
antibody assumes its three dimensional configuration in an aqueous
environment. The remainder of the amino acids in the antigen
binding domains, referred to as "framework" regions, show less
inter-molecular variability. The framework regions largely adopt a
.beta.-sheet conformation and the CDRs form loops which connect,
and in some cases form part of, the .beta.-sheet structure. Thus,
framework regions act to form a scaffold that provides for
positioning the CDRs in correct orientation by inter-chain,
non-covalent interactions. The antigen binding domain (i.e.,
antibody combining site) formed by the positioned CDRs defines a
surface complementary to the epitope on the immunoreactive antigen.
This complementary surface promotes the non-covalent binding of the
antibody to its cognate epitope. The amino acids comprising the
CDRs and the framework regions, respectively, can be readily
identified for any given heavy or light chain variable region by
one of ordinary skill in the art, since they have been precisely
defined (see, "Sequences of Proteins of Immunological Interest,"
Kabat, E., et al., U.S. Department of Health and Human Services,
(1983); and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987),
which are herein incorporated by reference). "Humanized antibody"
or "chimeric antibody" includes antibodies in which CDR sequences
derived from the germline of another mammalian species, such as a
mouse, have been grafted onto human framework sequences.
[0129] The terms "T lymphocyte," "T cell," "T cells," and "T cell
population", are used interchangeably herein to refer to a cell or
cells which display on their surface one or more antigens
characteristic of T cells, for example, CD3 and CD11b. The term
includes progeny of a T cell or T cell population. A "T lymphocyte"
or "T cell" includes a cell which expresses CD3 on its cell surface
and a T cell antigen receptor (TCR) capable of recognizing antigen
when displayed on the surface of autologous cells, or any
antigen-presenting matrix, together with one or more MHC molecules
or one or more non-classical MHC molecules. The term "T cells" may
refer to any T cells, including for example, lymphocytes that are
phenotypically CD3.sup.+ i.e., express CD3 on the cell surface.
[0130] As used herein, CD3, is used to refer individually or
collectively to a molecule expressed as part of the T cell receptor
and having a meaning as typically ascribed to it in the art. In
humans, the term CD3 encompasses all known CD3 subunits, for
example CD3 delta, CD3 epsilon, CD3 gamma, and CD3 zeta (TCR zeta),
as well as CD3 alpha (TCR alpha), and CD3 beta (TCR beta) in
individual or independently combined form.
[0131] The term "peptibody" refers to a peptide or polypeptide
which comprises less than a complete, intact antibody. A peptibody
can be an antibody Fc domain attached to at least one peptide. A
peptibody does not include antibody variable regions, an antibody
combining site, CH1 domains, or Ig, light chain constant region
domains.
[0132] The term "naturally occurring" when used in connection with
biological materials such as a nucleic acid molecules,
polypeptides, host cells, and the like refers to those which are
found in nature and not modified by a human being.
[0133] The term "domain" as used herein refers to a part of a
molecule or structure that shares common physical or chemical
features, for example hydrophobic, polar, globular, helical domains
or properties, e.g., a protein binding domain, a DNA binding domain
or an ATP binding domain. Domains can be identified by their
homology to conserved structural or functional motifs.
[0134] A "conservative amino acid substitution" is one in which one
amino acid residue is replaced with another amino acid residue
having a similar side chain. Families of amino acid residues
having, similar side chains have been defined in the art,
including, basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine). For example,
substitution of a phenylalanine for a tyrosine is a conservative
substitution. In some embodiments, conservative substitutions in
the sequences of the polypeptides and antibodies of the invention
do not abrogate the binding of the polypeptide or antibody
containing the amino acid sequence to the antigen(s) to which the
polypeptide or antibody binds. Methods of identifying nucleotide
and amino acid conservative substitutions and non-conservative
substitutions which do not eliminate polypeptide or antigen binding
are, well-known in the art (see, e.g., Brummell et al., Biochem.
32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884
(1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417
(1997)).
[0135] A "modular recognition domain" (MRD) or "target binding
peptide" is a molecule, such as a protein, glycoprotein and the
like, that can specifically (non-randomly) bind to a target
molecule. The amino acid sequence of a MRD can typically tolerate
some degree of variability and still retain a degree of capacity to
bind the target molecule. Furthermore, changes in the sequence can
result in changes in the binding specificity and in the binding
constant between a preselected target molecule and the binding
site. In one embodiment, the MRD is an agonist of the target it
binds. An MRD agonist refers to a MRD that in some way increases or
enhances the biological activity of the MRD's target protein or has
biological activity comparable to a known agonist of the MRD's
target protein. In another embodiment, the MRD is an antagonist of
the target it binds. An MRD antagonist refers to an MRD that blocks
or in some way interferes with the biological activity of the MRD's
target protein or has biological activity comparable to a known
antagonist or inhibitor of the MRD's target protein.
[0136] "Cell surface receptor" refers to molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
cell surface receptor of the present invention is an activated
integrin receptor, for example, an activated .alpha.v.beta.3
integrin receptor on a metastatic cell. As used herein, "cell
surface receptor" also includes a molecule expressed on a cell
surface that is capable of being bound by an MRD containing
antibody of the invention.
[0137] As used herein, a "target binding site" or "target site" is
any known, or yet to be defined, amino acid sequence having the
ability to selectively bind a preselected agent. Exemplary
reference target sites are derived from the RGD-dependent integrin
ligands, namely fibronectin, fibrinogen, vitronectin, von
Willebrand factor and the like, from cellular receptors such as
ErbB2, VEGF, vascular homing peptide or angiogenic cytokines, from
protein hormones receptors such as insulin-like growth factor-I
receptor, epidermal growth factor receptor and the like, and from
tumor antigens.
[0138] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of any molecule
capable of being recognized and specifically bound by a particular
binding agent (e.g., an antibody or an MRD). When the recognized
molecule is a polypeptide, epitopes can be formed from contiguous
amino acids and noncontiguous amino acids and/or other chemically
active surface groups of molecules (such as carbohydrates)
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids are typically retained upon protein
denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0139] An antibody, MRD, antibody-containing MRD, or other molecule
is said to "competitivety inhibit" binding of a reference molecule
to a given epitope if it binds to that epitope to the extent that
it blocks, to some degree, binding of the reference molecule to the
epitope. Competitive inhibition may be determined by any method
known in the art, for example, competition ELISA assays. As used
herein, an antibody, MRD, antibody-containing MRD, or other
molecule may be said to competitively inhibit binding of the
reference molecule to a given epitope, for example, by at least
90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0140] The term "protein" is defined as a biological polymer
comprising units derived from amino acids linked via peptide bonds;
a protein can be composed of two or more chains.
[0141] A "fusion polypeptide" is a polypeptide comprised of at
least two polypeptides and optionally a linking sequence to
operatively link the two polypeptides into one continuous
polypeptide. The two polypeptides linked in a fusion polypeptide
are typically derived from two independent sources, and therefore a
fusion polypeptide comprises two linked polypeptides not normally
found linked in nature. The two polypeptides may be operably
attached directly by a peptide bond or may be linked indirectly
through a linker described herein or otherwise known in the
art.
[0142] The term "operably linked," as used herein, indicates that
two molecules are attached so as to each retain functional
activity. Two molecules are "operably linked" whether they are
attached directly (e.g., a fusion protein) or indirectly (e.g., via
a linker).
[0143] The term "linker" refers to a peptide located between the
antibody and the MRD or between two MRDs. Linkers can have from
about 1 to 20 amino acids, about 2 to 20 amino acids, or about 4 to
15 amino acids. One or more of these amino acids may be
glycosylated, as is well understood by those in the art. In one
embodiment, the 1 to 20 amino acids are selected from glycine,
alanine, proline, asparagine, glutamine, and lysine. In another
embodiment, a linker is made up of a majority of amino acids that
are sterically unhindered, such as glycine and alanine. Thus, in
some embodiments, the linker is selected from polyglycines (such as
(Gly).sub.5, and (Gly).sub.8), poly(Gly-Ala), and polyalanines. The
linker can also be a non-peptide linker such as an alkyl linker, or
a PEG linker. For example, alkyl linkers such as
--NH--(CH.sub.2)s--C(O)--, wherein s=2-20 can be used. These alkyl
linkers may further be substituted by any non-sterically hindering
group such as lower alkyl (e.g., C.sub.1-C.sub.6) lower acyl,
halogen (e.g., Cl, Br), CN, NH.sub.2, phenyl, etc. An exemplary
non-peptide linker is a PEG linker. In certain embodiments, the PEG
linker has a molecular weight of about 100 to 5000 kDa, or about
100 to 500 kDa. The peptide linkers may be altered to form
derivatives. In some embodiments, the linker is a non-peptide
linker such as an alkyl linker, or a PEG linker. In further
embodiments, the linker is a "cleavable linker" facilitating
release of an MRD or cytotoxic agent within a cell or in the
proximity of the cell.
[0144] "Target cell" refers to any cell in a subject (e.g., a human
or animal) that can be targeted by a multispecific and multivalent
composition (e.g., an antibody-containing MRD) or MRD of the
invention. The target cell can be a cell expressing or
overexpressing the target binding site, such as an activated
integrin receptor.
[0145] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells, or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0146] As used herein, the term "effector cell" refers to an immune
cell which is involved in the effector phase of an immune response,
as opposed to the cognitive and activation phases of an immune
response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells
including cytolytic T cells (CTLs)), killer cells, natural killer
cells, macrophages, monocytes, eosinophils, neutrophils,
polymorphonuclear cells, granulocytes, mast cells, and basophils).
Some effector cells express specific Fc receptors and carry out
specific immune functions. In certain embodiments, an effector cell
is capable of inducing antibody-dependent cell-mediated
cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC.
For example, monocytes and macrophages, which express FcR, are
involved in specific killing of target cells and presenting
antigens to other components of the immune system, or binding to
cells that present antigens. In other embodiments, an effector cell
can phagocytose a target antigen or target cell. The expression of
a particular FcR on an effector, cell can be regulated by humoral
factors such as cytokines. For example, expression of Fc alpha RI
has been found to be up-regulated by G-CSF or GM-CSF. This enhanced
expression increases the effector function of Fc alpha RI-bearing
cells against targets. Exemplary functions of an effector cell
include the phagocytosing or lysing of a target antigen, or a
target cell.
[0147] "Target cell" refers to any cell or pathogen whose
elimination would be beneficial in a patient (e.g., a human or
animal) and that can be targeted by a composition (e.g., antibody)
of the invention.
[0148] "Patient," "subject," "animal" or "mammal" are used
interchangeably and refer to mammals such as human patients and
non-human primates, as well as experimental animals such as
rabbits, rats, and mice, and other animals. Animals include all
vertebrates, e.g., mammals and non-mammals, such as sheep, dogs,
cows, chickens, amphibians, and reptiles. In some embodiments, the
patient is a human.
[0149] "Treating" or "treatment" includes the administration of the
antibody comprising an MRD of the present invention to prevent or
delay the onset of the symptoms, complications, or biochemical
indicia of a disease, condition, or disorder, alleviating the
symptoms or arresting or inhibiting further development of the
disease, condition, or disorder. Treatment can be prophylactic (to
prevent or delay the onset of the disease, or to prevent the
manifestation of clinical or subclinical symptoms thereof) or
therapeutic suppression or alleviation of symptoms after the
manifestation of the disease, condition, or disorder. Treatment can
be with the antibody-MRD composition alone, the MRD alone, or in
combination of either with one or more additional therapeutic
agents.
[0150] As used herein, the terms "pharmaceutically acceptable," or
"physiologically tolerable" and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to or upon a human without the production of
therapeutically prohibitive undesirable physiological effects such
as nausea, dizziness, gastric upset and the like.
[0151] "Modulate," means adjustment or regulation of amplitude,
frequency, degree, or activity. In another related aspect, such
modulation may be positively modulated (e.g., an increase in
frequency, degree, or activity) or negatively modulated (e.g., a
decrease in frequency, degree, or activity).
[0152] "Cancer," "tumor," or "malignancy" are used as synonymous
terms and refer to any of a number of diseases that are
characterized by uncontrolled, abnormal proliferation of cells, the
ability of affected cells to spread locally or through the
bloodstream and lymphatic system to other parts of the body
(metastasize) as well as any of a number of characteristic
structural and/or molecular features. A "cancerous tumor," or
"malignant cell" is understood as a cell having specific structural
properties, lacking differentiation and being capable of invasion
and metastasis. Examples of cancers that may be treated using the
antibody-MRD fusions of the invention include solid tumors and
hematologic cancers. Additional, examples of cancers that may be
treated using the antibody-MRD fusions of the invention include
breast, lung, brain, bone, liver, kidney, colon, head and neck,
ovarian, hematopoietic (e.g., leukemia), and prostate cancer.
Further examples of cancer that may be treated using the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) include, but are not limited to, carcinoma, lymphoma,
blastoma, sarcoma, and leukemia. More particular examples of such
cancers include squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer, adenocarcinoma of the lung, squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
cancer, liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma and various types of head and neck
cancers. Other types of cancer and tumors that may be treated using
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) are described herein or otherwise known in the art.
[0153] An "effective amount" of an antibody, MRD, or MRD-containing
antibody as disclosed herein is an amount sufficient to carry out a
specifically stated purpose such as to bring about an observable
change in the level of one or more biological activities related to
the target to which the antibody, MRD, or MRD-containing antibody
binds. In certain embodiments, the change increases the level of
target activity. In other embodiments, the change decreases the
level of target activity. An "effective amount" can be determined
empirically and in a routine manner, in relation to the stated
purpose.
[0154] The term "therapeutically effective amount" refers to an
amount of an antibody, MRD, MRD-containing antibody, other
multivalent and multispecific drug of the invention, or other drug
effective to "treat" a disease or disorder in a patient or mammal.
In the case of cancer, the therapeutically effective amount of the
drug can reduce angiogenesis and neovascularization; reduce the
number of cancer cells; reduce the tumor size; inhibit (i.e., slow
to some extent or stop) cancer cell infiltration into peripheral
organs; inhibit (i.e., slow to some extent or stop) tumor
metastasis; inhibit, to some extent, tumor growth or tumor
incidence; stimulate immune responses against cancer cells and/or
relieve to some extent one or more of the symptoms associated with
the cancer. See the definition herein of "treating". A
"therapeutically effective amount" also may refer to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result. A therapeutically effective amount of
a composition of the invention may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the composition to elicit a desired response in the
individual. A therapeutically effective amount is also one in which
any toxic or detrimental effects of the therapeutic composition are
outweighed by the therapeutically beneficial effects.
[0155] To the extent the drug can prevent growth and/or kill
existing cancer cells, it can be cytostatic and/or cytotoxic. A
"prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically, but not necessarily, since
a prophylactic dose is used in subjects (patients) prior to or at
an earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0156] Where embodiments of the invention are described in terms of
a Markush group or other grouping of alternatives, the present
invention encompasses not only the entire group, listed as a whole,
but also each member of the group individually and all possible
subgroups of the main group, and also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the disclosed and/or claimed invention.
II. MODULAR RECOGNITION DOMAINS (MRDS)
[0157] The present invention describes an approach based on the
adaptation of target binding peptides or modular recognition
domains (MRDs) as fusions to catalytic or non-catalytic
antibodies.
[0158] In certain embodiments, where the antibody component of the
MRD-antibody fusion is a catalytic antibody, the MRD-antibody
fusions provide for effective targeting to tumor cells or soluble
molecules while leaving the prodrug activation capability of the
catalytic antibody intact. MRDs can also emend the binding capacity
of non-catalytic antibodies providing for an effective approach to
extend the binding functionality of antibodies, particularly for
therapeutic purposes.
[0159] One aspect of the present invention relates to development
of a full-length antibody comprising at least one modular
recognition domain (MRD). In another non-exclusive embodiment, the
full-length antibody comprises more than one MRD, wherein the MRDs
have the same or different specificities. In addition, a single MRD
may be comprised of a tandem repeat of the same or different amino
acid sequence that can allow for the binding of a single MRD to
multiple targets and/or to a repeating epitope on a given
target.
[0160] The interaction between a protein ligand and its target
receptor site often takes place at a relatively large interface.
However, only a few key residues at the interface contribute to
most of the binding. The MRDs can mimic ligand binding. In certain
embodiments, the MRD can mimic the biological activity of a ligand
(an agonist MRD) or through competitive binding inhibit the
bioactivity of the ligand (an antagonist MRD). MRDs in multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
can also affect targets in other ways, e.g., by neutralizing,
blocking, stabilizing, aggregating, or crosslinking the MRD
target.
[0161] It is contemplated that MRDs of the present invention will
generally contain a peptide sequence that binds to target sites of
interests and have a length of about 2 to 150 amino acids, about 2
to 125 amino acids, about 2 to 100 amino acids, about 2 to 90 amino
acids, about 2 to 80 amino acids, about 2 to 70 amino acids, about
2 to 60 amino acids, about 2 to 50 amino acids, about 2 to 40 amino
acids, about 2 to 30 amino acids, or about 2 to 20 amino acids. It
is also contemplated that MRDs have a length of about 10 to 150
amino acids, about 10 to 125 amino acids, about 10 to 100 amino
acids, about 10 to 90 amino acids, about 10 to 80 amino acids,
about 10 to 70 amino acids, about 10 to 60 amino acids, about 10 to
50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino
acids, or about 10 to 20 amino acids. It is further contemplated
that MRDs have a length of about 20 to 150 amino acids, about 20 to
125 amino acids, about 20 to 100 amino acids, about 20 to 90 amino
acids, about 20 to 80 amino acids, about 20 to 70 amino acids,
about 20 to 60 amino acids, about 20 to 50 amino acids, about 20 to
40 amino acids, or about 20 to 30 amino acids. In certain
embodiments, the MRDs have a length of about 2 to 60 amino acids.
In other embodiments, the MRDs have a length of about 10 to 60
amino acids. In other embodiments, the MRDs have a length of about
10 to 50 amino acids. In additional embodiments, the MRDs have a
length of about 10 to 40 amino acids. In additional embodiments,
the MRDs have a length of about 10 to 30 amino acids.
[0162] In some embodiments, one or more of the MRD components of
the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd of
less than 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 m 5.times.10.sup.-15
M, or 10.sup.-15 M. In one embodiment, one or more of the MRD
components of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd less
than 5.times.10.sup.-5 M. In another embodiment, one or more of the
MRD components of the multivalent and multispecific compositions
(e.g., MRD-containing antibodies) have a dissociation constant or
Kd less than 5.times.10.sup.-8 M. In another embodiment, one or
more of the MRD components of the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) have a dissociation
constant or Kd less than 5.times.10.sup.-9 M. In another
embodiment, one or more of the MRD components of the multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
have a dissociation constant or Kd less than 5.times.10.sup.-10 M.
In another embodiment, one or more of the MRD components of the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) have a dissociation constant or Kd less than
5.times.10.sup.-11 M. In another embodiment, one or more of the MRD
components of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd less
than 5.times.10.sup.-12 M.
[0163] In specific embodiments, one or more of the MRD components
of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) bind their targets with an off rate
(k.sub.off) of less than 5.times.10.sup.-2 sec.sup.-1, 10.sup.-2
sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1, or 10.sup.-3 sec.sup.-1.
More preferably, one or more of the MRD components of the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) bind their targets with an off rate (k.sub.off) of less
than 5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1,
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1, or 10.sup.-7 sec.sup.-1.
[0164] In other specific embodiments, one or more of the MRD
components of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) bind their targets with an on rate
(k.sub.on) of greater than 10.sup.3 M.sup.-1sec.sup.-1,
5.times.10.sup.3 M.sup.-1sec.sup.-1, 10.sup.4 M.sup.-1sec.sup.-1,
or 5.times.10.sup.4 M.sup.-1sec.sup.-1. More preferably, one or
more of the MRD components of the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) bind their targets
with an on rate (k.sub.on) of greater than 10.sup.5
M.sup.-1sec.sup.-1, 5.times.10.sup.5 M.sup.-sec.sup.-1, 10.sup.6
M.sup.-1sec.sup.-1, or 5.times.10.sup.6 M.sup.-1sec.sup.-1, or
10.sup.7 M.sup.-1sec.sup.-1.
[0165] In some embodiments, the MRDs are affibodies. Affibodies
represent a class of affinity proteins based on a 58-amino acid
residue protein domain derived from one of the IgG-binding domains
of staphylococcal protein A. This three helix bundle domain has
been used as a scaffold for the construction of combinatorial
phagemid libraries, from which affibody variants that bind a
desired target molecule, such as one or more of the targets
disclosed herein, can routinely be selected using phage display
technology (see, e.g., Nord et al., Nat. Biotechnol. 15:772-7
(1997), and Ronmark et al., A, Eur. J. Biochem. 2002; 269:2647-55).
Further details of Affibodies and methods of production thereof are
provided by reference to U.S. Pat. No. 5,831,012, which is herein
incorporated by reference in its entirety.
[0166] In other embodiments, an MRD of the invention (e.g., an MRD
on an MRD-containing antibody) contains one or more amino acid
residues or sequences of amino acid residues (including
derivatives, analogs, and mimetics thereof) that are preferentially
targeted by chemistries or other processes that covalently or
non-covalently link a molecular entity to the MRD, as compared to,
the MRD without the preferentially targeted sequences or the
antibody component of the MRD-containing antibody. For example, in
some embodiments, the amino acid sequence of the MRD contains one
or more residues having a reactive side chain (e.g. cysteine or
lysine) that allows for selective or preferential linkage of the
MRD to cytotoxic agents (e.g., drug and prodrug conjugates, toxins,
and bioactive ligands) or imaging agents.
[0167] The use of these "linking" MRDs to arm an MRD-comprising
antibody with a "payload" overcomes many of the issues associated
with antibody destabilization and reduction in antibody activity
that have frequently been observed using conventional methods for
generating immunotoxins. The "payload" component of an
MRD-comprising antibody complex of the invention can be any
composition that confers a beneficial therapeutic, diagnostic, or
prognostic effect, or that provide an advantage in manufacturing,
purifying or formulating an MRD-containing antibody. In some
embodiments, the payload is a chemotherapeutic drug, or a prodrug,
such as, doxorubicin or a maytansinoid-like drug. In additional
embodiments, the payload is another MRD, a toxin, a
chemotherapeutic drug, a catalytic enzyme, a prodrug, a radioactive
nuclide, a chelator (e.g., for the attachment of lanthanides) or
another component of the multivalent and multispecific compositions
of the invention as described herein.
[0168] In nonexclusive embodiments, the MRD does not contain an
antigen binding domain, or another antibody domain such as a
constant region, a variable region, a complementarity determining
region (CDR), a framework region, an Fc domain, or a hinge region.
In one non-exclusive embodiment, the MRD does not contain an
antigen binding domain. In another non-exclusive embodiment, the
MRD does not contain three CDRs. In another non-exclusive
embodiment, the MRD does not contain CDR1 and CDR2. In yet another
non-exclusive embodiment, the MRD does not contain CDR1. In one
nonexclusive embodiment, the MRD is not derived from a natural
cellular ligand. In another nonexclusive embodiment, the MRD is not
a radioisotope. In another nonexclusive embodiment, the MRD is not
a protein expression marker such as glutathione S-transferase
(GST), His-tag, Flag, hemagglutinin (HA), MYC or a fluorescent
protein (e.g., GFP or RFP). In another nonexclusive embodiment, the
MRD does not bind serum albumin. In an additional nonexclusive
embodiment, the MRD is not a small molecule that is a cytotoxin. It
yet another nonexclusive embodiment, the MRD does not have
enzymatic activity. In another non-exclusive embodiment, the MRD
has a therapeutic effect when administered alone and/or when fused
to an Fc in a patient or animal model. In another non-exclusive
embodiment, the MRD has a therapeutic effect when repeatedly
administered alone and/or when fused to an Fc in a patient or
animal model (e.g., 3 or more times over the course of at least six
months).
[0169] In some embodiments, the MRD is conformationally
constrained. In other embodiments, the MRD is not conformationally
constrained. In some embodiments, the MRD contains one cysteine
residue. The cysteine residue in the MRD can form an interchain
bond (e.g., between cysteines within the same MRD, different
peptide linked MRDs, and an MRD and a peptide linked
immunoglobulin). In some embodiments, the MRD(s) participating in
the interchain bond is/are associated with a single core
target-binding domain. In other embodiments, the MRD(s)
participating in the interchain bond is/are associated with
multiple core target-binding domains. In an alternative embodiment,
the cysteine residue in the MRD can form an interchain bond (e.g.,
between cysteines of non-peptide linked MRDs or an MRD and an
immunoglobulin that are not linked by a peptide bind). In some
embodiments, the MRD(s) associated with the interchain bond is/are
associated with a single core target-binding domain (i.e., 2 MRDs
located on different polypeptide chains form one or more interchain
bonds and collectively form one target binding site). Thus, for
example, the invention encompasses MRD-containing antibodies
wherein MRDs located on the carboxyl terminus of the heavy chain
interact (e.g., via disulfide bond) so as to form a single target
binding site. In other embodiments, the MRD(s) associated with the
interchain bond is/are associated with multiple core target-binding
domains. Alternatively, as discussed herein, the MRD can contain
one or more cysteine residues (or other residue having a reactive
side chain (e.g., lysine)) that allows for selective or
preferential linkage of the MRD to a cytotoxic agent.
[0170] In some embodiments, the MRD contains two cysteine residues
outside the core target-binding domain. In some embodiments, the
MRD contains two cysteine residues located within the core
target-binding domain at each end of the target-binding domain. In
some embodiments, a first cysteine is located near the terminus of
the molecule (i.e. at the C-terminus of an MRD on the C-terminus of
a linker or antibody chain or at the N-terminus of an MRD on the
N-terminus of a linker or antibody chain). Thus, in some
embodiments, a first cysteine is located within one amino acid,
within two amino acids, within three amino acids, within four amino
acids, within five amino acids, or within six amino acids of the
terminus, of the molecule. In some embodiments, a second cysteine
is located near the MRD fusion location (i.e. at the N-terminus of
an MRD on the C-terminus of a linker or antibody chain or at the
C-terminus of an MRD on the N-terminus of a linker or antibody
chain). Thus, in some embodiments, a second cysteine is located
within one amino acid, within two amino acids, within three amino
acids, within four amino acids, within five amino acids, within 10
amino acids, or within 15 amino acids from the MRD fusion.
[0171] In some embodiments, the MRD is capped with stable residues.
In some embodiments, the MRD is disulfide capped. In some
embodiments, the MRD does not contain cleavage sites.
[0172] In some embodiments, the MRD has been selected to not
contain known potential human T-cell epitopes.
[0173] In some particular embodiments, the MRD has a particular
hydrophobicity. For example, the hydrophobicity of MRDs can be
compared on the basis of retention times determined using
hydrophobic interaction chromatography or reverse phase liquid
chromatography.
[0174] The MRD target can be any molecule that it is desirable for
an MRD-containing antibody to interact with. For example, the MRD
target can be a soluble factor or a transmembrane protein, such as
a cell surface receptor. The MRI) target can also be an
extracellular component or an intracellular component. In certain
non-exclusive embodiments, the MRD target is a factor that
regulates cell proliferation, differentiation, or survival. In
other nonexclusive embodiments, the MRD target is a cytokine. In
another nonexclusive embodiment, the MRD target is a factor that
regulates angiogenesis. In another nonexclusive embodiment, the MRD
target is a factor that regulates cellular adhesion and/or
cell-cell interaction. In certain non-exclusive embodiments, the
MRD target is a cell signaling molecule. In another nonexclusive
embodiment, the MRD target is a factor that regulates one or more
immune responses, such as, autoimmunity, inflammation and immune
responses against cancer cells. In another nonexclusive embodiment,
the MRD target is a factor that regulates cellular adhesion and/or
cell-cell interaction. In an additional nonexclusive embodiment,
the MRD target is a cell signaling molecule. In another embodiment,
an MRD can bind a target that is itself an MRD. The ability of MRDs
to bind a target and block, increase, or interfere with the
biological activity of the MRD target can be determined using or
routinely modifying assays, bioassays, and/or animal models known
in the art for evaluating such activity.
[0175] The MRDs are able to bind their respective target when the
MRDs are attached to an antibody. In some embodiments, the MRD is
able to bind its target when not attached to an antibody. In some
embodiments, the MRD is a target agonist. In other embodiments, the
MRD is a target antagonist. In certain embodiments, the MRD can be
used to localize an MRD-containing antibody to an area where the
MRD target is located.
[0176] The sequence of the MRD can be determined several ways. For
example, MRD sequences can be derived from natural ligands or known
sequences that bind to a specific target binding site.
Additionally, phage display technologies have emerged as a powerful
method in identifying peptides which bind to target receptors and
ligands. In peptide phage display libraries, naturally occurring
and non-naturally occurring (e.g., random peptide) sequences can be
displayed by fusion with coat proteins of filamentous phage. The
methods for elucidating binding sites on polypeptides using phage
display vectors has been previously described, in particular in
WO94/18221, which is herein incorporated by reference. The methods
generally involve the use of a filamentous phage (phagemid) surface
expression vector system for cloning and expressing polypeptides
that bind to the pre-selected target site of interest.
[0177] The methods of the present invention for preparing MRDs
include the use of phage display vectors for their particular
advantage of providing a means to screen a very large population of
expressed display proteins and thereby locate one or more specific
clones that code for a desired target binding reactivity. The
ability of the polypeptides encoded by the clones to bind a target
and/or alter the biological activity of the target can be
determined using or routinely modifying assays and other
methodologies described herein or otherwise known in the art. For
example, phage display technology can be used to identify and
improve the binding properties of MRDs. See, e.g., Scott et al.,
Science 249:386 (1990); Devlin et al., Science 249:404 (1990); U.S.
Pat. Nos. 5,223,409, 5,733,731, 5,498,530, 5,432,018, 5,338,665,
5,922,545; and Int. Appl. Publ. Nos. WO96/40987 and WO98/15833;
which are herein incorporated by reference. In peptide phage
display libraries, natural and/or non-naturally occurring peptide
sequences can be displayed by fusion with coat proteins of
filamentous phage. The displayed peptides can be affinity-eluted
against a target of interest if desired. The retained phage may be
enriched by successive rounds of affinity purification and
repropagation. The best binding peptides may be sequenced to
identify key residues within one or more structurally related
families of peptides. See, e.g., Cwirla et al., Science 276:1696-9
(1997), in which two distinct families were identified. The peptide
sequences may also suggest which residues may be safely replaced by
alanine scanning or by mutagenesis at the DNA level. Mutagenesis
libraries may be created and screened to further optimize the
sequence of the best binders. Lowman, Ann. Rev. Biophys. Biomol.
Struct. 26:401-424 (1997).
[0178] Structural analysis of protein-protein interaction may also
be used to suggest peptides that mimic the binding activity of
large protein ligands. In such an analysis, the crystal structure
may suggest the identity and relative orientation of critical
residues of the large protein ligand, from which a peptide such as
an MRD may be designed. See, e.g., Takasaki et al., Nature Biotech.
15:1266-1270 (1997). These analytical methods may also be used to
investigate the interaction between a target and an MRD selected by
phage display, which can suggest further modification of the MRDs
to increase binding affinity.
[0179] Other methods known in the art can be used to identify MRDs.
For example, a peptide library can be fused to the carboxyl
terminus of the lac repressor and expressed in E. coil. Another E.
coli-based method allows display on the cell's outer membrane by
fusion with as peptidoglycan-associated lipoprotein (PAL). These
and related methods are collectively referred to as "E. coli
display." In another method, translation of random RNA is halted
prior to ribosome release, resulting in a library of polypeptides
with their associated RNA still attached. This and related methods
are collectively referred to as "ribosome display." Other known
methods employ chemical linkage of peptides to RNA. See, for
example, Roberts and Szostak, Proc. Natl. Acad. Sci. USA
94:12297-12303 (1997). This and related methods are collectively
referred to as "RNA-peptide screening, RNA display and mRNA
display." Chemically derived peptide libraries have been developed
in which peptides are immobilized on stable, non-biological
materials, such as polyethylene rods or solvent-permeable resins.
Another chemically derived peptide library uses photolithography to
scan peptides immobilized on glass slides. These and related
methods are collectively referred to as "chemical-peptide
screening." Chemical-peptide screening may be advantageous in that
it allows use of D-amino acids and other unnatural analogues, as
well as non-peptide elements. Both biological and chemical methods
are reviewed in Wells and Lowman, Curr. Opin. Biotechnol. 3:355-362
(1992). Furthermore, constrained libraries, linear libraries,
and/or focused libraries (comprised of structurally related domains
that share significant primary sequence homology) can be used to
identify, characterize, and modify MRDs
[0180] An improved MRD that specifically binds a desired target can
also be prepared based on a known MRD sequence. For example, at
least one, two, three, four, five, or more amino acid mutations
(e.g., conservative or non-conservative substitutions), deletions
or insertions can be introduced into a known MRD sequence and the
resulting MRD can be screened for binding to the desired target and
biological activity, such as the ability to antagonize target
biological activity or to agonize target biological activity. In
another embodiment, the sites selected for modification are
affinity matured using phage display techniques known in the art.
See, e.g., Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-4 24
(1997).
[0181] Any technique for mutagenesis known in the art can be used
to modify individual nucleotides in a DNA sequence, for purposes of
making amino acid addition(s), substitution(s) or deletion(s) in
the antibody sequence, or for creating/deleting restriction sites
and sequences coding for desired amino acids (e.g., cysteine) to
facilitate further manipulations. Such techniques include, but are
not limited to, chemical mutagenesis, in vitro site-directed
mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985);
Hutchinson et al., J. Biol. Chem. 253:6551 (1978)),
oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet.
19:423-463 (1985); Hill et al., Methods Enzymol. 155:558-568
(1987)), PCR-based overlap extension (Ho et al., Gene 77:51-59
(1989)), PCR-based megaprimer mutagenesis (Sarkar et al.,
Biotechniques 8:404-407 (1990)), etc. Modifications can be
confirmed by DNA sequencing.
[0182] Additional fusion proteins can be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling") DNA shuffling can be employed to alter the activities
of SYNAGIS.RTM. or fragments thereof (e.g., an antibody or a
fragment thereof with higher affinities and lower dissociation
rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238,
5,830,721, 5,834,252, and 5,837,458, and Patten et al, Curr.
Opinion Biotechnol. 8:724-33 (1997); Harayama et al., Trends
Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol.
287:265-76 (1999); Lorenzo et al., Biotechniques 24(2):308-313
(1998); U.S. Appl. Publ. Nos. 20030118592 and 200330133939; and
Int. Appl. Publ. No. WO02/056910; each of which is herein
incorporated by reference in its entirety.
[0183] Additionally, MRDs can be identified based on their effects
in assays that measure particular pathways or activities. For
example, assays that measure signaling pathways (e.g.,
phosphorylation studies or multimerization), ion channel fluxes,
intracellular cAMP levels, cellular activities such as migration,
adherence, proliferation, or apoptosis, and viral entry,
replication, budding, or integration can be used to identify,
characterize, and improve MRDs.
[0184] Variants and derivatives of the MRDs that retain the ability
to bind the target antigen are included within the scope of the
present invention. Included within variants are insertional,
deletional, and substitutional variants, as well as variants that
include MRDs presented herein with additional amino acids at the N-
and/or C-terminus, including from about 0 to 50, 0 to 40, 0 to 30,
0 to 20 amino acids and the like. It is understood that a
particular MRD of the present invention may be modified to contain
one, two, or all three types of variants. Insertional and
substitutional variants may contain natural amino acids,
unconventional amino acids, or both. In some embodiments, the MRD
contains a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, or 20 amino acid differences when compared to an MRD
sequence described herein. In some embodiments, the amino acid
differences are substitutions. These substitutions can be
conservative or non-conservative in nature and can include
unconventional or non-natural amino acids. In other embodiments the
MRD contains a sequence that competitively inhibits the ability of
an MRD-containing sequence described herein to bind with a target
molecule. The ability of an MRD to competitively inhibit another
MRD-containing sequence can be determined using techniques known in
the art, including ELISA and BIAcore analysis.
[0185] The ability of an MRD to bind its target can be assessed
using any technique that assesses molecular interaction. For
example, MRD-target interaction can be assayed as described in the
Examples below or alternatively, using in vitro or in vivo binding
assays such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, protein A
immunoassays, and immunohistochemistry (IHC). Assays evaluating the
ability of an MRD to functionally affect its target (e.g., assays
to measure signaling, proliferation, migration etc) can also be
used to indirectly assess MRD-target interaction.
[0186] An improved MRD that has a particular half-life in vivo can
also be prepared based on a known MRD sequence. For example, at
least one, two, three, four, five, or more amino acid mutations
(e.g., conservative or non-conservative substitutions), deletions
or insertions can be introduced into a known MRD sequence and the
resulting MRD can be screened for increased half-life. Thus,
variants and derivatives, of the MRDs that retain the ability to
bind the target and have an increased half-life can be included in
multivalent and multispecific compositions (e.g., MRD-containing
antibodies). Thus, in some embodiments, an MRD in an MRD-containing
antibody has a half-life of at least about 5, at least about 10, at
least about 15, at least about 20, at least about 25, at least
about 30, at least about 35, at least about 40, at least about 45,
at least about 50, at least about 55, at least about 60, at least
about 65, at least about 70, at least about 75, at least about 80,
at least about 85, at least about 90, at least about 95, at least
about 100, at least about 110, at least about 120, at least about
130, at least about 140, or at least about 150 hours. In some
embodiments, an MRD in an MRD-containing antibody has a half-life
of at least about 5, at least about 10, at least about 15, at least
about 20, at least about 25, at least about 30, at least about 35,
at least about 40, at least about 45, at least about 50, at least
about 55, at least about 60, at least about 65, at least about 70,
at least, about 75, at least about 80, at least about 85, at least
about 90, at least about 95, at least about 100, at least about
110, at least about 120, at least about 130, at least about 140, or
at least about 150 hours.
[0187] Once the sequence of the MRD has been elucidated, the
peptides may be prepared by any of the methods known in the art.
For example, the MRD peptides can be chemically synthesized and
operably attached to the antibody or can be synthesized using
recombinant technology. For example, MRDs can be synthesized in
solution or on a solid support using known techniques. Various
automatic synthesizers are commercially available and can be used
in accordance with known protocols. See, for example, Tam et al.,
J. Am. Chem. Soc. 105:6442 (1983); Merrifield, Science 232:341-347
(1986); Barany and Merrifield, The Peptides, Goss and Meienhofer,
eds, Academic Press, New York, 1-284; Barany et al., Int. J. Pep.
Protein Res., 30:705-739 (1987); and U.S. Pat. No. 5,424,398, each
of which is incorporated herein by reference in its entirety.
[0188] MRDs can be synthesized with covalently attached molecules
that are not amino acids but aid in the purification,
identification, and/or tracking of an MRD in vitro or in vivo.
(e.g., biotin for reacting with avidin or avidin-labeled
molecules).
[0189] The following MRD targets are described in more detail by
way of example only.
[0190] In some embodiments described herein, the MRD targets an
integrin. The role of integrins such as .alpha.v.beta.3 and
.alpha.v.beta.5 as tumor-associated markers has been well
documented. A recent study of 25 permanent human cell lines
established from advanced ovarian cancer demonstrated that all
lines were positive for .alpha.v.beta.5 expression and many were
positive for .alpha.v.beta.3 expression. Studies have also shown
that .alpha.v.beta.3 and .alpha.v.beta.5 is highly expressed on
malignant human cervical tumor tissues. Integrins have also
demonstrated therapeutic effects in animal models of Kaposi's
sarcoma, melanoma, and breast cancer.
[0191] A number of integrin .alpha.v.beta.3 and .alpha.v.beta.5
antagonists are in clinical development. These include cyclic RGD
peptides and synthetic small molecule RGD mimetics. Two
antibody-based integrin antagonists are currently in clinical
trials for the treatment of cancer. The first is VITAXIN.RTM.
(MEDI-522 Abegrein), the humanized form of the murine anti-human
.alpha.v.beta.3 antibody LM609. A dose-escalating phase I study in
cancer patients demonstrated that VITAXIN.RTM. is safe for use in
humans. Another antibody in clinical trials is CNT095, a fully
human Ab that recognizes .alpha.v integrins. A Phase I study of
CNT095 in patients with a variety of solid tumors has shown that it
is well tolerated. Cliengitide (EMD 121974), a peptide antagonist
of .alpha.v.beta.3 and .alpha.v.beta.5, has also proven safe in
phase I trials. Furthermore, there have been numerous drug
targeting and imaging studies based on the use of ligands for these
receptors. These preclinical and clinical observations demonstrate
the importance of targeting .alpha.v.beta.3 and .alpha.v.beta.5 and
studies involving the use of antibodies in this strategy have
consistently reported that targeting through these integrins is
safe.
[0192] Clinical trials are also ongoing for antagonists targeting
.alpha.5v.beta.1 for treating metastatic melanoma, renal cell
carcinoma, and non-small cell lung cancer (M200 (volociximab) and
malignant glioma (ATN-161).
[0193] integrin-binding MRDs containing one or more RGD tripeptide
sequence motifs represent an example of MRDs of the invention.
Ligands having the RGD motif as a minimum recognition domain and
from which MRDs of the invention can be derived are well known, a
partial list of which includes, with the corresponding integrin
target in parenthesis fibronectin (.alpha.3.beta.1,
.alpha.5.beta.1, .alpha.v.beta.1, .alpha.11b.beta.3,
.alpha.v.beta.3, and .alpha.3.beta.1) fibrinogen (.alpha.M.beta.2
and .alpha.11b.beta.1) on Willebrand factor (.alpha.11b.beta.3 and
.alpha.v.beta.3), and vitronectin (.alpha.11b.beta.3,
.alpha.v.beta.3 and .alpha.b.beta.5).
[0194] In one embodiment, the RGD containing targeting MRD is a
member selected from the group consisting of: YCRGDCT (SEQ ID
NO:3); PCRGDCL (SEQ ID NO:4); TCRGDCY (SEQ ID NO:5); and LCRGDCF
(SEQ ID NO:6).
[0195] A MRD that mimics a non-RGD-dependent binding site on an
integrin receptor and having the target binding specificity of a
high affinity ligand that recognizes the selected integrin is also
contemplated in the present invention. MRDs that bind to an
integrin receptor and disrupt binding and/or signaling activity of
the integrin are also contemplated.
[0196] In some embodiments, the MRD targets an angiogenic molecule,
Angiogenesis is essential to many physiological and pathological
processes. Ang2 has been shown to act as a proangiogenic molecule.
Administration of Ang2-selective inhibitors is sufficient to
suppress both tumor angiogenesis and corneal angiogenesis.
Therefore, Ang2 inhibition alone or in combination with inhibition
of other angiogenic factors, such as VEGF, can represent an
effective antiangiogenic strategy for treating patients with solid
tumors.
[0197] It is contemplated that MRDs useful in the present invention
include those that bind to angiogenic receptors, angiogenic
factors, and/or Ang2. In a specific embodiment, an MRD of the
invention binds Ang2. In further embodiments, the TIE2 binding
component comprises a fragment of ANG2 that binds TIE2. In
particular embodiments, compositions of the invention bind TIE2 and
comprise amino acids 283-449 of the human ANG2 disclosed in NCBI
Ref. Seq. No. NP.sub.--001138.1.
[0198] In one embodiment, an MRD and/or -MRD-containing antibody
binds Ang2 and contains a sequence selected from the group
consisting of: GAQTNFMPMDDLEQRLYEQFILQQGLE (SEQ ID NO:9) (ANGa);
LWDDCYFFPNPPHCYNSP (SEQ ID NO:11) (ANGb); LWDDC YSYPNPPHCYNSP (SEQ
ID NO:12) (ANGc); LWDDCYSFPNPPPICYNSP (SEQ ID NO: 15) (ANGd);
DCAVYPNPPWCYKMEFGK (SEQ ID NO:16) (ANGe); PHEECYFYPNPPFICYT MS (SEQ
ID NO:17) (ANGf); and PHEECYSYPNPPHCYTMS (SEQ ID NO:18) (ANGg).
[0199] in an additional embodiment, an MRD and/or -MRD-containing
antibody binds Ang2 and contains a sequence selected from the group
consisting of GAQTNFMPMDDLEQRLYEQPILQ OGLE (SEQ ID NO:9) (ANGa);
LWDDCYFFPNPPHCYNSP (SEQ ID NO:11) (ANGb); LWDDCYSYPNPPHCYNSP (SEQ
ID NO:12 (ANGc); LWDDCYSFPNPPHCYNSP (SEQ ID NO:15) (ANGd);
DCANTYPNPPWCYKMEFGK (SEQ ID NO:16) (ANGe); PHEECYFYPNPP HCYTMS (SEQ
ID NO:17) (ANGf); and PHEECYSYPNPPFICYTMS (SEQ ID NO:18)
(ANGg).
[0200] ANG-2 binding peptides disclosed in U.S. Pat. Nos.
7,309,483, 7,205,275, 7,138,370 7,063,965, 7,063,840, 7,045,302,
7,008,781, 6,825,008, 6,645,484, 6,627,415, 6,455,035, 6,441,137,
6,433,143, 6,265,564, 6,166,185, 5,879,672, 5,814,464, 5,681,714,
5,650,490, 5,643,755 and 5,521,073; and U.S. Appl. Publ. Nos.
2007/0225221, 2007/0093419, 2007/0093418, 2007/0072801,
2007/0025993, 2006/0122370, 2005/0186665, 2005/0175617,
2005/0106099, 2005/0100906, 2003/0236193, 2003/0229023,
2003/0166858, 2003/0166857, 2003/0162712, 2003/0109677,
2003/0092891, 2003/0040463, 2002/0173627 and 2002/0039992, and
Intl. Appl. Publ. Nos. WO2006/005361, WO/2006/002854,
WO2004/092215, WO/2004/076650, WO2003/057134, WO/2000/075323,
WO2000/065085, WO/1998/018914 and WO1995/021866, the disclosures of
each of which is herein incorporated by reference in its
entirety.
[0201] In some embodiments, the MRD targets vascular endothelial
growth factor (VEGF). In one embodiment, the antibody-MRD fusion
comprises an MRD with the sequence ATWLPPP (SEQ ID NO:71), which
inhibits VEGF-mediated angiogenesis. Binetruy-Tournaire et al.,
EMBO J. 19:1525-1533 (2000). In additional embodiments, an
anti-VEGF antibody containing an MRD that targets VEGF is
contemplated in the present invention, Anti-VEGF antibodies can be
found for example in Presta et al., Cancer Research 57:4593-4599
(1997); and Fuh et al., J. Biol. Chem. 281:10 6625 (2006), each of
which is herein incorporated by reference in its entirety.
[0202] Insulin-like growth factor-I receptor-specific MRDs can also
be used in the present invention.
[0203] Vascular homing-specific MRDs are also contemplated for use
in the present invention. A number of studies have characterized
the efficacy of linking the vascular homing peptide to other
proteins like IL12 or drugs to direct their delivery in live
animals.
[0204] Numerous other target binding sites are contemplated as
being the target of the antibody-MRD fusions of the present
invention, including for example, FGFR1, FGFR2, EGFR, ErbB2, ErbB3,
ErbB4, CD20, insulin-like growth factor-I receptor, and hepatocyte
growth factor receptor. MRDs can be directed towards these target
binding sites or the corresponding ligands.
[0205] In one embodiment, the MRD binds to IL6. In one embodiment,
the MRD binds to IL6R.
[0206] In one embodiment, the MRD binds to HER2/3.
[0207] In another embodiment, the MRD binds ErbB2.
[0208] In some embodiments, the MRD binds to a human protein. In
some embodiments, the MRD binds to both a human protein and its
ortholog in mouse, rat, rabbit, or hamster.
III. ANTIBODIES
[0209] The antibody in the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) described herein can
be any suitable antigen-binding immunoglobulin. In certain
embodiments, the MRD-containing antibody molecules described herein
retain the structural and functional properties of traditional
monoclonal antibodies. Thus, the antibodies retain their epitope
binding properties, but advantageously also incorporate one or more
additional target-binding specificities.
[0210] Antibodies that can be used in the multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
include, but are not limited to, monoclonal, multispecific, human,
humanized, primatized, and chimeric antibodies. Immunoglobulin or
antibody molecules of the invention can be of any type (e.g., IgG,
IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2) or subclass of immunoglobulin molecule. In specific
embodiments, the antibodies are IgG1. In other specific
embodiments, the antibodies are IgG3.
[0211] Antibodies that can be used as part of the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) can be
naturally derived or the result of recombinant engineering (e.g.,
phage display antibodies can include xenomouse and synthetic). The
modifications, for example, to enhance half-life or to increase or
decrease antibody dependent cellular cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) activity. Antibodies can be
from or derived from any animal origin including birds and mammals
or generated synthetically. In some embodiments, the antibodies are
human, murine, donkey, rabbit, goat, guinea pig, camel, llama,
horse, or chicken antibodies. In specific embodiments, the
antibodies are human.
[0212] In certain embodiments, the heavy chain portions of one
polypeptide chain of a multimer are identical to those on a second
polypeptide chain of the multimer. In alternative embodiments, the
heavy chain portion-containing monomers of the invention are not
identical. For example, each monomer may comprise a different
target binding site, forming, for example, a bispecific
antibody.
[0213] Bispecific, bivalent antibodies, and methods of making them,
are described, for instance in U.S. Pat. Nos. 5,731,168, 5,807,706,
5,821,333, and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537;
each of which is herein incorporated by reference in its entirety.
Bispecific tetravalent antibodies, and methods of making them are
described, for instance, in Int. Appl. Publ. Nos. WO02/096948 and
WO00/44788, the disclosures of both of which are herein
incorporated by reference in its entirety. See generally, Int.
Appl. Publ. Nos. WO93/17715, WO92/08802, WO91/00360, and
WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat.
Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and
Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0214] The heavy chain portions of the antibody component of the
MRD-antibody fusions for use in the methods disclosed herein may be
derived from different immunoglobulin molecules. For example, a
heavy chain portion of a polypeptide may comprise a CH1 domain
derived from an IgG1 molecule and a hinge region derived from an
IgG3 molecule. In another example, a heavy chain portion can
comprise a hinge region derived, in part, from an IgG1 molecule
and, in part, from an IgG3 molecule. In another example, a heavy
chain portion can comprise a chimeric hinge region derived, in
part, from an IgG1 molecule and, in part, from an IgG4
molecule.
[0215] In some embodiments, the antigen binding domains of the
antibody component of the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) bind to their target
with a dissociation constant or Kd of less than 5.times.10.sup.-3
M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5
M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7
M, 10 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 10.sup.-10 M,
10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M. In one
embodiment, the antibody component of the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) have a
dissociation constant or Kd of less than 5.times.10.sup.-5 M. In
another embodiment, antigen binding of the antibody component of
the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) has a dissociation constant or Kd of
less than 5.times.10.sup.-8 M. In another embodiment, antigen
binding of the antibody component of the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) has a
dissociation constant or Kd of less than less than
5.times.10.sup.-9 M. In another embodiment, the antibody component
of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd of
less than 5.times.10.sup.-10 M. In another embodiment, the antibody
component of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd of
less than 5.times.10.sup.-11 M. In another embodiment, the antibody
component of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) have a dissociation constant or Kd of
less than 5.times.10.sup.-12 M.
[0216] In specific embodiments, the antibody component of the
MRD-containing antibody binds its target with an off rate
(k.sub.off) of less than 5.times.10.sup.-2 sec.sup.-1, 10.sup.-2
sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1, or 10.sup.-3 sec.sup.-1.
More preferably, the antibody component of the MRD-containing
antibody binds its target with an off rate (k.sub.off) of less than
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1,
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1, or 10.sup.-7 sec.sup.-1.
[0217] In other specific embodiments, the antibody component of the
MRD-containing antibody binds its target with an on rate (k.sub.on)
of greater than 10.sup.3 M.sup.-1sec.sup.-1, 5.times.10.sup.3
M.sup.-1 sec.sup.-1, 10.sup.4 M.sup.-1sec.sup.-1, or
5.times.10.sup.4 M.sup.-1sec.sup.-1. More preferably, the antibody
component of the MRD-containing antibody binds its target with an
on rate (k.sub.on) of greater than 10.sup.5 M.sup.-1 sec.sup.-1,
5.times.10.sup.5 M.sup.-1sec.sup.-1, 10.sup.6 M.sup.-1sec.sup.-1,
or 5.times.10.sup.6 M.sup.-1sec.sup.-1, or 10.sup.7
M.sup.-1sec.sup.-1.
[0218] Affinity maturation strategies and chain shuffling
strategies (e.g., gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling") are known in the art and can be employed to generate
high affinity and/or to alter the activities (e.g., ADCC and CDC)
of multivalent and multispecific compositions (e.g., multivalent
and multispecific compositions (e.g., MRD-containing antibodies)).
See, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,
5,834,252 and 5,837,458; and Patten et al., Curr. Opinion
Biotechnol. 8:724-733 (1997), Harayama, Trends Biotechnol.
16(2):76-82 (1998), Hansson et al., J. Mol. Biol. 287:265-276
(1999) and Lorenzo and Blasco, Biotechniques 24(2):308-313 (1998),
each of which is herein incorporated by reference in its entirety.
Advantageously, affinity maturation strategies and chain shuffling
strategies can routinely be applied to generate multivalent and
multispecific compositions (e.g., MRD-containing antibodies) can
also include variants and derivatives that improve antibody
function and/or desirable pharmacodynamic properties.
[0219] Accordingly, certain embodiments of the invention include an
antibody-MRD fusion, in which at least a fraction of one or more of
the constant region domains has been altered so as to provide
desired biochemical characteristics such as reduced or increased
effector functions, the ability to non-covalently dimerize,
increased ability to localize at the site of a tumor, reduced serum
half-life, or increased serum half-life when compared with an
unaltered antibody of approximately the same immunoreactivity. The
alterations of the constant region domains can be amino acid
substitutions, insertions, or deletions.
[0220] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) expressed on certain cytotoxic cells (e.g.,
Natural Killer (NK) cells, neutophils, and macrophages) enables
these cytotoxic effector cells to localize to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
Specific high-affinity IgG antibodies directed to the surface of
target cells "arm" the cytotoxic cells and are required for such
killing. Lysis of the target cell is extracellular, requires
contact or close proximity between the cytotoxic cells and target
cells, and does not involve complement.
[0221] As used herein, the term "enhances ADCC" (e.g., referring to
cells) is intended to include any measurable increase in cell lysis
when contacted with a variant MRD-containing antibody as compared
to the cell killing of the same cell in contact with a
MRD-containing antibody that has not been so modified in a way that
alters ADCC in the presence of effector cells (for example, at a
ratio of target cells:effector cells of 1:50), e.g., an increase in
cell lysis by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 150%, 200%, 250%, 300%, or 325%.
[0222] In certain embodiments, the antibody component of the
antibody-MRD fusion has been modified to increase antibody
dependent cellular cytotoxicity (ADCC) (see, e.g., Bruhns et al.,
Blood 113:3716-3725 (2009); Shields et al., J. Biol. Chem.
276:6591-6604 (2001); Lazar et al., Proc. Natl. Acad. Sci. USA
103:4005-4010 (2006); Stavenhagen et al., Cancer Res., 67:8882-8890
(2007); Horton et al., Cancer Res. 68:8049-8057 (2008); Zalevsky et
al., Blood 113:3735-3743 (2009); Bruckheimer et al., Neoplasia
11:509-517 (2009); Allan et al., WO2006/020114; Strohl, Curr. Op.
Biotechnol. 20:685-691 (2009); and Watkins et al., WO2004/074455,
each of which is herein incorporated by reference in its entirety).
Examples of Fc sequence engineering modifications contained in the
antibody component of the antibody-MRD fusions that increases ADCC
include one or more modifications corresponding to: IgG1-S298A,
E333A, K334A; IgG1-S239D, 1332E; IgG1-S239D, A330L, I332E;
IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or
V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and
IgG1-F243L, R292P, Y300L, V305I, P396L; wherein the numbering of
the residues in the Fc region is that of the EU index as in
Kabat.
[0223] In one embodiment, an Fc variant protein has enhanced ADCC
activity relative to a comparable molecule. In a specific
embodiment, an Fc variant protein has ADCC activity that is at
least 2 fold, or at least 3 fold, or at least 5 fold or at least 10
fold or at least 50 fold or at least 100 fold greater than that of
a comparable molecule. In another specific embodiment, an Fc
variant protein has enhanced binding to the Fc receptor Fc gamma
RIIIA and has enhanced ADCC activity relative to a comparable
molecule. In other embodiments, the Fc variant protein has both
enhanced ADCC activity and enhanced serum half-life relative to a
comparable molecule.
[0224] The ability of any particular Fc variant protein to mediate
lysis of the target cell by ADCC can be assayed using techniques
known in the art. For example, to assess ADCC activity a
multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) can be added to target cells in
combination with immune effector cells, which can be activated by
the antigen antibody complexes resulting in cytolysis of the target
cell. Cytolysis is generally detected by the release of label
(e.g., radioactive substrates, fluorescent dyes or natural
intracellular proteins) from the lysed cells. Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Specific examples of in vitro ADCC
assays are described in Wisecarver et al., J Immunol Methods
79:277-282 (1985); Bruggemann et al., J. Exp. Med. 166:1351-1361
(1987); Wilkinson et al., J. Immunol. Methods 258:183-191 (2001);
Patel et al., J. Immunol. Methods 184:29-38 (1995). Alternatively,
or additionally, ADCC activity of the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) can be
assessed in vivo, e.g., in an animal model such as that disclosed
in Clynes et al., PNAS USA 95:652-656 (1998), and U.S. Pat. No.
7,662,925.
[0225] In certain embodiments, the antibody component of the
antibody-MRD fusion has been modified to decrease ADCC (see, e.g.,
Idusogie et al., J. Immunol. 166:2571-2575 (2001); Sazinsky et al.,
Proc. Natl. Acad. Sci. USA 105:20167-20172 (2008); Davis et al., J.
Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J. Immunol.
23:403-411 (1993); Alegre et al., Transplantation 57:1537-1543
(1994); Xu et al., Cell Immunol. 200:16-26 (2000); Cole et al.,
Transplantation 68:563-571 (1999); Hutchins et al., Proc. Natl.
Acad. Sci. USA 92:11980-11984 (1995); Reddy et al., J. Immunol.
164:1925-1933 (2000); Int. Appl. Publ. No. WO1997/11971, and
WO2007/106585; U.S. Appl. Publ. 2007/0148167A1; McEarchern et al.,
Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol.
20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol.
47:1489-1497 (2007), each of which is herein incorporated by
reference in its entirety). Examples of Fc sequence engineering
modifications contained in the antibody component of the
antibody-MRD fusions that decreases ADCC include one or more
modifications corresponding to: IgG1-K326W, E333S; IgG2-E333S;
IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A,
G237A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118-260; IgG4-EU
sequence 261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C220S,
C226S, C229S, P238S; IgG1-C226S, C229S, E233P, L234V, L235A; and
IgG1-L234F, L235E, P331S.
[0226] In certain embodiments, the antibody component of the
antibody-MRD fusion has been modified to increase
antibody-dependent cell phagocytosis (ADCP); (see, e.g., Shields et
al., J. Biol. Chem. 276:6591-6604 (2001); Lazar et al., Proc. Natl.
Acad. Sci. USA 103:4005-4010 (2006); Stavenhagen et al., Cancer
Res., 67:8882-8890 (2007); Richards et al., Mol. Cancer. Ther.
7:2517-2527 (2008); Horton et al., Cancer Res. 68:8049-8057 (2008),
Zalevsky et al., Blood 113:3735-3743 (2009); Bruckheimer et al.,
Neoplasia 11:509-517 (2009); Allan et al., WO2006/020114; Strohl,
Curr. Op. Biotechnol. 20:685-691 (2009); and Watkins et al.,
WO2004/074455, each of which is herein incorporated by reference in
its entirety.). Examples of Fc sequence engineering modifications
contained in the antibody component of the antibody-MRD fusions
that increases ADCP include one or more modifications corresponding
to: IgG1-5298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L,
1332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without
S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L,
P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; IgG1-G236A, S239D,
I332E.
[0227] In certain embodiments, the antibody component of the
antibody-MRD fusion has been modified to decrease ADCP (see, e.g.,
Sazinsky et al., Proc. Natl. Acad. Sci. USA 105:20167-20172 (2008);
Davis et al., J. Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur.
J. Immunol. 23:403-411 (1993); Alegre et al., Transplantation
57:1537-1543 (1994); Xu et al., Cell Immunol. 200:16-20 (2000);
Cole et al., Transplantation 68:563-571 (1999); Hutchins et al.,
Proc. Natl. Acad. Sci. USA 92:11980-11984 (1995); Reddy et al., J.
Immunol. 164:1925-1933 (2000); Intl. Appl. Publ. Nos. WO1997/11971
and WO2007/106585; U.S. Appl. Publ. 2007/0148167; McEarchern et
al., Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol.
20:685-691 (2009); and Kumagai et al., J. Clin. Pharmacol.
47:1489-1497 (2007), each of which is herein incorporated by
reference in its entirety). Examples of Fc sequence engineering
modifications contained in the antibody component of the
antibody-MRD fusions that decreases ADCC include one or more
modifications corresponding to: IgG1-N297A; IgGL234A, L235A;
IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E;
IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q,
V309L, A330S, A331S; IgG1-C220S, C226S, C229S, P238S; IgG1-C226S,
C229S, E233P, L234V, L235A; and IgG1-L234F, L235E, P331S.
[0228] "Complement dependent cytotoxicity" and "CDC" refer to the
lysing of a target cell in the presence of complement. The
complement activation pathway is initiated by the binding of the
first component of the complement system (C1q) to a molecule, an
antibody for example, complexed with a cognate antigen. To assess
complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be
performed. In one embodiment, an Fc variant protein has enhanced
CDC activity relative to a comparable molecule. In a specific
embodiment, an Fc variant protein has CDC activity that is at least
2 fold, or at least 3 fold, or at least 5 fold, or at least 10
fold, or at least 50 fold, or at least 100 fold greater than that
of a comparable molecule. In other embodiments, the Fc variant
protein has both enhanced CDC activity and enhanced serum half-life
relative to a comparable molecule.
[0229] In certain embodiments, the antibody component of the
antibody-MRD fusions have been modified to increase
complement-dependent cytotoxicity (CDC) (see, e.g., (see, e.g.,
Idusogie et al., J. Immunol. 166:2571-2575 (2001); Strohl, Curr.
Op. Biotechnol. 20:685-691 (2009); and Natsume et al., Cancer Res.
68:3863-3872 (2008), each of which is herein incorporated by
reference in its entirety). Examples of Fc sequence engineering
modifications contained in the antibody component of the
antibody-MRD fusions that increases CDC include one or more
modifications corresponding to: IgG1-K326A, E333A; and IgG1-K326W,
E333S, IgG2-E333S.
[0230] In one embodiment, the present invention provides
formulations, wherein the Fc region comprises a non-naturally
occurring amino acid residue at one or more positions selected from
the group consisting of 234, 235, 236, 239, 240, 241, 243, 244,
245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296,
297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334
as numbered by the EU index as set forth in Kabat. Optionally, the
Fe region can comprise a non-naturally occurring amino acid residue
at additional and/or alternative positions known to one skilled in
the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, and
6,737,056; and Int. Appl. Publ. Nos. WO01/58957, WO02/06919,
WO04/016750, WO04/029207, WO04/035752 and WO05/040217).
[0231] In specific embodiments MRD-containing antibodies of the
invention contain an Fc variant comprising at least one non
naturally occurring amino acid residue selected from the group
consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V,
2341, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H,
235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T,
239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241 R,
243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T,
256E, 262I, 262A. 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I,
264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y,
265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q,
267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T,
296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F,
299I, 299L, 299A, 299S, 299V, 200H, 299F, 299E, 313F, 325Q, 325L,
325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L,
328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H,
328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V,
330I, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q,
332T, 332H, 332Y, and 332A as numbered by the EU index as set forth
in Kabat. Optionally, the Fc region can comprise additional and/or
alternative non-naturally occurring amino acid residues known to
one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821,
6,277,375, and 6,737,056; and Int. Appl. Publ. Nos. WO01/58957.
WO02/06919, WO04/016750, WO04/029207, WO04/035752 and
WO05/040217).
[0232] In certain embodiments, the multivalent and monovalent
multispecific composition is an antibody-MRD fusions wherein the
antibody component has been modified to increase inhibitory binding
to Fc gamma RIIb receptor (see, e.g., Chu et al., Mol. Immunol.
45:3926-3933 (2008)). An example of Fc sequence engineering
modifications contained in the antibody component of the
antibody-MRD fusions that increases binding to inhibitory Fc gamma
RIIb receptor is IgG1-S267E, L328F.
[0233] In certain embodiments, the antibody component of the
antibody-MRD fusions have been modified to decrease CDC (see, e.g.,
Int. Appl. Publ. Nos. WO1997/11971 and WO2007/106585; U.S. Appl.
Publ. No 2007/0148167A1; McEarchern et al., Blood 109:1185-1192
(2007); Hayden-Ledbetter et al., Clin. Cancer 15:2739-2746 (2009);
Lazar et al, Proc. Natl. Acad. Sci. USA 103:4005-4010 (2006);
Bruckheimer et al., Neoplasia 11:509-517 (2009); Stohl, Curr. Op.
Biotechnol. 20:685-691 (2009); and Sazinsky et al., Proc. Natl.
Acad. Sci. USA 105:20167-20172 (2008); each of which is herein
incorporated by reference in its entirety). Examples of Fc sequence
engineering modifications contained in the antibody component of
the antibody-MRD fusions that decreases CDC include one or more
modifications corresponding to: IgG1-S239D, A330L, I332E; IgG2 EU
sequence 118-260; IgG4-EU sequence 261-447; IgG2-H268Q, V309L,
A330S, A331S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234F,
L235E, P331S; and IgG1-C226S, P230S.
[0234] The half-life on an IgG is mediated by its pH-dependent
binding to the neonatal receptor FcRn. In certain embodiments the
antibody component of the antibody-MRD fusion has been modified to
enhance binding to FcRn (see, e.g., Petkova et al., Int. Immunol.
18:1759-1769 (2006); Dall'Acqua et al., J. Immunol. 169:5171-5180
(2002); Oganesyan et al., Mol. Immunol. 46:1750-1755 (2009);
Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006), Hinton et
al., J. Immunol. 176:346-356 (2006); Datta-Mannan et al., Drug
Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem.
282:1709-1717 (2007); Int. Appl. Publ. No. WO2006/130834; Strohl,
Curr. Op. Biotechnol. 20:685-691 (2009); and Yeung et al., J.
Immunol. 182:7663-7671 (2009); each of which is herein incorporated
by reference in its entirety).
[0235] In additional embodiments, the antibody of the antibody-MRD
fusion has been modified to selectively bind FcRn at pH6.0, but not
pH 7.4. Examples of Fc sequence engineering modifications contained
in the antibody component of the antibody-MRD fusions that
increases half-life include one or more modifications corresponding
to: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y;
IgG1-N434A; and IgG1-T307A, E380A, N434A.
[0236] In other embodiments the antibody component of the
antibody-MRD fusion has been modified to decrease binding to FcRn
(see, e.g., Petkova et al., Int. Immunol. 18:1759-1769 (2006);
Datta-Mannan et al., Drug Metab. Dispos. 35:86-94 (2007);
Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); Strohl,
Curr. Op. Biotechnol. 20:685-691 (2009); and Vaccaro et al., Nat.
Biotechnol. 23:1283-1288 (2005), each of which is herein
incorporated by reference in its entirety). Examples of Fc sequence
engineering modifications contained in the antibody component of
the antibody-MRD fusions that decrease half-life include one or
more modifications corresponding to: IgG1-M252Y, S254T, T256E;
H433K, N434F, 436H; IgG1-1253A; and IgG1-P2571, N434H or D376V,
N434H.
[0237] In some embodiments, the antibody-MRD fusions have been
glyocoengineered or the Fc portion of the MRD-containing antibody
has been mutated to increase effector function using techniques
known in the art. For example, the inactivation (through point
mutations or other means) of a constant region domain may reduce Fc
receptor binding of the circulating modified antibody thereby
increasing tumor localization. In other cases it may be that
constant region modifications consistent with the instant invention
moderate complement binding and thus reduce the serum half-life and
nonspecific association of a conjugated cytotoxin. Yet other
modifications of the constant region may be used to modify
disulfide linkages or oligosaccharide moieties that allow for
enhanced localization due to increased antigen specificity or
antibody flexibility. The resulting physiological profile,
bioavailability and other biochemical effects of the modifications,
such as tumor localization, biodistribution and serum half-life,
can easily be measured and quantified using well know immunological
techniques without undue experimentation.
[0238] Methods for generating antibodies containing non-naturally
occurring Fc regions are known in the art. For example, amino acid
substitutions and/or deletions can be generated by mutagenesis
methods, including, but not limited to, site-directed mutagenesis
(Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR
mutagenesis (Higuchi, in "PCR Protocols: A Guide to Methods and
Applications", Academic Press, San Diego, pp. 177-183 (1990)), and
cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)).
Site-directed mutagenesis can be performed by the overlap-extension
PCR method (Higuchi, in "PCR Technology: Principles and
Applications for DNA Amplification", Stockton Press, New York, pp.
61-70 (1989)). Alternatively, the technique of overlap-extension
PCR (Higuchi, ibid.) can be used to introduce any desired
mutation(s) into a target sequence (the starting DNA). Other
methods useful for the generation of antibodies containing
non-naturally occurring Fc regions are known in the art (see, e.g.,
U.S. Pat. Nos. 5,624,821, 5,885,573, 5,677,425, 6,165,745,
6,277,375, 5,869,046, 6,121,022, 5,624,821, 5,648,260, 6,528,624,
6,194,551, 6,737,056, 6,821,505 and 6,277,375; U.S. Appl. Publ. No.
2004/0002587 and Int Appl. Publ. Nos. WO94/29351, WO99/58572,
WO00/42072, WO02/060919, WO04/029207, WO04/099249 and
WO04/063351).
[0239] Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) used according to the methods of the
invention also include derivatives that are modified, e.g., by the
covalent attachment of any type of molecule to the antibody such
that covalent attachment does not prevent the antibody from
specifically binding to its cognate, epitope. For example, but not
by way of limitation, the antibody derivatives include antibodies
that have been modified, e.g., by glycosylation, acetylation,
pegylation, phosphorylation, amidation, or derivatization by known
protecting/blocking groups. Any of numerous chemical modifications
may be carried out by known techniques, including, but not limited
to acetylation, formylation, etc. Additionally, the derivative may
contain one or more non-classical amino acids.
[0240] According to some embodiments the antibody component of
compositions of the invention is engineered to contain one or more
free cysteine amino acids having a thiol reactivity within a
desirable range (e.g. 0.6 to 1.0), wherein the cysteine engineered
antibody is prepared by a process comprising replacing one or more
amino acid residues of a parent antibody by cysteine. In some
embodiments one or more free cysteine amino acid residues are
located in a light chain. In additional embodiments one or more
free cysteine amino acid residues are located in a heavy chain. In
additional embodiments one or more free cysteine amino acid
residues are located in a both the heavy and light chain. In some
embodiments, the cysteine engineered MRD-containing antibody
contains a free cysteine amino acid having a thiol reactivity value
in the range of 0.6 to 1.0, and a sequence modification in the
light chain or the heavy chain that is disclosed in U.S. Pat. No.
7,855,275. In other embodiments, the cysteine engineered antibody
contains a free cysteine amino acid having a thiol reactivity value
in the range of 0.6 to 1.0, and a sequence modification in the
light chain or the heavy chain that is not disclosed in U.S. Pat.
No. 7,855,275, the contents of which are herein incorporated by
reference in its entirety.
[0241] In additional embodiments, the MRD-containing antibody is
engineered to contain one or more free selenocysteine amino acids
or another non-natural amino acid capable of forming disulfide
bonds. Antibodies containing the same and methods for making such
antibodies are known in the art. See, e.g., Hofer et al., Proc.
Natl. Acad. Sci. 105(34):12451-12456 (2008); and Hofer et al.,
Biochem. 48(50):12047-12057 (2009), each of which is herein
incorporated by reference in its entirety. In some embodiments one
or more free selenocysteine amino acid residues are located in a
light chain. In additional embodiments one or more free
selenocysteine amino acid residues are located in a heavy chain. In
additional embodiments one or more free selenocysteine amino acid
residues are located in a both the heavy and light chain.
[0242] In certain embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) have been modified
so as to not elicit a deleterious immune response in the animal to
be treated, e.g., in a human. In one embodiment, the antibody is
modified to reduce immunogenicity using art-recognized techniques.
For example, antibody components of the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) can be
humanized, primatized, deimmunized, or chimerized. These types of
antibodies are derived from a non-human antibody, typically a
murine or primate antibody, that retains or substantially retains
the antigen-binding properties of the parent antibody, but which is
less immunogenic in humans. This may be achieved by various
methods, including (a) grafting the entire non-human variable
domains onto human constant regions to generate chimeric
antibodies; (b) grafting at least a part of one or more of the
non-human complementarity determining regions (CDRs) into human
frameworks and constant regions with or without retention of
critical framework residues; or (c) transplanting the entire
non-human variable domains, but "cloaking" them with human-like
sections by replacement of surface residues. Such methods are
disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855
(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen
et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.
28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and
U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, each
of which is herein incorporated by reference in its entirety.
[0243] De-immunization can also be used to decrease the
immunogenicity of an MRD-containing antibody. As used herein, the
term "de-immunization" includes alteration of an MRD-containing
antibody to modify T cell epitopes (see, e.g., Int. Appl. Pub.
WO9852976A1, and WO0034317A2, each if which is herein incorporated
by reference in its entirety). For example, VH and VL sequences
from the starting antibody are analyzed and a human T cell epitope
"map" is generated from each V region showing the location of
epitopes in relation to complementarity-determining regions (CDRs)
and other key residues within the sequence. Individual T cell
epitopes from the T cell epitope map are analyzed in order to
identify alternative amino acid substitutions with a low risk of
altering activity of the final antibody. A range of alternative VH
and VL sequences are designed comprising combinations of amino acid
substitutions and these sequences are subsequently incorporated
into a range of antibodies for use in the diagnostic and treatment
methods disclosed herein, which are then tested for function.
Typically, between 12 and 24 variant antibodies are generated and
tested. Complete heavy and light chain genes comprising modified V
and human C regions are then cloned into expression vectors and the
subsequent plasmids introduced into cell lines for the production
of whole antibody. The antibodies are then compared in appropriate
biochemical and biological assays, and the optimal variant is
identified.
[0244] Many different antibody components of the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) can be
used in the methods described herein. It is contemplated that
catalytic and non-catalytic antibodies can be used in the present
invention. For example, Antibody 38C2 is an antibody-secreting
hybridoma and has been previously described in Int. Appl. Pub.
WO97/21803. 38C2 contains an antibody combining site that catalyzes
the aldol addition reaction between an aliphatic donor and an
aldehyde acceptor. In a syngeneic mouse model of neuroblastoma,
systemic administration of an etoposide prodrug and intra-tumor
injection of Ab 38C2 inhibited tumor growth.
[0245] The antibody target of the MRD-containing antibody (i.e.,
the target of the antigenic binding domain) can be any molecule
that it is desirable for a MRD-antibody fusion to interact with.
For example, the antibody target can be a soluble factor or the
antibody target can be a transmembrane protein, such as a cell
surface receptor. The antibody target can also be an extracellular
component or an intracellular component. In certain embodiments,
the antibody target is a factor that regulates cell proliferation,
differentiation, or survival. In other embodiments, the antibody
target is a cytokine. In another nonexclusive embodiment, the
antibody target is a factor that regulates angiogenesis. In another
nonexclusive embodiment, the antibody target is a factor that
regulates one or more immune responses, such as, autoimmunity,
inflammation and immune responses against cancer cells. In another
nonexclusive embodiment, the antibody target is a factor that
regulates cellular adhesion and/or cell-cell interaction. In
certain nonexclusive embodiments, the antibody target is a cell
signaling molecule. The ability of an antibody to bind to a target
and to block, increase, or interfere with the biological activity
of the antibody target can be determined using or routinely
modifying assays, bioassays, and/or animal models known in the art
for evaluating such activity.
[0246] In some embodiments the antibody target of the
MRD-containing antibody is a disease-related antigen. The antigen
can be an antigen characteristic of a particular cancer, and/or of
a particular cell type (e.g., a hyperproliferative cell), and/or of
a particular pathogen (e.g., a bacterial cell (e.g., tuberculosis,
smallpox, anthrax), a virus (e.g., HIV), a parasite (e.g., malaria,
leichmaniasis), a fungal infection, a mold, a mycoplasm, a pr on
antigen, or an antigen associated with a disorder of the immune
system.
[0247] In some embodiments, the antibody target of the
MRD-containing antibody is a target that has been validated in an
animal model or clinical setting.
[0248] In other embodiments, the antibody target of the
MRD-containing antibody is a cancer antigen.
[0249] In one embodiment, the antibody target of the MRD-containing
antibody is: PDGFRA, PDGFRB, PDGF-A, PDGF-B, PDGF-CC, PDGF-C,
PDGF-D, VEGFR1, VEGFR2, VEGFR3, VEGFC, VEGFD, neuropilin 2 (NRP2),
betacellulin, PLGF, RET (rearranged during transfection), TIE1,
TIE2 (TEK), CA125, CD3, CD4, CD7, CD10, CD13, CD25, CD32, CD32b,
CD44, CD49e (integrin alpha 5), CD55, CD64, CD90 (THY1), CD133
(prominin 1), CD147, CD166, CD200, ALDH1, ESA, SHH, DHH, IHH,
patched1 (PTCH1), smoothened (SMO), WNT1, WNT2B, WNT3A, WNT4,
WNT4A, WNT5A, WNT5B, WNT713, WNT8A, WNT10A, WNT10B, WNT16B, LRP5,
LRP6, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1,
Notch3, Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1
(TNFb, LTa), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6
(Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF7 (CD27
Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8
(CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF12 (TWEAK),
TNFRSF12 (TWEAKR), TNFSF13 (APRIL), TNFSF13B (BLYS), TNFRSF13B
(TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A), TNFRSF17 (BCMA),
TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), TNFRSF25 (DR3),
ANG1 (ANGPT1), ANG3 (ANGPTL1), ANG4 (ANGPT4), IL1 alpha, IL1 beta,
IL1R1, IL1R2, IL2, IL2R, IL5, IL5R, IL6, IL6R, IL8, IL8R, IL10,
IL10R, IL12, IL12R, IL13, IL13R, IL15, IL15R, IL18, IL18R, IL19,
IL19R, IL21R, IL23, IL23R, mif, XAG1, XAG3, REGIV, FGF1, FGF2,
FGF3, FGF4, FGFR1, FGFR2, FGFR3, ALK, ALK1, ALK7, ALCAM, Artemin,
Axl, TGFb, TGFb2, TGFb3, TGFBR1, IGFIIR, BMP2, BMP5, BMP6, BMPR1,
GDF3, GDF8, GDF9, N-cadherin, E-cadherin, VE-cadherin, NCAM, L1CAM
(CD171), ganglioside GM2, ganglioside GD2, calcitonin, PSGR, DCC,
CDCP1, CXCR2, CXCR7, CCR3, CCR5, CCR7, CCR10, CXCL1, CXCL5, CXCL6,
CXCL8, CXCL12, CCL3, CCL4, CCL5, CCL11, Claudin1, Claudin2,
Claudin3, Claudin4, TMEFF2, neuregulin, MCSF, CSF, CSFR (fms),
GCSE, GCSFR, BCAM, HPV, hCG, SR1F, PSA, FOLR2 (folate receptor
beta), BRCA1, BRCA2, HLA-DR, ABCC3, ABCB5, HM1.24, LFA1, LYNX,
S100A8, S100A9, SCF, Von Willebrand factor, Lewis Y6 receptor,
Lewis Y, CA G250 (CA9), integrin avb3 (CNTO95), integrin avb5,
activity B1 alpha, leukotriene B4 receptor (LTB4R), neurotensin NT
receptor (NTR), 5T4 oncofetal antigen, Tenascin C, MMP, MMP2, MMP7,
MMP9, MMP12, MMP14, MMP26, cathepsin G, cathepsin H, cathepsin L,
SULF1, SULF2, MET, UPA, MHC1, MN (CA9), TAG-72, TM4SF1, Heparanase
(HPSE), syndecan (SDC1), Ephrin B2, Ephrin B4, or relaxin2. An MRD
that binds to one of the above targets is encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are
also encompassed by the invention. The above antibody and MRD
targets and those otherwise described herein are intended to be
illustrative and not limiting.
[0250] In another embodiment, the antibody target of the
MRD-containing antibody is CD19, CD22, CD30, CD33, CD38, CD44v6,
TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), CD52, CD54 (ICAM), CD74,
CD80, CD200, EPCAM (EGP2), neuropilin 1 (NRP1), TEM1, mesothelin,
TGFbeta 1, TGFBRII, phosphatidlyserine, folate receptor alpha
(FOLR1), TNFRSF10A (TRAIL R1 DR4), TNFRSF10B (TRAIL R2 DR5), CXCR4,
CCR4, CCL2, HGF, CRYPTO, VLA5, TNFSF9 (41BB Ligand), TNFRSF9
(41BB), CTLA4, HLA-DR, IL6, TNFSF4 (OX40 Ligand), TNFRSF4 (OX40),
MUC1, MUC18, mucin CanAg, ganglioside GD3, EGFL7, PDGFRa, IL21,
IGF1, IGF2, CD117 (cKit), PSMA, SLAMF7, carcinoembryonic antigen
(CEA), FAP, integrin avb3, or integrin .alpha.5.beta.3. An MRD that
binds to one of the above targets are encompassed by the invention.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that bind to 1,
2, 3, 4, 5, 6, or more of the above targets are also encompassed by
the invention.
[0251] In particular embodiments, the antibody of the
MRD-containing antibody competes for target binding with an
antibody selected from: siplizumab CD2 (e.g., MEDI-507, MedImmune),
blinatumomab CD19 CD3 (e.g., MT103, Micromet/MedImmune);
XMAB.RTM.5574 CD19 (Xencor), SGN-19A CD19 (Seattle Genetics),
ASG-5ME (Agenesys and Seattle Genetics), MEDI-551 CD19 (MedImmune),
epratuzumab CD22 (e.g., hLL2, Immunomedics/UCB), inotuzumab
ozogamicin CD22 (Pfizer), iratumumab CD30 (e.g., SGN-30 (Seattle
Genetics) and MDX-060 (Medarex)), XMAB.RTM.2513 CD30 (Xencor),
brentuximab vedotin CD30 (e.g., SGN-35, Seattle Genetics),
gemtuzumab ozogamicin CD33 (e.g., MYLOTARG.RTM., Pfizer),
lintuzumab CD33 (e.g., antibody of Seattle Genetics), MOR202, CD38
(MorphoSys), daratumumab CD38 (e.g., Genmab antibody), CP870893
CD40 (Pfizer), dacetuzumab CD40 (e.g., SGN40, Seattle Genetics),
ANTOVA.RTM. CD40 (Biogen Idec), lucatumun ab CD40 (e.g., HCD122.
Novartis) XMAB.RTM.5485 CD40 (Xencor), teneliximub, ruplizumab
CD40L (e.g., ANTOVA.RTM.) bivatuzumab mertansine CD44v6,
alemtuzumab CD52 (e.g., CAMPATH.RTM./MABCAMPATH.RTM.,
Genzyme/Bayer), BI505 ICAM1 (Bioinvent), milatuzumab CD74 (e.g.,
antibody of Immunomedics), galiximab CD80 (Biogen Idec), BMS663513
4-1BB (Bristol-Myers Squibb), Alexion CD200 antibody (Alexion),
edrecolomab EPCAM (e.g., MAb17-1A, PANOREX.RTM. (GlaxoSmithKline),
AT003 EPCAM (Affitech)), adecatumumab EPCAM (e.g., MT201,
Micromet), oportuzumab monatox EPCAM, Genentech anti-NRP1 antibody,
MORAB004 TEM1 (Morphotek), MORAB009 mesothelin (Morphotek),
lerdelimumab TGFb1 (e.g., CAT-152, Cambridge Antibody Technology),
metelimumab TGFb1 (e.g., CAT-192, Cambridge Antibody Technology),
ImClone anti-TGFBRII antibody, bavituximab phosphatidylserine
(e.g., antibody of Peregrine (Peregrine Pharmaceuticals)), AT004
phosphatidylserine (Affitech), AT005 phosphatidylserine (Affitech),
MORAB03 folate receptor alpha (Morphotek), farletuzumab folate
receptor alpha cancer (e.g., MORAB003, Morphotek), CS1008 DR4
(Sankyo), mapatumumab DR4 (e.g., HGS-ETR1, Human Genome Sciences),
LBY135 DR5 (Novartis), AMG66 DR5 (Amgen), Apomab DR5 (Genentech),
PRO95780 (Genentech), lexatumumab DR5 (e.g., HGS-ETR2, Human Genome
Sciences), conatumumab DR5 (e.g. AMG655, Amgen), tigatuzumab DR5
(e.g., CS-1008), AT009 CXCR4 (Affitech), AT008 CCR4 (Affitech),
CNTO-888 CCL2 (Centocor), AMG102 HGF (Amgen), CRYPTO antibody
(Biogen Idec), M200 antibody VLA5 (Biogen Idec), ipilimumab CTLA4
(e.g., MDX-010, Bristol-Myers Squibb/Medarex), belatacept CTLA4 ECD
(e.g., CP-675,206, Pfizer), IMMU114 HLA-DR (Immunomedics),
apolizumab HLA-DR, toclizumab IL6R (e.g.,
ACTEMR.RTM.A/ROACTREMRA.RTM., Hoffman-La Roche), OX86 OX40,
pemturnomab PEM/MUC1 (Theragyn), ABX-MA1 MUC-18 (Abgenix),
clivatuzumab MUC-18 (e.g., hPAM4, Immunomedics), cantuzumab
mertansine mucin CanAg, ecromeximab (Ludwig Institute), Genentech
anti-EGFL7 antibody, AMG820 CSFR (Amgen), olaratumab PDGFRa (e.g.,
antibody of Imclone (Imclone)), IL21 antibody Zymogenetics
(Zymogenetics), MEDI-573 IGF1/IGF2 (Medlmmune), AMG191 cKit
(Amgen), etaracizumab (e.g., MEDI-522, MedImune), and MLN591 PSMA
(Millennium Pharmaceuticals), elotuzumab SLAMF7 (e.g., HuLuc63,
BMS), labetuzumab CEA (CEA-CIDE.RTM., Immunomedics), sibrotuzumab
FAP, CNTO95 integrin avb3 (Centocor), VITAXIN.RTM. integrin avb3
(MedImmune), and voloximab .alpha.5.beta.1 (antibody targets are
italicized). MRDs that compete for target binding with one of the
above antibodies are encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1, 2, 3, 4, 5, 6, or more of the above antibodies are also
encompassed by the invention.
[0252] In additional embodiments, the antibody of the
MRD-containing antibody competes for target binding with an
antibody selected from: MDX-1342 CD19 (BMS), SGN-CD19A CD19
(Seattle Genetics), an anti-CD20 antibody described in U.S. Pat.
No. 5,500,362, ofatumumab CD20 (e.g., ARZERRA.RTM., GENMAB),
veltuzumab CD20 (hA20, Takeda and Nycomed), PRO70769 CD20
(Genentech; see e.g., Intl. Appl. No. PCT/US2003/040426), AMG780
Tie2/Ang1 (Amgen), REGN910 ANG2 (Regeneron), and anti-CD22 antibody
described in U.S. Pat. No. 5,789,554 (Immunomedics), lumiliximab
CD23 (e.g., IDEC152, Biogen), IDEC-152 CD23 (Biogen), MDX-1401 CD30
(BMS), HeFi-1 CD30 (NCl), daratumumab CD38, an anti CD-40 antibody
described in Intl. Appl. Publ. No. WO2007124299 (Novartis),
IDEC-131 CD40L (Biogen), MDX-1411 CD70 (BMS), SGN-75 CD70 ADC
(Seattle Genetics), HuMax-CD74.TM. CD74 ADC (Genmab), IDEC-114 CD80
(Biogen), TRC105 CD105/endoglin (Tracon), ABX-CBL CD147 (Amgen),
RG1HuMax-TF.TM. Tissue Factor (TF)(Genmab), HuMax-Her2.TM. ErbB2
(Genmab), Trastuzumab-DM1 ErbB2-DM1 (Genentech), AMG888 HER3 (Amgen
and Daiichi Sankyo), HuMV833 VEGF (Tsukuba Research Lab, see, e.g.,
Intl. Appl. Publ. No. WO/2000/034337), IMC-18F1 VEGFR1 (Imclone),
IMC-1C11 VEGFR (Imclone), DC101 VEGFR2 (Imclone), KSB-102 EGFR (KS
Biomedix), mAb-806 EGFR (Ludwig Institute for Cancer Research),
MR1-1 EGFRvIII toxin (IVAX, National Cancer Institute), HuMax-EGFR
EGFR (Genrnab, see, e.g., U.S. application Ser. No. 10/172,317),
IMC-11F8 EGFR (Imclone), CDX-110 EGFRvIII (AVANT
Immunotherapeutics), zalumumab EGFR (Genmab), 425, EMD55900 and
EMD62000 EGFR (Merck KGaA, see, e.g., U.S. Pat. No. 5,558,864),
ICR62EGFR (Institute of Cancer Research, see, e.g., Intl. Appl.
Publ. No. WO95/20045), SC100 EGFR (Seance11 and ISU Chemical),
MOR201 FGFR-3 (Morphosys), ARGX-111 c-Met (arGEN-X), HuMax-cMet.TM.
cMet (Genmab), GC-1008 TGFb1 (Genzyme), MDX-070 PMSA (BMS), huJ591
PSMA (Cornell Research Foundation), muJ591 PSMA (Cornell Research
Foundation), GC1008 TGFb (Genzyme), NG-1 Ep-CAM (Xoma), MOR101
ICAM-1 (CD54) (Morphosys), MOR102 ICAM-1 (CD54)(Morphosys), ABX-MA1
MUC18 (Abgenix), HumaLYM (Intracel), HumaRAD-HN (Intracel),
HumaRAD-OV (Intracel), ARGX-110 and ARGX-111 (arGEN-X),
HuMax-Lymphoma (Genrnab and Amgen), Milatuzumab CD74 (e.g.,
IMMU-115, IMMU-110; Immunomedics), HuMax-Cancer Heparanase I
(Genmab), Hu3S193 Lewis (y) (Wyeth, Ludwig Institute of Cancer
Research), RAV12 N-linked carbohydrate epitope (Raven), nimotuzumab
(TheraCIM, hR3; YM Biosciences, see, e.g., U.S. Pat. Nos. 5,891,996
and 6,506,883), BEC2 GD3 (Imclone), .sup.90Ytacatuzumab tetraxetan
alpha fetoprotein (e.g., FP-CIDE.RTM., Immunomedics), KRN330
(Kirin), huA33 A33 (Ludwig Institute for Cancer Research), mAb 216
B cell glycosylated epitope (NCl), REGN421 DLL4 (Regeneron),
ASG-5ME SLC44A4 ADC (AGS-5), ASG-22ME Nectin-4 ADC, CDX-1307
(MDX-1307), hCGb (Celldex), parathyroid hormone-related protein
(PTH-rP)(UCB), MT293 cleaved collagen (TRC 093/D93, Tracon),
KW-2871 GD3 (Kyowa), KIR (1-7F9) KIR (Novo), A27.15 transferrin
receptor (Salk Institute, see, e.g., Intl. Appl. Publ. No.
WO2005/111082) and E2.3 transferrin receptor (Salk Institute). MRDs
that compete for target binding with one of the above antibodies
are encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4,
5, 6, or more of the above antibodies are also encompassed by the
invention. In additional embodiments, one of the above-described
antibodies is the antibody of the MRD-containing antibody.
[0253] In particular embodiments, the antibody of the
MRD-containing antibody is an antibody selected from siplizumab CD2
(e.g., MEDI-507, MedImmune), blinatumomab CD19 CD3 (e.g., MT103,
Micromet/MedImmune); XMAB.RTM.5574 CD19, (Xencor), SGN-19A CD19
(Seattle Genetics), ASG-5ME (Agenesys and Seattle Genetics),
MEDI-551 CD19 (MedImmune), epratuzumab CD22 (e.g., hLL2,
Immunomedics/UCB), inotuzumab ozogamicin CD22, iratumumab CD30
(e.g., SGN-30 (Seattle Genetics) and MDX-060 (Medarex)),
XMAB.RTM.2513 CD30 (Xencor), brentuximab vedotin CD30 (e.g.,
SGN-35, Seattle Genetics), gemtuzumab ozogamicin CD33 (e.g.,
MYLOTARG.RTM., Pfizer), lintuzumab CD33 (e.g., antibody of Seattle
Genetics), MOR202 CD38 (MorphoSys), daratumumab CD38 (e.g., Genmab
antibody), CP870893 CD40 (Pfizer), dacetuzumab CD40 (e.g., SGN40,
Seattle Genetics), ANTOVA.RTM. CD40 (Biogen Idec), lucatumumab CD40
(e.g., HCD122, Novartis) XMAB.RTM.5485 CD40 (Xencor), teneliximab,
ruplizumab CD40L (e.g., ANTOVA.RTM.), bivatuzumab mertansine
CD44v6, alemtuzumab CD52 (e.g., CAMPATH.RTM./MABCAMPATH.RTM.,
Genzyme/Bayer), BI505 ICAM1 (Bioinvent), milatuzumab CD74 (e.g.,
antibody of Immunomedics), galiximab CD80 (Biogen Idec), BMS663513
4-1BB (Bristol-Myers Squibb), Alexion CD200 antibody (Alexion),
edrecolomab EPCAM (e.g., MAb17-1A, PANOREX.RTM. (GlaxoSmithKline),
AT003 EPCAM (Affitech)), adecatumumab EPCAM (e.g., MT201,
Micromet), oportuzumab monatox EPCAM, Genentech anti-NRP1 antibody,
MORAB004 TEM1 (Morphotek), MORAB009 mesothelin (Morphotek),
lerdelimumab TGFb1 (e.g., CAT-152, Cambridge Antibody Technology),
metelimumab TGFb1 (e.g., CAT-192, Cambridge Antibody Technology),
ImClone anti-TGFBRII antibody, bavituximab phosphatidylserine
(e.g., antibody of Peregrine (Peregrine Pharmaceuticals)), AT004
phosphatidylserine (Affitech), AT005 phosphatidylserine (Affitech),
MORAB03 folate receptor alpha (Morphotek), farletuzumab folate
receptor alpha cancer (e.g., MORAB003, Morphotek), CS1008 DR4
(Sankyo), mapatumumab DR4 (e.g., HGS-ETR1, Human Genome Sciences),
LBY135 DR5 (Novartis), AMG66 DR5 (Amgen), Apomab DR5 (Genentech),
PRO95780 (Genentech), lexatumumab DR5 (e.g., HGS-ETR2, Human Genome
Sciences), conatumumab DR5 (e.g., AMG655, Amgen), tigatuzumab
(e.g., CS-1008), AT009 CXCR4 (Affitech), AT008 CCR4 (Affitech),
CNTO-888 CCL2 (Centocor), AMG102 HGF (Amgen), CRYPTO antibody
(Biogen Idec), M200 antibody VLA5 (Biogen Idec), ipilimumab CTLA4
(e.g., MDX-010, Bristol-Myers Squibb/Medarex), belatacept CTLA4 ECD
(e.g., CP-675,206, Pfizer), IMMU114 HLA-DR (Immunomedics),
apolizumab HLA-DR, toclizumab IL6R (e.g.,
ACTEMR.RTM.A/ROACTREMRA.RTM., Hoffman-La Roche) OX86 OX40,
pemtumomab PEM/MUC1 (Theragyn), ABX-MA1 MUC-18 (Abgenix),
cantuzumab mertansine mucin CanAg, ecromeximab (Ludwig Institute),
Genentech anti-EGFL7 antibody, AMG820 CSFR (Amgen), olaratumab
PDGFRa (e.g., antibody of Imclone (Imclone)), IL21 antibody
Zymogenetics (Zymogenetics), MEDI-573 IGF1/IGF2 (MedImmune), AMG191
cKit (Amgen), etaracizumab (e.g., MEDI-522, MedImmuune), MLN591
PSMA (Millennium Pharmaceuticals), elotuzumab SLAMF7 (e.g.,
HuLuc63, PDL), labetuzumab CEA (CEA-CIDE.RTM., Immunomedics),
sibrotuzumab FAP, CNTO95 integrin avb3 (Centocor), VITAXIN.RTM.
integrin avb3 (MedImmune), and voloximab .alpha.5.beta.1 (e.g.,
M200, PDL and Biogen Idec).
[0254] In an additional embodiment, the antibody target of the
MRD-containing antibody is ALK1. In one embodiment, the antibody is
PF-3,446,962 (Pfizer). In another embodiment, the antibody binds to
the same epitope as PF-3,446,962. In a further embodiment, the
antibody competitively inhibits binding, of PF-3,446,962 to ALK1.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
ALK1 binding with PF-3,446,962 are also encompassed by the
invention.
[0255] In an additional embodiment, the antibody target of the
MRD-containing antibody is CD22. In one embodiment, the antibody is
inotuzumab (e.g., inotuzumab ozogamicin CMC-544, PF-5,208,773;
Pfizer). In one embodiment, the antibody binds to the same epitope
as inotuzumab. In another embodiment, the antibody competitively
inhibits binding of inotuzumab to CD22. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for CD22 binding with
inotuzumab are also encompassed by the invention.
[0256] In an additional embodiment, the antibody target of the MRD
containing antibody is CRYPTO. In one embodiment, the antibody is
the Biogen CRYPTO antibody that has advanced to phase I clinical
trials (Biogen Idec). In another embodiment, the antibody binds to
the same epitope as, the Biogen CRYPTO antibody. In a further
embodiment, the antibody competitively inhibits binding of the
Biogen CRYPTO antibody to CRYPTO. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for CRYPTO binding with the Biogen
CRYPTO antibody are also encompassed by the invention.
[0257] In an additional embodiment, the antibody target of the
MRD-containing antibody is TNFSF5 (CD40 LIGAND). In one embodiment,
the antibody is the Biogen CD40L antibody that has advanced to
phase I clinical trials (Biogen Idec). In another embodiment, the
antibody binds to the same epitope as the Biogen CD40L antibody. In
a further embodiment, the antibody competitively inhibits binding
of the Biogen CD40L antibody to CD40L. Multivalent and
multispecific con positions (e.g., MRD-containing antibodies)
having 1, 2, 3, 4, 5, 6, or more MRDs that compete for CD40L
binding with the Biogen CD40L antibody are also encompassed by the
invention.
[0258] In an additional embodiment, the antibody target of the
MRD-containing antibody is CD80. In one embodiment, the antibody is
galiximab (Biogen Idec). In another embodiment, the antibody binds
to the same epitope as galiximab. In a further embodiment, the
antibody competitively inhibits binding of galiximab to CD80.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
CD80 binding with galiximab are also encompassed by the
invention.
[0259] In additional embodiments, an MRD-containing antibody binds
CD80 and a target selected from: CD2, CD3, CD4, CD19, CD20, CD22,
CD23, CD30, CD33, TNFRSF5 (CD40), CD52, CD74, TNFRSF10A (DR4),
TNFRSF10B (DR5), VEGFR1, VEGFR2 and VEGF. In additional
embodiments, an MRD-containing antibody binds CD80 and a target
selected from: CD3, CD4 and NKG2D. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind CD80 and
also at least bind 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds CD80. In further
embodiments, the antibody component of the MRD-containing antibody
is galiximab.
[0260] In an additional embodiment, the antibody target of the
MRD-containing antibody is MCSF. In one embodiment, the antibody is
PD-360,324 (Pfizer). In another embodiment, the antibody binds to
the same epitope as PD-360,324. In a further embodiment, the
antibody competitively inhibits binding PD-360,324 to MCSF.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
MCSF binding with PD-360,324 are also encompassed by the
invention.
[0261] In an additional embodiment, the antibody target of the
MRD-containing antibody is CD44. In one embodiment, the antibody is
PF-3,475,952 (Pfizer). In another embodiment, the antibody binds to
the same epitope as PF-3,475,952. In a further embodiment, the
antibody competitively inhibits binding of PF-3,475,952 to CD44.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
CD44 binding with PF-3,475,952 are also encompassed by the
invention.
[0262] In an additional embodiment, the antibody target of the
MRD-containing antibody is p-cadherin (CDH3). In one embodiment,
the antibody is PF-3,732,010 (Pfizer). In another embodiment, the
antibody binds to the same epitope as PF-3,732,010. In a further
embodiment, the antibody competitively inhibits binding of
PF-3,732,010 to p-cadherin. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for p-cadherin binding with
PF-3,732,010 are also encompassed by the invention.
[0263] In another embodiment, the antibody target of the
MRD-containing antibody is ANG2 (ANGPT2). In one embodiment, the
antibody is MEDI3617 (MedImmune). In one embodiment, the antibody
binds to the same epitope as MEDI3617. In another embodiment, the
antibody competitively inhibits binding of MEDI3617 to ANG2.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
ANG2 binding with MEDI3617 are also encompassed by the
invention.
[0264] In other embodiments, the antibody component of the
MRD-containing antibody is an ANG-2 binding antibody disclosed in
U.S. Pat. Nos. 7,063,965, 7,063,840, 6,645,484, 6,627,415,
6,455,035, 6,433,143, 6,376,653, 6,166,185, 5,879,672, 5,814,464,
5,650,490, 5,643,755, 5,521,073; U.S. Appl. Publ. Nos. 2011/0158978
(e.g., H4L4), 2006/0246071, 2006/0057138, 2006/0024297,
2006/0018909, 2005/0100906, 2003/0166858, 2003/0166857,
2003/0124129, 2003/0109677, 2003/0040463 and 2002/0173627; or Intl.
Appl. Publ. Nos. WO2006/020706, WO2006/045049, WO2006/068953, or
WO2003/030833 (the disclosure of each of which is herein
incorporated by reference in its entirety). Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for ANG2 binding with
these antibodies are also encompassed by the invention.
[0265] In another embodiment, an MRD-containing antibody binds ANG2
and additionally binds a target selected from: VEGF (i.e., VEGFA),
VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g.,
FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, TNF, FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL1, IL1beta, IL6,
IL8, IL18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4,
PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7,
.alpha.2.beta.1 integrin, .alpha.4.beta.1 integrin, .alpha.5.beta.1
integrin, .alpha.v.beta.3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12,
PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind ANG2 and
at least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In further embodiments, the antibody
component of the MRD-containing antibody is MEDI3617, AMG780 or
REGN910. In further embodiments, the antibody component of the
MRD-containing antibody is H4L4.
[0266] In particular embodiments, the MRD-containing antibody binds
ANG2 and TNF. In additional embodiments, the MRD-containing
antibody binds ANG2 and IL6. In other embodiments, the
MRD-containing antibody binds ANG2 and IL1. In further embodiments,
the administered MRD-containing antibody binds ANG2, IL6 and TNF.
In further embodiments, the administered MRD-containing antibody
binds ANG2, IL1 and TNF. In further embodiments, the MRD-containing
antibody binds ANG2, IL1, IL6 and TNF.
[0267] In particular embodiments, the MRD-containing antibody binds
ANG2 and TNF and the antibody component of the MRD-containing
antibody is adalimumab. In another embodiment, the MRD-containing
antibody competes with adalimumab for binding to TNF.
[0268] In additional embodiments, the antibody component of the
MRD-containing antibody binds ANG2. In further embodiments, the
antibody component of the MRD-containing antibody is an ANG2
binding antibody selected from SAITAng-2-1, SAITAng-2-2,
SAITAng-2-3, SAITAng-2-4 or another antibody disclosed in Intl.
Appl. Publ. No. WO2009/142460. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having an antibody
and/or 1, 2, 3, 4, 5, 6, or more MRDs that compete for ANG2 binding
with one or more of these antibodies are also encompassed by the
invention.
[0269] In additional embodiments, the antibody component of the
MRD-containing antibody binds TIE2. In further embodiments, the
antibody component of the MRD-containing antibody is a TIE2 binding
antibody disclosed in U.S. Pat. Nos. 6,365,154 and 6,376,653; U.S.
Appl. Publ. Nos. 2007/0025993, 2006/0057138 and 2006/0024297; or
Intl. Appl. Publ. Nos. WO2006/020706, WO2000/018437 and
WO2000/018804 (the disclosure of each of which is herein
incorporated by reference in its entirety). Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
an antibody and/or 1, 2, 3, 4, 5, 6, or more MRDs that compete for
TIE binding with one or more these antibodies are also encompassed
by the invention.
[0270] In certain embodiments, the antibody target of the
MRD-containing antibody is EGFR(ErbB1), ErbB2, ErbB3, ErbB4, CD20,
insulin-like growth factor-I receptor, prostate specific membrane
antigen, an integrin, or cMet.
[0271] In one embodiment, the antibody in the MRD-containing
antibody specifically binds EGFR(ErbB1). In a specific embodiment,
the antibody is ERBITUX.RTM. (IMC-C225). In one embodiment, the
antibody binds to the same epitope as ERBITUX.RTM.. In another
embodiment, the antibody competitively inhibits binding of
ERBITUX.RTM. to EGFR. In another embodiment, the antibody in the
MRD-containing antibody inhibits EGFR dimerization. In another
specific embodiment, the antibody is matuzumab (e.g., EMD 72000,
Merck Serono) or panitumumab (e.g., VECTIBIX.RTM., Amgen). In
another embodiment, the antibody binds to the same epitope as
matuzumab or panitumumab. In another embodiment, the antibody
competitively inhibits binding of matuzumab or panitumumab to EGFR.
In another embodiment, the antibody is ABX-EGF (Immunex) or
MEDX-214 (Medarex). In another embodiment, the antibody binds to
the same epitope as ABX-EGF or MEDX-214. In another embodiment, the
antibody competitively inhibits binding of ABX-EGF or MEDX-214 to
EGFR. In another specific embodiment, the antibody is zalutumumab
(Genmab) or nimotazumab (Biocon). In an additional embodiment, the
antibody binds to the same epitope as zalutumumab (Genmab) or
nimotuzumab (Biocon). In another embodiment, the antibody
competitively inhibits binding of zalutumumab (Genmab) or
nimotuzumab (Biocon) to EGFR.
[0272] In one embodiment, an MRD-containing antibody binds
EGFR(ErbB1) and a target selected from: HGF, CD64, CDCP1, RON,
cMET, ErbB2, ErbB3, IGF1R, PLGF, RGMa, PDGFRa, PDGFRb, VEGFR1,
VEGFR2, TNFRSF10A (DR4), TNFRSF10B (DR5), IGF1,2, IGF2, CD3, CD4,
NKG2D and tetanus toxoid. In some embodiments, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibodies) binds at least 1, 2, 3, 4, 5 or more of these targets.
In specific embodiments, the antibody component of the
MRD-containing antibody binds EGFR. In further embodiments, the
antibody component of the MRD-containing antibody is matuzumab,
panitumumab, MEDX-214, or ABX-EGF. In further embodiments, the
antibody component of the MRD-containing antibody is nimotuzumab
(Biocon) or zalutumumab. In specific embodiments, the antibody
component of the MRD-containing antibody is Erbitux.RTM..
[0273] In specific embodiments, the MRD containing antibody binds
ErbB1 and additionally binds ErbB3. In some embodiments, the
antibody component of the MRD-containing antibody binds ErbB1 and
an MRD of the MRD-containing antibody binds ErbB3. In a particular
embodiment, the antibody component of the MRD-containing antibody
is cetuximab. In additional embodiments, the antibody component of
the MRD-containing antibody competes for ErbB1-binding with
cetuximab. In another embodiment, the antibody in the
MRD-containing antibody is an ErbB1-binding antibody selected from:
nimotuzumab (Biocon), matuzumab (Merck KGaA), panitumumab (Amgen),
zalutumumab (Genmab), MEDX-214, and ABX-EGF. In additional
embodiments, the antibody component, MRD component and/or
MRD-containing antibody competes for ErbB1-binding with an antibody
selected from nimotuzumab, matuzumab, panitumumab, and zalutumumab.
In other embodiments, the antibody component of the MRD-containing
antibody binds ErbB3 and an MRD of the MRD-containing antibody
binds ErbB1 In additional embodiments, the antibody component of
the MRD-containing antibody is an ErbB3-binding antibody selected
from MM121 (Merrimack), 8B8 (Genentech), AV203 (Aveo), and AMG888
(Amgen). In additional embodiments, the antibody component, MRD
component and/or MRD-containing antibody competes for ErbB3 binding
with an antibody selected from MM121, 8B8, AV203, and AMG888.
[0274] In one embodiment the MRD-containing antibody specifically
binds ErbB2 (Her2). In a specific embodiment, the antibody is
trastuzumab (e.g., HERCEPTIN.RTM., Genentech/Roche). In one
embodiment, the antibody binds to the same epitope as trastuzumab.
In another embodiment, the antibody competitively inhibits binding
of trastuzumab to ErbB2. An MRD that competes for target binding
with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2, 3, 4, 5, 6, or more of
the above antibodies are also encompassed by the invention Thus,
the invention encompasses MRD-containing antibodies comprising at
least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with at least 1, 2, 3, 4, 5, 6 of the above antibodies.
[0275] In other embodiments, the antibody in the MRD-containing
antibody specifically binds to ErbB2. In one embodiment, the
antibody in the MRD-containing antibody is an antibody that
specifically binds to the same epitope as the anti-ErbB2 antibody
trastuzumab (e.g., HERCEPTIN.RTM., Genentech). In another
embodiment, the antibody in the MRD-containing antibody is an
antibody that competitively inhibits ErbB2-binding by the
anti-ErbB2 antibody trastuzumab. In yet another embodiment, the
antibody in the MRD-containing antibody is the anti-ErbB2 antibody
trastuzumab. In another embodiment, the antibody in the
MRD-containing antibody inhibits HER2 dimerization. In another
embodiment, the antibody in the MRD-containing antibody inhibits
HER2 heterodimerization with HER3 (ErbB3). In a specific
embodiment, the antibody is pertuzumab (e.g., OMNITARG.RTM. and
phrMab2C4, Genentech). In another embodiment, the antibody
specifically binds to the same epitope as pertuzumab. In another
embodiment, the antibody in the MRD-containing antibody is an
antibody that competitively inhibits binding of ErbB2 by
pertuzumab. An MRD that competes for target binding with one of the
above antibodies is also encompassed by the invention. Multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with 1, 2 or more of the above antibodies are also
encompassed by the invention. Accordingly, in one embodiment the
antibody in the MRD-containing antibody is trastuzumab and 1, 2, 3,
4, 5, 6, or more MRDs in the MRD-containing antibody competitively
inhibit binding of ErbB2 by pertuzumab.
[0276] In another embodiment, the antibody in the MRD-containing
antibody is an ErbB2-binding antibody selected from the group:
MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics), MGAH22
(MacroGenics), and pertuzumab (OMNITARG.TM., 2C4; Genentech). An
MRD that competes for target binding with one of the above
antibodies is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1, 2, 3, or 4 of the above antibodies are also encompassed by the
invention. Thus, the invention encompasses MRD-containing
antibodies comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that
compete for target binding with at least 1, 2, 3 or 4 of the above
antibodies.
[0277] In specific embodiments, the MRD containing antibody binds
ErbB2 and additionally binds ErbB3. In some embodiments, the
antibody component of the MRD-containing antibody binds ErbB2 and
an MRD of the MRD-containing antibody binds ErbB3. In a particular
embodiment, the antibody component of the MRD-containing antibody
is trastuzumab. In additional embodiments, the antibody component,
MRD component and/or MRD-containing antibody competes for
ErbB2-binding with trastuzumab. In another embodiment, the antibody
in the MRD-containing antibody is an ErbB2-binding antibody
selected from MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics),
MGAH22 (MacroGenics), and pertuzumab (OMNITARG.TM.). In additional
embodiments, the antibody component, MRD component and/or
MRD-containing antibody competes for ErbB2-binding with an antibody
selected from MDX-210, tgDCC-E1A, MGAH22, and pertuzumab. In other
embodiments, the antibody component of the MRD-containing antibody
binds ErbB3 and an MRD of the MRD-containing antibody binds
ErbB2.
[0278] In some embodiments, the antibody in the MRD-containing
antibody comprises the CDRs of the anti-ErbB2 antibody trastuzumab.
The CDR, VH, and VL sequences of trastuzumab are provided in Table
1.
TABLE-US-00001 TABLE 1 CDR Sequence VL-CDR1 RASQDVNTAVAW (SEQ ID
NO: 59) VL-CDR2 SASFLYS (SEQ ID NO: 60) VL-CDR3 QQHYTTPPT (SEQ ID
NO: 61) VH-CDR1 GRNIKDTYIH(SEQ ID NO: 62) VH-CDR2
RIYPTNGYTRYADSVKG(SEQ ID NO: 63) VH-CDR3 WGGDGFYAMDY(SEQ ID NO: 64)
VL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQK
PGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL
QPEDFATYYCQQHYTTPPTFGQGTKVEIKRT (SEQ ID NO: 65) VH
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQ
APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT
AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VSS (SEQ ID NO: 66)
[0279] In one embodiment the MRD-containing antibody specifically
binds ErbB3 (Her3). In a specific embodiment, the antibody is MM121
(Merrimack Pharmaceuticals) or AMG888 (Amgen). In one embodiment,
the antibody binds to the same epitope as MM121 or AMG888. In
another embodiment, the antibody competitively inhibits binding of
MM121 or AMG888 to ErbB3. In, another specific embodiment, the
antibody is AV-203 (AVEO). In one embodiment, the antibody binds to
the same epitope as AV-203. In another embodiment, the antibody
competitively inhibits binding of AV-203. An MRD that competes for
target binding with one of the above antibodies is also encompassed
by the invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1 or both of the above
antibodies are also encompassed by the invention
[0280] In one embodiment the MRD-containing antibody specifically
binds VEGF (VEGFA). In a specific embodiment, the antibody is
bevacizumab (e.g., AVASTIN.RTM., Genentech/Roche). In one
embodiment, the antibody binds to the same epitope as bevacizumab.
In another embodiment, the antibody competitively inhibits binding
of bevacizumab to VEGFA. In another embodiment the MRD-containing
antibody is AT001 (Affitech). In one embodiment, the antibody binds
to the same epitope as AT001. In another embodiment, the antibody
competitively inhibits binding of AT001 to VEGFA. An MRD that
competes for target binding with one of the above antibodies is
also encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1 or both
of the above antibodies are also encompassed by the invention.
[0281] In some embodiments, the antibody in the MRD-containing
antibody comprises the CDRs of the anti-VEGF antibody bevacizumab.
The CDR, VH, and VL sequences of bevacizumab are provided in Table
2.
TABLE-US-00002 TABLE 2 CDR Sequence VL-CDR1 SASQDISNYLN (SEQ ID NO:
72) VL-CDR2 FTSSLHS (SEQ ID NO: 73) VL-CDR3 QQYSTVPWT (SEQ ID NO:
74) VH-CDR1 GYTFTNYGMN (SEQ ID NO: 75) VH-CDR2 WINTYTGEPTYAADFKR
(SEQ ID NO: 76) VH-CDR3 YPHYYGSSHWYFDV (SEQ ID NO: 77) VL
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP
GKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYSTVPWTFGQGTKVEIKR (SEQ ID NO: 78) VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA
PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY
LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT VSS (SEQ ID NO: 79)
[0282] In other specific embodiments, the antibody in the
MRD-containing antibody specifically binds VEGF. In a specific
embodiment, the antibody is bevacizumab (e.g., AVASTIN.RTM.,
Genentech). In one embodiment, the antibody binds to the same
epitope as bevacizumab. In another embodiment, the antibody
competitively inhibits binding of bevacizumab to VEGF. In another
specific embodiment, the antibody is r84 (Peregrine) or 2C3
(Peregrine). In another embodiment, the antibody binds to the same
epitope as r84 or 2C3. In another embodiment, the antibody
competitively inhibits VEGF binding by r84 or 2C3. An MRD that
competes for target binding with one of the above antibodies is
also encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2, or 3
of the above antibodies are also encompassed by the invention.
[0283] In one embodiment, an MRD-containing antibody binds VEGF and
additionally binds an angiogenic target selected from: VEGFB, FGF1,
FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2
(e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TNFSF2
(TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF, TIE2,
PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSE, CXCR4, PTPRC, TIE2, VEGFR1,
VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, .alpha.2.beta.1 integrin,
.alpha.4.beta.1 integrin, .alpha.5.beta.1 integrin, .alpha.v.beta.3
integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and
PDGFRB. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind VEGF and at least 1, 2, 3, 4,
5 or more of these targets are also encompassed by the invention.
In specific embodiments, the antibody component of the
MRD-containing antibody binds VEGF. In further embodiments, the
antibody component of the MRD-containing antibody is r85, 2C3 or
AT001. In a specific embodiment, the antibody component of the
MRD-containing antibody is bevacizumab.
[0284] In one embodiment, an MRD-containing antibody binds VEGF and
additionally binds a target selected from: IL1 beta,
phosphatidylserine, TNFSF11 (RANKL), TNFSF12 (TWEAK), IGF1,2, IGF2,
IGF1, DKK1, SDF2, CXC3CL1 (fractalkine), sclerostin and tetanus
toxoid and HGF. In another embodiment, an MRD-containing antibody
binds VEGF and additionally binds a target selected from: ErbB3,
EGFR, cMet, VEGF, RON (MST1R), DLL4, CDCP1 CD318), NRP1, ROBO4,
CD13, CTLA4 (CD152), ICOS (CD278), CD20, CD22, CD30, CD33, CD80 and
IL6R. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind VEGF and at least 1, 2, 3, 4,
5 or more of these targets are also encompassed by the invention.
In specific embodiments, the antibody component of the
MRD-containing antibody binds VEGF. In further embodiments, the
antibody component of the MRD-containing antibody is r85, 2C3 or
AT001. In a specific embodiment, the antibody component of the
MRD-containing antibody is bevacizumab.
[0285] In another embodiment, the MRD-containing antibody
specifically binds VEGFR1. In one embodiment, the antibody
competitively inhibits binding of Aflibercept (Regeneron) to
VEGFR1. In another embodiment, the antibody in the MRD-containing
antibody inhibits VEGFR1 dimerization. An MRD that competes for
target binding with Aflibercept is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with Aflibercept are also
encompassed by the invention.
[0286] In another embodiment, the MRD-containing antibody
specifically binds VEGFR2. In a specific embodiment, the antibody
is ramucirumab (e.g., IMC1121B and IMC1C11, ImClone). In another
embodiment, the antibody in the MRD-containing antibody inhibits
VEGFR2 dimerization. In one embodiment, the antibody binds to the
same epitope as ramucirumab. In another embodiment, the antibody
competitively inhibits binding of ramucirumab to VEGFR2. In another
embodiment, the antibody competitively inhibits binding of
Aflibercept to VEGFR2. An MRD that competes for target binding with
ramucirumab is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
ramucirumab or Aflibercept are also encompassed by the
invention.
[0287] In other embodiments, the antibody in the MRD-containing
antibody specifically binds to an FGF receptor (e.g., FGFR1, FGFR2,
FGFR3, or FGFR4). In one embodiment, the antibody in the
MRD-containing antibody is an antibody that specifically binds to
FGFR1 (e.g., FGFR1-IIIC). In a specific embodiment, the antibody is
IMC-A1 (lineIone), IL one embodiment, the antibody binds to the
same epitope as IMC-A1. In another embodiment, the antibody
competitively inhibits binding of IMC-A1 to FGFR1. In an additional
embodiment, the antibody competitively inhibits binding of FP-1039
(Five Prime) to an FGF ligand of FGFR1. In another embodiment, the
antibody in the MRD-containing antibody is an antibody that
specifically binds to FGFR2 (e.g., FGFR2-IIIB and FGFR2-IIIC). In a
further embodiment, the antibody in the MRD-containing antibody is
an antibody that specifically binds to FGFR3. In a specific
embodiment, the antibody is IMC-A1 (Imclone). In one embodiment,
the antibody binds to the same epitope as PRO-001 (ProChon
Biotech), R3Mab (Genentech), or 1A6 (Genentech). In another
embodiment, the antibody competitively inhibits binding of PRO-001
(ProChon Biotech), R3Mab (Genentech), or 1A6 (Genentech). An MRD
that competes for target binding with one of the above antibodies
or ligand traps is also encompassed by the invention. Multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with 1 or more of the above antibodies or ligand traps are
also encompassed by the invention.
[0288] In one embodiment, the antibody in the MRD-containing
antibody specifically binds CD20. In a specific embodiment the
antibody is rituximab (e.g., RITUXAN.RTM./MABTHERA.RTM.,
Genentech/Roche/Biogen Idec). In one embodiment, the antibody binds
to the same epitope as rituximab. In another embodiment, the
antibody competitively inhibits binding of rituximab to CD20. In an
additional embodiment, the antibody is GA101 (Biogen
Idec/Roche/Glycart). In one embodiment, the antibody binds to the
same epitope as GA101. In another embodiment, the antibody
competitively inhibits binding of GA101 to CD20. In an additional
embodiment, the antibody is PF-5,230,895 (SBI-087; Pfizer). In one
embodiment, the antibody binds to the same epitope as PF-5,230,895.
In another embodiment, the antibody competitively inhibits binding
of PF-5,230,895 to CD20. In another specific embodiment, the
antibody is ocrelizumab (e.g., 2H7; Genentech/Roche/Biogen Idec).
In one embodiment, the antibody binds to the same, epitope as
ocrelizumab. In another embodiment, the antibody competitively
inhibits binding of ocrelizumab to CD20. In another specific
embodiment, the MRD-containing antibody is selected from:
obinutuzumab (e.g., GA101; Biogen Idec/Roche/Glycart), ofatumumab
(e.g., ARZERRA.RTM. and HuMax-CD20 Genmab), veltuzumab (e.g.,
IMMU-160, Immunomedics), AME-133 (Applied Molecular Evolution),
SGN35 (Millennium), TG-20 (GTC Biotherapeutics), afutuzumab
(Hoffman-La Roche) and PRO131921 (Genentech). In another
embodiment, the antibody binds to the same epitope as an antibody
selected from: obinutuzumab, ofatumumab, veltuzumab, AME-133,
SGN35, TG-20 and PRO131921. In another embodiment, the antibody
competitively, inhibits CD20 binding by an antibody selected from
obinutuzumab, ofatumumab, veltuzumab, AME-133, SGN35, TG-20,
afutuzumab, and PRO131921. An MRD that competes for target binding
with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2, 3, 4, 5, 6, or more of
the above antibodies are also encompassed by the invention. Thus,
the invention encompasses MRD-containing antibodies comprising at
least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with at least 1, 2, 3, 4, 5, 6 of the above antibodies.
[0289] In additional embodiments, an MRD-containing antibody binds
CD20 and a target selected from: CD19, CD22, CD30, TNFRSF5 (CD40),
CD52, CD74, CD80, CD138, VEGFR1, VEGFR2, EGFR, TNFRSF10A (DR4),
TNFRSF10B (DR5), TNF, NGF, VEGF, IGF1,2, IGF2, IGF1 and RANKL. In,
additional embodiments, an MRD-containing antibody binds CD20 and a
target selected from: CD3, CD4 and NKG2D. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind CD20 and also bind 2, 3, 4, 5 or more of these targets are
also encompassed by the invention. In specific embodiments, the
antibody component of the MRD-containing antibody binds CD20. In
further embodiments, the antibody component of the MRD-containing
antibody is an antibody selected from: rituximab, GA101,
PF-5,230,895, ocrelizumab obinutuzumab, ofatumumab, veltuzumab,
AME-133, SGN35, TG-20, afutuzumab and PRO131921.
[0290] In one embodiment the MRD-containing antibody specifically
binds IGF1R. In a specific embodiment, the antibody is selected
from cixutumumab (e.g., IMC-A12, Imclone), figitumumab (e.g.,
CP-751,871, Pfizer), AMG479 (Amgen), BIIB022 (Biogen Idec), SCH
717454 (Schering-Plough), and R1507 (Hoffman La-Roche). In one
embodiment, the antibody binds to the same epitope as an antibody
selected from cixutumumab, figitumumab, AMG479, BIIB022, SCH
717454, and R1507. In another embodiment, the antibody
competitively inhibits IGF1R binding by an antibody selected from
cixutumumab, figitumumab, AMG479, BIIB022, SCH 717454, and R1507.
In a specific embodiment, the antibody is figitumumab. In another
specific embodiment, the antibody binds to the same epitope as
figitumumab. In a further specific embodiment, the antibody
competitively inhibits IGF1R binding by figitumumab. In an
additional specific embodiment, the antibody is BIIB022. In another
specific embodiment, the antibody binds to the same epitope as
BIIB022. In a further specific embodiment, the antibody
competitively inhibits IGF1R binding by BIIB022. In another
embodiment, the antibody in the MRD-containing antibody inhibits
IGF1R dimerization. An MRD that competes for target binding with
one of the above antibodies is also encompassed by the invention.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
IGF1R binding with 1, 2, 3, 4, 5, 6, or more of the above
antibodies are also encompassed by the invention. Thus, the
invention encompasses MRD-containing antibodies comprising at least
1, 2, 3, 4, 5, 6, or more MRDs that compete for IGF1R binding with
at least 1, 2, 3, 4, 5, 6 of the above antibodies.
[0291] In additional embodiments, an MRD-containing antibody binds
IGF1R and a target selected from: EGFR, ErbB2, ErbB3, PDGFRa,
PDGFRb, cMet, TNFRSF10A (DR4), TNFRSF10B (DR5), CD20, NKG2D, VEGF,
PGE2, IGF1, IGF2 and IGF1,2. In additional embodiments, an
MRD-containing antibody binds IGF1R and a target selected from:
CD3, CD4 and NKG2D. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind IGF1R and bind at 1, 2,
3, 4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds IGF1R. In further embodiments, the
antibody component of the MRD-containing antibody is selected from:
cixutumumab, figitumumab, AMG479, BIIB022, SCH 717454, and
R1507.
[0292] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) binds a
target (e.g., ligand, receptor, or accessory protein) associated
with an endogenous blood brain barrier (BBB) receptor mediated
transport system (e.g., the insulin receptor, transferrin receptor,
leptin receptor, lipoprotein receptor, and the IGF receptor
mediated transport systems) and is capable of crossing to the brain
side of the BBB. In some embodiments, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) has 2, 3, 4, 5, or more binding sites (i.e., is capable
of multivalently binding) a target antigen (e.g., ligand, receptor,
or accessory protein) associated with an endogenous BBB receptor
mediated transport system (e.g., the insulin receptor, transferrin
receptor, leptin receptor, lipoprotein receptor, and the IGF
receptor mediated transport systems). In additional embodiments,
the multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) has, a single binding site for a target
associated with an endogenous BBB receptor mediated transport
system. In further embodiments, the multivalent and monovalent
multispecific composition has 2, 3, 4, 5, or more single binding
sites for a target associated with an endogenous BBB receptor
mediated transport system. In further embodiments, the
MRD-containing antibody binds 1, 2, 3, 4, 5, or more targets
located on the brain (cerebrospinal fluid) side of the BBB. In
further embodiments, the MRD-containing antibody additionally binds
1, 2, 3, 4, 5, or more targets located on the brain (cerebrospinal
fluid) side of the BBB. In particular embodiments, the
MRD-containing antibody binds 1, 2, 3, 4, 5, or more targets
associated with a neurological disease or disorder. In particular
embodiments, the neurological disease or disorder is selected from
brain cancer, a neurodegenerative disease, schizophrenia, epilepsy,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
ALS, multiple sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g.,
HIV-associated dementia). Accordingly, the invention encompasses
methods of treating a patient by administering a therapeutically
effective amount of a multivalent and monovalent multispecific
composition to treat a neurological disease or disorder selected
from brain cancer, a neurodegenerative disease, schizophrenia,
epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's
disease, ALS, multiple sclerosis, Neuromyelitis optica and
Neuro-AIDS (e.g., HIV-associated dementia). In another embodiment,
the multivalent and monovalent multispecific composition is
administered to a patient to treat a brain cancer, metastatic
cancer of the brain, or primary cancer of the brain. In additional
embodiments, the multivalent and monovalent multispecific
composition is administered to a patient to treat a neurological
tumor such as, a glioma (e.g., a glioblastoma, glioblastoma
multiforme (GBM), and astrocytoma), ependyrnoma, oligodendroglioma,
neurofibroma, sarcoma, medulloblastoma, primitive neuroectodermal
tumor, pituitary adenoma, neuroblastoma or cancer of the meninges
(e.g., meningioma, meningiosarcoma and gliomatosis). In particular
embodiments the invention encompasses methods of treating a patient
by administering a therapeutically effective amount of a
multivalent and monovalent multispecific composition to treat a
neurodegenerative disease.
[0293] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) binds an
endogenous BBB receptor mediated transport system selected from the
insulin receptor, transferrin receptor, leptin receptor,
lipoprotein receptor, and the IGF receptor mediated transport
systems.
[0294] In some embodiments, the multivalent and multispecific
composition (e.g., MRD-containing antibody) binds transferrin
receptor. In additional embodiments, the MRD containing antibody
binds a target selected from: low-density lipoprotein receptor 1
(LRP-1), a LRP-1 ligand or a functional fragment or variant thereof
that binds LRP-1, Low-density lipoprotein receptor 2 (LRP-2), a
LRP-2 ligand or a functional fragment or variant thereof that binds
LRP-1, a transferrin protein or a functional fragment or variant
thereof, insulin receptor, TMEM30A, leptin receptor, IGF receptor,
an IGFR ligand or a functional fragment or variant thereof,
diphtheria receptor, a diphtheria receptor ligand or a functional
fragment or variant thereof, choline transporter, a complex that
binds choline receptor, an amino acid transporter (e.g., LAT1/CD98,
SLC3A2, and SLC7A5), an amino acid transporter ligand or a
functional fragment or variant thereof, RAGE, a RAGE ligand or a
functional fragment or variant thereof, SLC2A1 and a SLC2A1 ligand
or a functional fragment or variant thereof.
[0295] In additional embodiments, the multivalent and multispecific
composition (e.g., MRD-containing antibody) binds RAGE. In further
embodiments, the multivalent and multispecific composition (e.g.,
MRD-containing antibody) binds RAGE and a target selected from:
Abeta, endothelin1, TNF, IL6, MCSF, an AGE, a S100 member, HMGIB1,
LPS and TLR2. Multivalent and multispecific compositions that bind
RAGE and also bind 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds RAGE.
[0296] In additional embodiments, the multivalent and multispecific
composition (e.g., MRD-containing antibody) binds a target antigen
associated with an endogenous blood brain barrier (BBB) receptor
mediated transport system and also binds a target antigen selected
from alpha-synuclein, RGM A, NOGO A, NgR, OMGp MAG, CSPG, neurite
inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4) an
ephrin, A-beta, AGE (S100 A, amphoterin), NGF, soluble A-B,
aggrecan, midkine, neurocan, versican, phosphacan, Te38, and PGE2,
ILL IL1R, IL6, IL6R, IL12, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5
(CD40), TNFSF5 (CD40 LIGAND), CD45RB, CD52, CD200, VEGF, VLA4, TNF
alpha, Interferon gamma, GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a
and MCP-1. In a further embodiment, the MRD-containing antibody has
a single binding site for a target associated with an endogenous
blood brain barrier (BBB) receptor mediated transport system and
further binds a target selected from alpha-synuclein, RGM A, NOGO
A, NgR, OMGp MAG, CSPG, neurite inhibiting semaphorins (e.g.,
Semaphorin 3A and Semaphorin 4) an ephrin, A-beta, AGE (S100 A,
amphoterin), NGF, soluble A-B, aggrecan, midkine, neurocan,
versican, phosphacan, Te38, PGE2, IL1, IL1R, IL6, IL6R, IL12, IL18,
IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND),
CD45RB, CD52, CD200, VEGF, VLA4, TNF alpha, Interferon gamma,
GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a and MCP-1.
[0297] In additional embodiments, the MRD-containing antibody is
administered to a patient to treat a neurological disease or
disorder selected from brain cancer, a neurodegenerative disease,
schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease,
Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica
and Neuro-AIDS (e.g., HIV-associated dementia). In one embodiment,
the multivalent and monovalent multispecific composition contains 2
binding sites for 2 or more of the above targets. In a further
embodiment, the multivalent and monovalent multispecific
composition contains 2 binding sites for 3 or more targets. In
additional embodiments, the targets bound by the multivalent and
monovalent multispecific composition are associated with cancer. In
a further embodiment the targets bound by the multivalent and
monovalent multispecific composition are associated with 1, 2, 3,
4, 5 or more different signaling pathways or modes of action
associated with cancer.
[0298] In one embodiment, the antibody in the MRD-containing
antibody specifically binds integrin. In a specific embodiment, the
antibody is selected from: MEDI-522 avb3 (VITAXIN.RTM., MedImmune),
CNTO 95 a5b3 (Centocor), JC7U .alpha.v.beta.3, and volociximab a5b1
(e.g., M200, PDL and Biogen Idec). In another embodiment, the
antibody binds to the same epitope as an antibody selected from:
MEDI-522, CNTO 95, JC7U .alpha.v.beta.3, and volociximab. In
another embodiment, the antibody competitively inhibits integrin
binding by an antibody selected from: MEDI-522, CNTO 95, JC7U, and
M200. In a specific embodiment, the antibody is natalizumab (e.g.,
TSABRI.RTM., Biogen Idec). In one embodiment, the antibody binds to
the same epitope as natalizumab. In another embodiment, the
antibody competitively inhibits integrin binding by natalizumab. An
MRD that competes for target binding with one of the above
antibodies is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1, 2, 3, 4, 5, 6, or more of the above antibodies are also
encompassed by the invention.
[0299] In one embodiment, the antibody in the MRD-containing
antibody specifically binds cMet. In a specific embodiment, the
antibody is selected from: MetMab (OA-5D5, Genentech), AMG-102
(Amgen) and DN30. In another embodiment, the antibody binds to the
same epitope as an antibody selected from MetMab), AMG-102 and
DN30. In another embodiment, the antibody competitively inhibits
cMET binding by an antibody selected from MetMab (OA-5D5), AMG-102
and DN30. An MRD that competes for target binding with one of the
above antibodies is also encompassed by the invention. Multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
having 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with 1, 2, or 3 of the above antibodies are also
encompassed by the invention.
[0300] In one embodiment, the antibody in the MRD-containing
antibody specifically binds cMet and the antibody is selected from:
11E1, CE-355621, LA480 and LMH87. In another embodiment, the
antibody binds to the same epitope as an antibody selected from
MetMab), AMG-102 and DN30. In another embodiment, the antibody
competitively inhibits cMET binding by an antibody selected from:
11E1, CE-355621, LA480 and LMH87. An MRD that competes for target
binding with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2, 3 or 4 of the above
antibodies are also encompassed by the invention.
[0301] In additional embodiments, an MRD containing antibody binds
cMET and a target selected from ErbB2, ErbB3, EGFR, IGF1R, NRP1,
RON, PDGFRa, PDGFRb, VEGF, VEGFR1, VEGFR2, TGF beta, TGF beta R2,
CD82, CD152, NGF, BMP2, BMP4, BMP5, BMP9, BMP10, BMPR-IA, ALK1,
a3b1 integrin and HGF. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind cMET and also bind at
least 1, 2, 3, 4, 5 or more of these targets are also encompassed
by the invention. In specific embodiments, the antibody component
of the MRD-containing antibody binds cMET. In further embodiments,
the antibody component of the MRD-containing antibody is an
antibody selected from MetMab, AMG-102 and DN30. In other
embodiments, the antibody component of the MRD-containing antibody
is an antibody selected from 11E1, CE-355621, LA480 and LMH87.
[0302] In additional embodiments, an MRD-containing antibody binds
MST1R (RON). In a specific embodiment, an MRD-containing antibody
binds RON and a target selected from EGFR, ErbB2, ErbB3, VEGFR1,
VEGFR2, cMET, CXCR4, VEGF, MST, MTSP1, CDCP1, EPHB2, NGF, CXCL12
and HGF (SF). Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind MST1R and also bind at least
1, 2, 3, 4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds MST1R.
[0303] In one embodiment, the antibody in the MRD-containing
antibody specifically binds HGF (SF). In a specific embodiment, the
antibody is AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). In
another embodiment, the antibody binds to the same epitope as
AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). In another
embodiment, the antibody competitively inhibits HGF binding by
AMG-102 (Amgen) or SCH 900105 (AV-229, AVEO). An MRD that competes
for target binding with AMG-102 (Amgen) or SCH 900105 (AV-229,
AVEO) is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1, 2, or 3 of the above antibodies are also encompassed by the
invention.
[0304] In a specific embodiment, an MRD-containing antibody binds
HGF and a target selected from: ErbB2, ErbB3, EGFR, IGF1R, NRP1,
RON, PDGFRa, PDGFRb, VEGF, VEGFR1, VEGFR2, TGF beta, TGF beta R2,
CD82, CD152, NGF, BMP2, BMP4, BMP5, BMP9, BMP10, BMPR-IA, ALK1,
a3b1 integrin, cMET, MST1R (RON), CXCR4, MST, MTSP1, CDCP1, EPHB2,
NGF, CXCL12 NRP1 and phosphatidylserine. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind HGF and also bind at least 1, 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds HGF. In further embodiments, the antibody component of the
MRD-containing antibody is AMG-102 or SCH 900105.
[0305] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds a5b1 integrin (VLA5). In
a specific embodiment, the antibody is volociximab (e.g., M200
Biogen Idec). In another embodiment, the antibody binds to the same
epitope as volociximab. In a further embodiment, the antibody
competitively inhibits a5b1 integrin binding by volociximab. An MRD
that competes for a5b1 integrin binding with volociximab is also
encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for a5b1 integrin binding with
volociximab are also encompassed by the invention.
[0306] In another embodiment, the antibody target of the
MRD-containing antibody is an antigen associated with an autoimmune
disorder, inflammatory or other disorder of the immune system or is
associated with regulating an immune response.
[0307] In another embodiment the MRD-containing antibody improves
the performance of antigen presenting cells (e.g., dendritic
cells). In one embodiment the antibody target of the MRD-containing
antibody is a member selecting from: CD19, CD20, CD21, CD22, CD23,
CD27, CD28, CD30, CD30L, TNFSF 14 (LIGHT, HVEM Ligand), CD70, ICOS,
ICOSL (B7-H2), CTLA4, PD-1, PDL1 (B7-H1), B7-H4, B7-H3, PDL2
(B7-DC), BTLA, CD46, CD80 (B7-1), CD86 (B7-2), HLA-DR, CD74, PD1,
TNFRSF4 (OX40), TNFRSF9 (41BB), TNFSF4 (OX40 Ligand), TNFSF9 (41BB
Ligand), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF13B
(TACT), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), TNFRSF18 (GITR), MHC-1,
TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), FcgammaRIIB, and IL4R.
[0308] In one embodiment the antibody target of the MRD-containing
antibody is an immunoinhibitory target selected from IL1, IL1 beta,
IL1Ra, L-5, IL6, IL6R, CD26L, CD28, CD80, FcRn, and Fc Gamma RIIB.
An MRD that binds to one of the above targets is encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that bind to 1, 2, 3, 4, 5, 6, or more of the above targets are
also encompassed by the invention.
[0309] In one embodiment, an MRD-containing antibody binds
prostaglandin E2 (PGE2). In a specific embodiment, an
MRD-containing antibody binds IL6R and a target selected from:
EGFR, IGF1R, IL6R, TNF, NGF, IL1 beta, IL6, IL17A, VEGF, IL15,
IL18, S1P and Abeta. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind PGE2 and also bind at
least 1, 2, 3, 4, 5 or more of these targets are also encompassed
by the invention. In specific embodiments, the antibody component
of the MRD-containing antibody binds PGE2.
[0310] In another embodiment the antibody target of the
MRD-containing antibody is an immunostimulatory target (e.g., an
agonist of a target associated immune cell activation (such as
TNFRSF9 (41BB) or TNFRSF5 (CD40)) or an antagonist of an inhibitory
immune checkpoint (such as CTLA-4)). In, one embodiment the
antibody target of the MRD-containing antibody is an
immunostimulatory target selected from CD25, CD28, CTLA-4, PD1,
PDL1, B7-H1, B7-H4, IL10, TGFbeta, TNFSF4 (OX40 Ligand), TNFRSF4
(OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), TNFSF9 (41BB Ligand),
TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM),
TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF18 (GITR Ligand) and TNFRSF18
(GITR). An MRD that binds to one of the above targets is
encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the
above targets are also encompassed by the invention. In specific
embodiments, the MRD-containing antibody binds 2, 3 or all 4
targets selected from CTLA-4, TNFRSF18 (GITR), 4-1BB, and TNFRSF5
(CD40). In one embodiment, the MRD-containing antibody binds CTLA-4
and TNFRSF9 (41BB). In another embodiment, the MRD-containing
antibody binds CTLA-4 and TNFRSF18 (GITR). In another embodiment,
the MRD-containing antibody binds CTLA-4 and TNFRSF5 (CD40). In
another embodiment, the MRD-containing antibody binds TNFRSF5
(CD40) and TNFRSF9 (41BB). In another embodiment, the
MRD-containing antibody binds TNFRSF4 (OX40) and TNFRSF9 (41BB). In
another embodiment, the MRD-containing antibody binds PD1 and
B7-H1. In an additional embodiment the MRD-containing antibody
enhances an immune response, such as the immune system's anti-tumor
response or an immune response to a vaccine.
[0311] In another embodiment the antibody target of the
MRD-containing antibody is a cytokine selected from: IL1 alpha, IL1
beta, IL18, TNFSF2 (TNFa), LTalpha, LT beta, TNFSF11 (RANKL),
TNFSF13B (13LYS), TNFSF13 (APRIL), IL6, IL7, IL10, IL12, IL15,
IL17A, IL23, OncoStatinM, TGFbeta, BMP2-15, PDGF (e.g., PDGF-A,
PDGF-B, PDGF-CC, PDGF-C, PDGF-D), an FGF family member (e.g., FGF1,
FGF2, FGF4, FGF7, FGF8b and FGF19), VEGF (e.g., VEGFA and VEGFB),
MIF, and a type I interferon. An MRD that binds to one of the above
targets is encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or
more of the above targets are also encompassed by the invention.
Thus, the invention encompasses MRD-containing antibodies
comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at
least 1, 2, 3, 4, 5, 6 of the above targets.
[0312] In another embodiment the antibody target of the
MRD-containing antibody is a cytokine selected from: TNF, CD25,
CD28, CTLA-4, PD1, PDL1, B7-H1, B7-H4, IL10, TGFbeta, TNFSF4 (OX40
Ligand), TNFRSF4 (OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40),
TNFSF9 (41BB Ligand), TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand),
TNFRSF14 (HVEM), TNFSF15 (TL1A), TNFRSF25 (DR3), TNFSF18 (GITR
Ligand), and TNFRSF18 (GITR). An MRD that binds to one of the above
targets is encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or
more of the above targets are also encompassed by the invention.
Thus, the invention encompasses MRD-containing antibodies
comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at
least 1, 2, 3, 4, 5, 6 of the above targets.
[0313] In one embodiment the antibody target of the MRD-containing
antibody is IL1Ra, IL1Rb, IL2, IL3, IL4, IL7, IL10, IL11, IL15,
IL16, IL17, IL17A, IL17F, IL18, IL19, IL25, IL32, IL33, interferon
beta, SCF, 13CA1/CXCL13, CXCL1, CXCL2, CXCL6, CXCL13, CXCL16, C3AR,
C5AR, CXCR1, CXCR2, CCR1, CCR3, CCR7, CCR8, CCR9, CCR10, ChemR23,
CCL3, CCL5, CCL11, CCL13, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22,
CCL24, CCL25, CCL26, CCL27, MPL, GP130, TLR2, TLR3, TLR4, TLR5,
TLR7, TLR8, TLR9, TREM1, TREM2, FcRn, FcGamma RIIB, oncostatin M,
lymphotoxin alpha (LTa), integrin beta 7 subunit, CD49a (integrin
alpha 1), integrin a5b3, MIF, ESM1, WIF1, cathepsin B, cathepsin D,
cathepsin K, cathepsin S, TNFSF2 (TNFa), TNFSF3 (LTb), TNFRSF3
(LTBR), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3),
TNFSF8 (CD30 Ligand), TNFRSF8 (CD30), TNFSF9 (41BB Ligand), TNFRSF9
(41BB), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF14 (LIGHT, HVEM
Ligand), TNFRSF14 (HVEM), TNFRSF16 (NGFR), TNFSF18 (GITR Ligand),
TNFRSF18 (GITR), TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6),
CD14, CD23 CD25, CD28, CD36, CD36L, CD39, CD52, CD91, CD153, CD164,
CD200, CD200R, BTLA, CD80 (B7-1), CD86 (B7-2), B7h, ICOS, ICOSL
(B7-H2), MHC, CD, B7-H3, B7-H4, B7x, SLAM, KIM-1, SLAMF2, SLAMF3,
SLAMF4, SLAMF5, SLAMF6, or SLAMF7. An MRD that binds to one of the
above targets is encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that, bind to 1, 2, 3, 4, 5, 6, or
more of the above targets are also encompassed by the invention.
Thus, the invention encompasses MRD-containing antibodies
comprising at least 1, 2, 3, 4, 5, 6, or more MRDs that bind to at
least 1, 2, 3, 4, 5, 6 of the above targets. The above antibody and
MRD targets and those otherwise described herein are intended to be
illustrative and not limiting.
[0314] In another embodiment, the antibody target of the
MRD-containing antibody is TNFSF1A (TNF/TNF-alpha), TNFRSF1A
(TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF7 (CD27 Ligand, CD70),
TNFRSF7 (CD27), TNFSF13B (BLYS), TNFSF13 (APRIL), TNFRSF13B (TACI),
TNFRSF13C (BAFFR), TNFRSF17 (BCMA), TNF SF15 (TL1A), TNFRSF25
(DR3), TNFSF12 (TWEAK), TNFRSF12 (TWEAKR), TNFSF4 (OX40 Ligand),
TNFRSF4 (OX40), TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), IL1, IL1
beta, IL1R, IL2R, IL4-Ra, IL5, IL5R, IL6, IL6R, IL9, IL12, IL13,
IL14, IL15, IL15R, IL17f, IL17R, IL17Rb, IL17RC, IL20, IL21,
IL22RA, IL23, IL23R, IL31, TSLP, TSLPR, interferon alpha,
interferon gamma, B7RP-1, cKit, GMCSF, GMCSFR, CTLA-4, CD2, CD3,
CD4, CD11a, CD18, CD20, CD22, CD26L, CD30, TNFRSF5 (CD40), CD80,
CD86, CXCR3, CXCR4, CCR2, CCR4, CCR5, CCR8, CCL2, CXCL10, PLGF,
PD1, B7-DC (PDL2), B7-H1 (PDLL), alpha4 integrin, A4B7 integrin,
C5, RhD, IgE, or Rh. An MRD that binds to one of the above targets
is encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that bind to 1, 2, 3, 4, 5, 6, or more of the
above targets are also encompassed by the invention. Thus, the
invention encompasses MRD-containing antibodies comprising at least
1, 2, 3, 4, 5, 6, or more MRDs that bind to at least 1, 2, 3, 4, 5,
6 of the above targets.
[0315] In particular embodiments, the antibody target of the
MRD-containing antibody competes for target binding with: SGN-70
CD70 (Seattle Genetics), SGN-75 CD70 (Seattle Genetics) Belimumab
BLYS (e.g., BENLYSTAR.RTM., Human Genome Sciences/GlaxoSmithKline),
Atacicept BLYS/APRIL (Merck/Serono), TWEAK (e.g., Biogen mAb), TL1A
antibodies of CoGenesys/Teva (e.g., hum11D8, hum25B9, and hum1B4
(U.S. Appl. Publ. No. 2009/0280116), OX40 mAb, humAb OX40L
(Genentech), rilonacept IL1 trap (e.g., ARCALYST.RTM., Regeneron),
catumaxomab IL1beta (e.g., REMOVAB.RTM., Fresenius Biotech GmbH),
Xoma052 IL1beta (Lilly), canakinumab IL1beta (e.g., ILARIS.RTM.
(Novartis) and ACZ885 (Novartis)), AMG108 IL1R (Amgen), daclizumab
IL2Ra (e.g., ZENAPAX.RTM., Hoffman-La Roche), basiliximab IL2Ra
(e.g., SIMULECT.RTM., Novartis), AMGN-317 IL4a (Amgen),
pascolizumab IL4 (PDL), mepolizumab IL5 (e.g., BOSATRIA.RTM.,
GlaxoSmithKline), reslizumab IL5 (e.g., SCH55700, Ception
Therapeutics), benralizumab IL5R (e.g., MEDI-563 MedImmune),
BIW-8405, IL5R (BioWa), etanercept TNFR2-fc (e.g., ENBREL.RTM.,
Amgen), siltuximab IL6 (e.g., CNTO328, Centocor), CNTO136 IL6
(Centocor), CDP-6038 IL6 (UCB), AMGN-220 IL6 (Amgen), REGN-88 IL6R
(Regeneron), tocilizumab IL6R (e.g., ACTEMRA.TM./ROACTEMRA.TM.,
Chugai/Roche), MEDI-528 IL9 (MedImmune), briakinumab IL12/13 (e.g.,
ABT-874, Abbott), ustekinumab IL12, IL23 (e.g., STELARA.RTM. and
CNTO 1275, Centocor), TNX-650 IL13 (Tanox), lebrikizamab IL13
(Genentech), tralokinumab IL13 (e.g., CAT354, e.g., Cambridge
Antibody Technology), AMG714 IL15 (Amgen), CRB-15 IL15R (Hoffman
La-Roche), AMG827 IL17R (Amgen), IL17RC antibody of
Zymogenetics/Merck Serono, IL20 antibody of Zymogenetics, IL20
antibody of Novo Nordisk, IL21 antibody of Novo Nordisk (e.g.,
NCT01038674), IL21 antibody Zymogenetics (Zymogenetics), IL22RA
antibody of Zymogenetics, IL31 antibody of Zymogenetics, AMG157
TSLP (Amgen), MEDI-545 interferon alpha (MedImmune), MEDI-546
interferon alpha receptor (MedImmune), AMG811 interferon gamma
(Amgen), INNO202 interferon gamma (Innogenetics/Advanced
Biotherapy), HuZAF interferon-gamma (PDL), AMG557 B7RP1 (Amgen),
AMG191 cKit (Amgen), MOR103 GMCSF (MorphoSys), mavrilimumab GMCSFR
(e.g., CAM-3001, MedImmune), tremelimumab CTLA4 (e.g., CP-675,206,
Pfizer), iplimumab CTLA4 (e.g., MDX-010, BMS/Medarex), alefacept
CD2 (e.g., AMEVIVE.RTM., Astellas), siplizumab CD2 (e.g., MEDI-507,
MedImmune), otelixizumab CD3 (e.g., TRX4, Tolerx/GlaxoSmithKline),
teplizumab CD3 (e.g., MGA031, MacroGenics/Eli Lilly), visilizumab
CD3 (e.g., NUVION.RTM., PDL), muromonab-CD3 CD3 (Ortho), ibalizumab
(e.g., TMB-355 and TNX-355, TaiMed Biologics), zanolimumab CD4
(e.g., HUMAX-CD4.RTM., Genmab), cedelizumab CD4 (Euroasian
Chemicals), keliximab CD4, priliximab CD4 (e.g., cMT412, Centocor),
BT-061 CD4 (BioTest AG), efalizumab CD11a (e.g.,
RAPTIVA.RTM./XANELIM.TM., Genentech/Roche/MerckSerono), MLN01 CD18
(Millennium Pharmaceuticals), epratuztumab CD22 (e.g., Amgen
antibody) and hLL2; (Immunomedics/UCB)), aselizumab CD26L,
iratumumab CD30 (e.g. SGN30 (Seattle (Genetics) and MDX-060
(Medarex), SGN40 CD40 (Seattle Genetics). ANTOVA.RTM.; CD40 ligand
(Biogen Idec), abatacept CD80 CD86 (e.g., ORENCIA.RTM..
Bristol-Myers Squibb), CT-011 PD1 (Cure Tech), GITR (e.g., TRX518,
(Tolerx), AT010 CXCR3 (Affitech), MLN1202 CCR2 (Millennium
Pharmaceuticals), AMG-761 CCR4 (Amgen), HGS004 CCR5 (Human Genome
Sciences), PRO 140 (Progenics), MDX-1338 CXCR4 (Medarex), CNTO-888
CCL2 (Centocor), ABN912 CCL2 (Novartis), MDX-1100 CXCL10 (Medarex),
TB-403 PLGF (BioInvent), natalizumab integrin Alpha4 subunit (e.g.,
TYSABRI.RTM., Biogen Idec/Elan), vedolizumab integrin A4B7 (e.g.,
MLN2, Millennium Pharmaceuticals/Takeda), eculizumab C5 Compliment
(e.g., SOLIRIS.RTM., Alexion), pexelizumab C5 Compliment (Alexion),
omalizumab IgE (e.g., XOLAIR.RTM., Genentech/Roche/Novartis),
talizumab (e.g., TNX-901, Tanox), toralizumab (IDEC 131, IDEC),
bertilimumab eotaxin (e.g., iCo-008, iCos Therapeutics Inc.),
ozrolimupab RhD (e.g., Sym001, Symphogen A/S), atorolimumab or
morolimumab (Rh factor). An MRD that competes for target binding
with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2, 3, 4, 5, 6, or more of
the above antibodies are also encompassed by the invention. Thus,
the invention encompasses MRD-containing antibodies comprising at
least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with at least 1, 2, 3, 4, 5 or 6 of the above
antibodies.
[0316] In particular embodiments, the antibody of the
MRD-containing antibody is: SGN-70 CD70 (Seattle Genetics), SGN-75
CD70 (Seattle Genetics), Belimumab BLYS (e.g., BENLYSTA.RTM., Human
Genome Sciences/GlaxoSmithKline), BIIB023 TWEAK (Biogen Idec), TL1A
antibodies of CoGenesys/Teva (e.g., 11D8, 25B9, and 1B4 (U.S. Appl.
Publ. No. 2009/0280116), OX40 mAb, humAb OX40L (Genentech),
catumaxomab IL1beta (e.g., REMOVAB.RTM., Fresenius Biotech GmbH),
canakinumab IL1beta (e.g., ILARIS.RTM. (Novartis) and ACZ885
(Novartis)), AMG108 IL1R (Amgen), daclizumab IL2Ra (e.g.,
ZENAPAX.RTM., Hoffman-La Roche), basiliximab IL2Ra (e.g.,
SIMULECT.RTM., Novartis), AMGN-317 IL4a (Amgen), pascolizumab IL4
(PDL), mepolizumab IL5 (e.g., BOSATRIA.RTM., GlaxoSmithKline),
reslizumab IL5 (e.g., SCH55700, Ception Therapeutics), benralizumab
IL5R (e.g., MEDI-563, Medlmmune), BIW-8405, IL5R (BioWa),
siltuximab IL6 (e.g., CNTO328, Centocor), CNTO-136 IL6 (Centocor),
CDP-6038 IL6 (UCB), AMGN-220 IL6 (Amgen), REGN-88 IL6R (Regeneron),
tocilizumab IL6R (e.g., ACTEMRA.TM./ROACTEMRA.TM., Chugai/Roche),
MEDI-528 IL9 (Medimmune), briakinumab IL12/13 (e.g., ABT-874,
Abbott), ustekinumab IL12, IL23 (e.g., CNTO 1275, Centocor),
lebrikizumab IL13 (Genentech), TNX-650 IL13 (Tanox), CAT354 IL13
(Cambridge Antibody Technology), AMG714 IL15 (Amgen), CRB-15 IL15R
(Hoffman La-Roche), AMG827 IL17R (Amgen), IL17RC antibody of
Zymogenetics/Merck Serono, IL20 antibody of Zymogenetics, IL20
antibody of Novo Nordisk. IL21 antibody of Novo Nordisk, IL21
antibody Zymogenetics (Zymogenetics), IL22RA, antibody of
Zymogenetics, IL31 antibody of Zymogenetics, AMG157 TSLP (Amgen),
MEDI-545 interferon alpha (Medimmune), MEDI-546 interferon alpha
receptor (Medimmune), AMG811 interferon gamma (Amgen), INNO202
interferon gamma (Innogenetics/Advanced Biotherapy), HuZAF
interferon-gamma (PDL), AMG557 B7RP1 (Amgen), AMG191 cKit (Amgen),
MOR103 GMCSF (MorphoSys), CAM-3001 GMCSFR (Medimmune), tremelimumab
CTLA4 (e.g., CP 675,206, Pfizer), iplimumab CTLA4
MDX-010BMS/Medarex), siplizumab CD2 MEDI-507, Medimmune),
otelixizumab CD3 (e.g., TRX4, Tolerx/GlaxoSmithKline),
muromonab-CD3 CD3 (Ortho), teplizumab CD3 MGA031, MacroGenics/Eli
Lilly), visilizumab CD3 (e.g., NUVION.RTM., PDL), zanolimumab CD4
(e.g., HUMAX-CD4.RTM., Genmab), cedelizumab CD4 (Euroasian
Chemicals), keliximab CD4, priliximab CD4 (e.g., cMT412, Centocor),
BT-061 CD4 (BioTest AG), ibalizumab (e.g., TMB-355 and TNX-355,
TaiMed Biologies), efalizamab CD11a (e.g.,
RAPTIVA.RTM./XANELIM.TM., Genentech/Roche/Merck-Serono), MLN01 CD18
(Millennium Pharmaceuticals), epratuzumab CD22 (e.g., Amgen
antibody) and hLL2 (Immunomedics/UCB)), aselizumab CD26L iratumumab
CD30 (e.g., SGN30 (Seattle Genetics) and MDX-060 (Medarex), SGN40
CD40 (Seattle Genetics), ANTOVA.RTM. CD40 ligand (Biogen Idec),
CT-011 PD1 (Cure Tech). AT010 CXCR3 (Affitech), MLN3897 CCR1
(Millennium Pharmaceuticals), MLN1202 CCR2 (Millennium
Pharmaceuticals), AMG-761 CCR4 (Amgen), HGS004 CCR5 (Human Genome
Sciences), PRO 140 (Progenies), MDX-1338 CXCR4 (Medarex), CNTO-888
CCL2 (Centocor), ABN912 CCL2 (Novartis), MDX-1100 CXCL10 (Medarex),
TB-403 PLGF (BioInvent), natalizumab integrin Alpha4 subunit (e.g.,
TYSABRI.RTM., Biogen Idec/Elan), vedolizumab integrin A4B7 (e.g.,
MLN02, Millennium Pharmaceuticals/Takeda), eculizumab C5 Compliment
(e.g., SOLIRIS.RTM., Alexion pharmaceuticals), omalizumab IgE
(e.g., XOLAIR.RTM., Genentech/Roche/Novartis), talizumab (e.g.,
TNX-901, Tanox), toralizumab (IDEC 131, DEC), bertilimumab eotaxin
(e.g., iCo-008, iCo Therapeutics Inc.), ozrolimupab RhD (e.g.,
Sym001, Symphogen A/S), atorolimumab or morolimumab (Rh
factor).
[0317] In additional embodiments, the antibody target of the
MRD-containing antibody competes for target binding with an
antibody selected from: oxelumab (e.g., RG4930; Genmab), AMG139
(Amgen), AMG181 (Amgen), CNTO 148 TNF (Medarex), an anti-TNF
antibody described in U.S. Pat. No. 6,258,562 (BASF), Humicade.RTM.
TNF (Celltech), HuM291CD3 fc receptor (PDL), Mik-beta-1 IL-2Rb
(CD122) (Hoffman LaRoche), REGN668 IL-4R (Regeneron), sarilumab
IL-6R (e.g., REGN 88, Regeneron), HuMax-Inflam IL-8 (e.g.,
HuMax-Inflam.TM./MDX-018; Genmab and Medarex), anti-IL-12 and/or
anti-IL-12p40 antibody disclosed in U.S. Pat. No. 6,914,128
(Abbott), HuMax-IL15 IL15 (Medarex and Genmab). ABX-IL8 IL8
(Abgenix), an anti-IL-18 antibody disclosed in US Appl. Pub. No.
2005/0147610 (Abbott), hCBE-11 LTBR (Biogen), HuMax-TAC IL-2Ra
CD25) (Genmab, see, e.g., Intl. Appl. Publ. No. WO2004045512, MLN01
Beta2 integrin (Xoma), D3H44 ATF (Genentech), MT203 GMCSF (Micromet
and Takeda), IFX1/CaCP29 (InflaRx GmbH), CAT-213 Eotaxin l
(Cambridge Antibody Technologies), MDDX-018 IL-8 (e.g.,
HuMax-Inflam.TM.; Medarex), REGN846 IL-4R (Regeneron, see, e.g., US
Appl. Pub. No. 20100291107), REGN728 (Regeneron), RGN846
(Regeneron), T2-18C3 IL1A (MABp1; XBiotech), RA-18C3 IL1A
(XBiotech) and CV-18C3 IL1A (XBiotech). An MRD that competes for
target binding with one of the above antibodies is also encompassed
by the invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2, 3, 4, 5, 6, or more of
the above antibodies are also encompassed by the invention. Thus,
the invention encompasses MRD-containing antibodies comprising at
least 1, 2, 3, 4, 5, 6, or more MRDs that compete for target
binding with at least 1, 2, 3, 4, 5, or 6 of the above
antibodies.
[0318] In additional embodiments, one of the above-described
antibodies is the antibody of the MRD-containing antibody.
[0319] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds CTLA4. In a specific
embodiment, the antibody is tremelimutnab (e.g., CP-675,206,
Pfizer). In another embodiment, the antibody binds to the same
epitope as tremelimumab. In a further embodiment, the antibody
competitively inhibits binding of tremelimumab to CTLA4. In an
additional specific embodiment, the antibody is ipilimumab (e.g.,
MDX-010, Bristol-Myers Squibb/Medarex). In one embodiment, the
antibody binds to the same epitope as ipilimumab. In a further
embodiment, the antibody competitively inhibits binding of
ipilimumab to CTLA4. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more
MRDs that compete for CTLA4 binding with tremelimumab or ipilimumab
are also encompassed by the invention.
[0320] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds TNFSF12 (TWEAK). In a
specific embodiment, the antibody is the TWEAK antibody of Biogen
that has advanced to Phase I clinical trials. In another
embodiment, the antibody binds to the same epitope as the Biogen
TWEAK antibody. In a further embodiment, the antibody competitively
inhibits binding of the Biogen TWEAK antibody to TWEAK. Multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
having 1, 2, 3, 4, 5, 6, or more MRDs that compete for TWEAK
binding with the Biogen TWEAK antibody are also encompassed by the
invention.
[0321] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds IL2Ra (CD25). In a
specific embodiment, the antibody is daclizumab (e.g.,
ZENAPAX.RTM.). In another embodiment, the antibody binds to the
same epitope as daclizumab. In a further embodiment, the antibody
competitively inhibits binding of daclizumab to IL2Ra (CD25).
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs that compete for
IL2Ra (CD25) binding with daclizumab are also encompassed by the
invention.
[0322] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds CD40 (TNFRSF5). In a
specific embodiment, the antibody is CP-870893 CD40 (Pfizer). In
another embodiment, the antibody binds to the same epitope as
CP-870893. In a further embodiment, the antibody competitively
inhibits binding, of CP-870893 to CD40. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for CD40 binding with
CP-870893 are also encompassed by the invention.
[0323] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds Alpha4 integrin. In a
specific embodiment, the antibody is natalizumab (e.g.,
TYSABRI.RTM.; Biogen Idec/Elan). In one embodiment, the antibody
binds to the same epitope as natalizumab. In a further embodiment,
the antibody competitively inhibits binding of natalizumab to
Alpha4 integrin. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that, compete for Alpha4 integrin binding with natalizumab are also
encompassed by the invention.
[0324] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds IL22. In a specific
embodiment, the antibody is PF-5,212,367 (ILV-094) (Pfizer). In
another embodiment, the antibody binds to the same epitope as
PF-5,212,367. In a further embodiment, the antibody competitively
inhibits binding of PF-5,212,367 to IL22. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for IL22 binding with
PF-5,212,367 are also encompassed by the invention.
[0325] In an additional embodiment, the antibody in the
MRD-containing antibody specifically binds MAdCAM. In a specific
embodiment, the antibody is PF-547,659 (Pfizer). In another
embodiment, the antibody binds to the same epitope as PF-547,659.
In a further embodiment, the antibody competitively inhibits
binding of PF-547,659 to MAdCAM. Multivalent and multispecific
compositions (e.g., MRD; containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for MAdCAM binding with PF-547,659
are also encompassed by the invention.
[0326] In one embodiment, the antibody in the MRD-containing
antibody specifically binds TNF. In a specific embodiment, the
antibody is adalimumab (e.g., HUMIRA.RTM./TRUDEXA.RTM., Abbott). In
one embodiment, the antibody binds to the same epitope as
adalimumab. In another embodiment, the antibody competitively
inhibits binding of adalimumab to TNF. In another specific
embodiment, the antibody is ATN-103 (Pfizer). In one embodiment,
the antibody binds to the same epitope as ATN-103. In another
embodiment, the antibody competitively inhibits binding of ATN-103
to INF. In another specific embodiment, the antibody is infliximab.
In one embodiment, the antibody binds to the same epitope as
infliximab. In another embodiment, the antibody competitively
inhibits binding of infliximab to TNF. In another specific
embodiment, the antibody is selected from certolizumab (e.g.,
CIMZIA.RTM., UCB), golimumab (e.g., SIMPONI.TM., Centocor), and
AME-527 (Applied Molecular Evolution). In one embodiment, the
antibody binds to the same epitope as certolizumab, golimumab, or
AME-527. In another embodiment, the antibody competitively inhibits
binding of certolizumab, golimumab, or AME-527, to TNF. An MRD that
competes for target binding with one of the above antibodies is
also encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2, 3, 4,
or 5, of the above antibodies are also encompassed by the
invention.
[0327] In some embodiments, the antibody in the MRD-containing
antibody comprises the CDRs of the anti-TNF antibody adalimumab.
The CDR, VH, and VL sequences of adalimumab are provided in Table
3.
TABLE-US-00003 TABLE 3 CDR Sequence VL-CDR1 RASQGIRNYLA (SEQ ID NO:
80) VL-CDR2 AASTLQS (SEQ ID NO: 81) VL-CDR3 RYNRAPYT (SEQ ID NO:
82) VH-CDR1 DYAMH (SEQ ID NO: 83) VH-CDR2 AITWNSGHIDYADSVEG (SEQ ID
NO: 84) VH-CDR3 VSYLSTASSLDY (SEQ ID NO: 85) VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQK
PGKAPKLLIYAASTLQSCIVPSRFSGSGSGTDFTLTISS
LQPEDVATYYCQRYNRAPYTFOQGTKVEIKR (SEQ ID NO: 86) VH
EVQLVESGGGLVQPGRSLRLSCAASGFTEDDYAMHWVRQ
APGKGLEWVSAITMTNSGHIDYADSVEGRFTISRDNAKN
SLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSS (SEQ ID NO: 87)
[0328] In one embodiment, an MRD-containing antibody binds TNF
(i.e., TNF alpha) and additionally binds a target selected from:
Te38, IL12, IL12p40, IL13, IL15, IL17, IL18, IL1beta, IL23, MIF,
PGE2, PGE4, VEGF, TNFSF11 (RANKL), TNFSF13B (BLYS), GP130, CD22 and
CTLA-4. In another embodiment, an MRD-containing antibody binds TNF
alpha, IL6, and TNFSF13B (BLYS). In another embodiment, an
MRD-containing antibody binds TNF alpha and TNFSF12 (TWEAK). In
additional embodiments, the MRD-containing antibody binds TNF and
INFSF15 (IL1A). In another embodiment, an MRD-containing antibody
binds TNF and additionally binds a target selected from NOF, SOST
(sclerostin), LPA, IL17A, DKK, alpha Vbeta3, IL23p19, IL2, IL2RA
(CD25), IL6, IL6R, IL12p40, IL6, IL10, IL21, IL22 and CD20 binds
TNF. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind TNF alpha and at least 1,2,3,
4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds TNF alpha. In further embodiments,
the antibody component of the MRD-containing antibody is
adalimumab, infliximab certolizumab golimumab, CNTO 148, AME-527 or
ATN-103.
[0329] In other embodiments, the target of the antibody of the
MRD-containing antibody is IL6. In some embodiments, the antibody
of the MRD-containing antibody is siltuximab (CNTO328, Centocor),
CNTO-136 (Centocor), CDP-6038 (UCB), or AMGN-220 (Amgen). In other
embodiments, the antibody of the MRD-containing antibody competes
with siltuximab (CNTO328, Centocor), CNTO-136 (Centocor), CDP-6038
(UCB), or AMGN-220 (Amgen) for binding to IL6. An MRD that competes
for target binding with one of the above antibodies is also
encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2, or
more of the above antibodies are also encompassed by the
invention.
[0330] In one embodiment, an MRD-containing antibody binds IL6. In
a specific embodiment, an MRD-containing antibody binds IL6 and a
target selected from: IL1, IL1 beta, IL1Ra, IL5, CD8, TNFRSF5
(CD40), PDL1, IL6R, IL17A, TNF, VEGF, TNFSF11 (RANKL) and PGE2.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind IL6 and also bind at least 1, 2, 3, 4, 5 or
more of these targets are also encompassed by the invention. In
specific embodiments, the antibody component of the MRD-containing
antibody binds IL6. In further embodiments, the antibody component
of the MRD-containing antibody is siltuximab, CNTO136, CDP-6038 or
AMGN-220.
[0331] In other embodiments, the target of the antibody of the
MRD-containing antibody is IL6R. In some embodiments, the antibody
of the MRD-containing antibody is REGN-88 (Regeneron) or
tocilizumab (ACTEMRA.TM./ROACTEMRA.TM., Chugai/Roche). In other
embodiments, the antibody of the MRD-containing antibody competes
with siltuximab, REGN-88 (Regeneron) or tocilizumab
(ACTEMRA.TM./ROACTEMRA.TM., Chugai/Roche) for binding to IL6R. An
MRD that competes for target binding with one of the above
antibodies is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1 or both of the above antibodies are also encompassed by the
invention.
[0332] In one embodiment, an MRD-containing antibody binds IL6R. In
a specific embodiment, an MRD-containing antibody binds IL6R and a
target selected from: CD8, TNFRSF5 (CD40), PDL1, IL6, IL17A, TNF,
VEGF, TNFSF11 (RANKL) and PGE2. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind IL6R and
also bind at least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds IL6R. In further
embodiments, the antibody component of the MRD-containing antibody
is REGN-88 or tocilizumab.
[0333] In some embodiments, an MRD-containing antibody binds
TNFSF15 (TL1A). In further embodiments, the MRD-containing antibody
binds TL1A and a target selected from TNF, IFN alpha, IFN gamma,
IL1, IL1beta, IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind TL1A and also bind at least 1, 2, 3, 4, 5 or
more of these targets are also encompassed by the invention. These
compositions have applications in treating diseases and disorders
including inflammatory bowel disease and autoimmune diseases such
as rheumatoid arthritis. In specific embodiments, the antibody
component of the MRD-containing antibody binds TL1a.
[0334] In some embodiments, an MRD-containing antibody binds
interferon alpha. In further embodiments, the MRD-containing
antibody binds interferon alpha and TNFSF13B (BLYS). In further
embodiments, the MRD-containing antibody binds interferon alpha,
TNFSF13B (BLYS), and a neutrophil extracellular trap (NET). These
compositions have applications in treating diseases and disorders
including autoimmune diseases such as rheumatoid arthritis and
systemic lupus erythematous. In specific embodiments, the antibody
component of the MRD-containing antibody binds interferon
alpha.
[0335] The multivalent and multispecific compositions of the
invention also have applications in treating neurologic diseases or
disorders including neurodegenerative diseases, pain and neural
injury or trauma. In particular embodiments, the target of the
antibody of the MRD-containing antibody is: amyloid beta (Abeta),
beta amyloid, complement factor D, PLP, ROBO4, ROBO, GDNF, NGF,
LINGO, or myostatin. In specific embodiments, the antibody in the
MRD-containing antibody is gantenerumab (e.g., R1450, Hoffman
La-Roche), bapineuzumab beta amyloid 9 (Elan and Pfizer),
solanezumab beta amyloid 9 (Eli Lilly), tanezumab NGF (e.g., RN624,
Pfizer), BIIIB033 LINGO (Biogen Idec), PF-3,446,879 myostatin
(Pfizer), or stamulumab myostatin (Wyeth). In another embodiment,
the antibody specifically binds to the same epitope as
gantenerumab, bapineuzumab, solarezumab, tanezumab, the Biogen
LINGO antibody, or stamulumab. In another embodiment, the antibody
in the MRD-containing antibody is an antibody that competitively
inhibits target binding by gantenerumab, bapineuzumab, solarezumab,
tanezumab, BIIIB033, or stamulumab. An MRD that competes for target
binding with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2 or more of the above
antibodies are also encompassed by the invention.
[0336] In an additional embodiment, the target of the antibody of
the MRD-containing antibody is beta amyloid. In a specific
embodiment, the antibody in the MRD-containing antibody is RN1219
(PF-4,360,365; Pfizer). In another embodiment, the antibody
specifically binds to the same epitope as RN1219. In a further
embodiment, the antibody in the MRD-containing antibody is an
antibody that competitively inhibits beta amyloid binding by RN
1219. An MRD that competes for beta amyloid binding with RN1219 is
also encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for beta amyloid binding with
RN1219 are also encompassed by the invention.
[0337] In an additional embodiment, the target of the antibody of
the MRD-containing antibody is NGF. In a specific embodiment, the
antibody in the MRD-containing antibody is tanezumab (e.g., RN624,
Pfizer). In another embodiment, the antibody specifically binds to
the same epitope as tanezumab. In a further embodiment, the
antibody in the MRD-containing antibody is an antibody that
competitively inhibits NGF binding by tanezumab. An MRD that
competes for NGF binding with tanezumab is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for NGF binding with tanezumab are also encompassed by
the invention.
[0338] In a specific embodiment, an MRD-containing antibody binds
NGF and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3,
TNFRSF21 (DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19,
CD22, TNFRSF5 (CD40), CD80, cMET, NRP1, TNF, LINGO, HGF, IGF1,
IGF1,2, IGF2, NGF, Te38, NogoA, RGM A, MAG, OMGp, NgR, TNFSF12
(TWEAK), PGE2, IL1 beta, Semaphorin 3A and Semaphorin 4.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind NGF and also bind at least 1, 2, 3, 4, 5 or
more of these targets are also encompassed by the invention. In
specific embodiments, the antibody component of the MRD-containing
antibody binds NGF. In further embodiments, the antibody component
of the MRD-containing antibody is tanezumab. In additional
embodiments, the antibody component of the MRD-containing antibody
competes for NGF binding with tanezumab. In further embodiments,
the antibody component of the MRD-containing antibody is MEDI-578.
In additional embodiments, the antibody component of the
MRD-containing antibody competes for NGF binding with MEDI-578.
[0339] In an additional embodiment, the target of the antibody of
the MRD-containing antibody is LINGO (e.g., LINGO1). In a specific
embodiment, the antibody in the MRD-containing antibody is BIIB033
(Biogen Idec). In another embodiment, the antibody specifically
binds to the same epitope as BIIB033. In a further embodiment, the
antibody in the MRD containing antibody is an antibody that
competitively inhibits LINGO binding by BIIB033. An MRD that
competes for LINGO binding with BIIB033 is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for LINGO binding with BIIB033 are also encompassed by
the invention.
[0340] In a specific embodiment, an MRD-containing antibody binds
LINGO and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3,
TNFRSF21 (DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19,
CD22, TNFRSF5 (CD40), CD80, cMET, NRP1, TNF, TNFSF12 (TWEAK), HGF,
IGF1, IGF1,2, IGF2, NGF, Te38, NogoA, RGM A, MAG, OMGp, NgR, NGF,
PGE2, IL1 beta, Semaphorin 3A and Semaphorin 4. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind LINGO and also bind at least 1, 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds LINGO. In further embodiments, the antibody component of, the
MRD-containing antibody is BIIB033.
[0341] In a specific embodiment, the target of an antibody of an
MRD-containing antibody is TNFSF12 (TWEAK). In another embodiment,
the antibody in the MRD-containing antibody binds TNFSF12 (TWEAK)
and a target selected from: MTX, NKG2D, RON, IL6R, ErbB3, TNFRSF21
(DR6), CD3, IGFR, DLL4, P1GF, CD20, EGFR, HER2, CD19, CD22, TNFRSF5
(CD40), CD80, cMET, NRP1, TNF, LINGO, HGF, IGF1, GF1,2, IGF2, NGF,
Te38, NogoA, RGM A, MAG, OMGp, NgR, NGF, PGE2, IL1 beta, Semaphorin
3A and Semaphorin 4. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind TNFSF12 (TWEAK) and
also bind at least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds TNFSF12 (TWEAK). In
further embodiments, the antibody component of the MRD-containing
antibody is BIIB023.
[0342] In another embodiment, the target of the antibody of the
MRD-containing antibody is: oxidized LDL, gpIIB, gpIIIa, PCSK9,
Factor VIII, integrin a2bB3, AOC3, or mesothelin. In specific
embodiments, the antibody in the MRD-containing antibody is BI-204
oxidized LDL (Bioinvent), abciximab gpIIB, gpIIIa (e.g., REOPRO,
Eli Lilly), AMG-145 PCSK9 (Amgen), TB-402 Factor VIII (Bioinvent),
vapaliximab, or tadocizumab integrin a2bB3 (Yamonochi Pharma). In
another embodiment, the antibody specifically binds to the same
epitope as BI-204, abciximab, AMG-145, TB-402, or tadocizumab. In
another embodiment, the antibody in the MRD-containing antibody is
an antibody that competitively inhibits binding of BI-204,
abciximab, AMG-145, TB-402, vapaliximab, or tadocizumab. An MRD
that competes for target binding with one of the above antibodies
is also encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2 or
more of the above antibodies are also encompassed by the
invention.
[0343] In other embodiments, the antibody of the MRD-containing
antibody is associated with bone growth and/or metabolism. In
certain embodiments the antibody target of the MRD-containing
antibody is TNFSF11 (RANKL). In other embodiments the antibody
target of the MRD-containing antibody is DKK1, osteopontin,
cathepsin K, TNFRSF19L (RELT), TNFRSF19 (TROY), or sclerostin
(CDP-7851 UCB Celltech). In another embodiment antibody target of
the MRD-containing antibody is TNFSF11 (RANKL). In a specific
embodiment, the antibody in the MRD-containing antibody is
denosumab (e.g., AMG-162, Amgen). In another embodiment, the
antibody specifically binds to the same epitope as denosumab. In
another embodiment, the antibody in the MRD-containing antibody is
an antibody that competitively inhibits binding of TNFSF11 (RANKL)
by denosumab. In another specific embodiment, the antibody is
AMG617 or AMG785 (e.g., CDP7851, Amgen). In another embodiment, the
antibody specifically binds to the same epitope as AMG617 or
AMG785. In another embodiment, the antibody in the MRD-containing
antibody is an antibody that competitively inhibits binding of
sclerostin by AMG617 or AMG785. An MRD that competes for target
binding with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2 or more of the above
antibodies are also encompassed by the invention.
[0344] In one embodiment, an MRD-containing antibody binds TNFSF11
(RANKL). In a specific embodiment, an MRD-containing antibody binds
TNFSF11 and a target selected from: sclerostin (SOST),
endothelin-1, DKK1, IL1, IL6, IL7, IL8, IL11, IL17A, MCSF, IGF1,
IGF2, IGF1,2 IGF1R, TNF, FGF1, FGF2, FGF4, FGF7, FGF8a, FGF8b,
FGF18, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa,
FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TGF beta, TGF beta R2, BMP2,
BMP4, BMP5, BMP9, BMP10, BMPR-IA, PDGF, PDGFRa, PDGFRb PTH, PTH
related protein (PTHrP), and PGE2. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind TNFSF11
and also bind at least 1, 2, 3, 4, 5 or more of these targets are
also encompassed by the invention. In specific embodiments, the
antibody component of the MRD-containing antibody binds TNFSF 11.
In further embodiments, the antibody component of the
MRD-containing antibody is denosumab, AMG617 or AMG785.
[0345] In additional embodiments, the antibody target of the
MRD-containing antibody is a bacterial antigen, a viral antigen, a
mycoplasm antigen, a prion antigen, or a parasite antigen (e.g.,
one infecting a mammal).
[0346] In other embodiments, the target of the antibody of the
MRD-containing antibody is a viral antigen. In one embodiment, the
target of the antibody of the MRD-containing antibody is anthrax,
hepatitis b, rabies, Nipah virus, west nile virus, a mengititis
virus, or CMV. In other embodiments, the antibody of the
MRD-containing antibody competes with antigen binding with
ABTHRAX.RTM. (Human Genome Sciences), exbivirumab, foravirumab,
libivimmab, rafivirumab, regavirumab, sevirumab (e.g., MSL-109,
Protovir), tuvirumab, raxibacumab, Nipah virus M102.4, or
MGAWN1.RTM. (MacroGenics) for target binding. An MRD that competes
for target binding with one of the above antibodies is also
encompassed by the invention. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) having 1, 2, 3, 4,
5, 6, or more MRDs that compete for target binding with 1, 2 or
more of the above antibodies are also encompassed by the invention.
An MRD that competes for target binding with one of the above
antibodies is also encompassed by the invention. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) having
1, 2, 3, 4, 5, 6, or more MRDs that compete for target binding with
1, 2 or more of the above antibodies are also encompassed by the
invention.
[0347] In other embodiments, the target of the antibody of the
MRD-containing antibody is RSV. In other embodiments, the antibody
of the MRD-containing antibody is motavizumab (e.g., NUMAX.RTM.,
MEDI-577; MedImmune) or palivizumab RSV fusion f protein (e.g.,
SYNAGIS.RTM., MedImmune). In other embodiments, the antibody of the
MRD-containing antibody competes with motavizumab or palivizumab
RSV fusion f protein, for target binding. In other embodiments, the
antibody of the MRD-containing antibody is felvizamab. In other
embodiments, the antibody of the MRD-containing antibody competes
with felvizumab for target binding. An MRD that competes for target
binding with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2 or more of the above
antibodies are also encompassed by the invention.
[0348] In other embodiments, the target of the antibody of the
MRD-containing antibody is a bacterial or fungal antigen. In other
embodiments, the antibody of the MRD-containing antibody competes
for antigen binding with nebacumab, edobacomab (e.g., E5),
tefibazumab (Inhibitex), panobacumab (e.g., KBPA101, Kenta),
pagibaximab (e.g., BSYX-A110, Biosynexus), urtoxazumab, or
efungumab (e.g., MYCOGRAB.RTM., Novartis). In other embodiments,
the antibody of the MRD-containing antibody is nebacumab,
eclobacomab, tefibazumab (Inhibitex), panobacumab, pagibaximab,
urtoxazumab, or efungumab, An MRD that competes for target binding
with one of the above antibodies is also encompassed by the
invention. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) having 1, 2, 3, 4, 5, 6, or more MRDs
that compete for target binding with 1, 2 or more of the above
antibodies are also encompassed by the invention.
[0349] In another specific embodiment, the antibody in the
MRD-containing antibody is the catalytic antibody 38C2. In another
embodiment, the antibody binds to the same epitope as 38C2. In
another embodiment, the antibody competitively inhibits 38C2.
[0350] Other antibodies of interest include A33 binding antibodies.
Human A33 antigen is a transmembrane glycoprotein of the Ig
superfamily. The function of the human A33 antigen in normal and
malignant colon tissue is not yet known. However, several
properties of the A33 antigen suggest that it is a promising target
for immunotherapy of colon cancer. These properties include (i) the
highly restricted expression pattern of the A33 antigen, (ii) the
expression of large amounts of the A33 antigen on colon cancer
cells, (iii) the absence of secreted or shed A33 antigen, (iv) the
fact that upon binding of antibody A33 to the A33 antigen, antibody
A33 is internalized and sequestered in vesicles, and (v) the
targeting of antibody A33 to A33 antigen expressing colon cancer in
preliminary clinical studies. Fusion of a MRD directed toward A33
to a catalytic or non-catalytic antibody would increase the
therapeutic efficacy of A33 targeting antibodies.
[0351] In some embodiments, the antibody in the MRD-containing
antibody binds to a human target protein. In some embodiments, the
MRD binds to both a human protein and its ortholog in mouse, rat,
rabbit, or hamster.
[0352] The antibodies in the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are able to bind
their respective targets when the MRDs are attached to the
antibody. In certain embodiments, the antibody binds its target
independently. In some embodiments, the antibody is a target
agonist. In other embodiments, the antibody is a target antagonist.
In certain embodiments, the antibody can be used to localize an
MRD-containing antibody to an area where the antibody target is
located.
[0353] It is contemplated that the antibodies used in the present
invention may be prepared by any method known in the art. For
example, antibody molecules and multivalent and multispecific
compositions (e.g., MRD-containing antibodies) can be
"recombinantly produced," i.e., produced using recombinant DNA
technology.
[0354] Monoclonal antibodies that can be used as the antibody
component of the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) can be prepared using hybridoma methods,
such as those described by Kohler and Milstein, Nature 256:495
(1975). Using the hybridoma method, a mouse, hamster, or other
appropriate host animal, is immunized as described above to elicit
the production by lymphocytes of antibodies that will specifically
bind to an immunizing antigen. Lymphocytes can also be immunized in
vitro. Following immunization, the lymphocytes are isolated and
fused with a suitable myeloma cell line using, for example,
polyethylene glycol, to form hybridoma cells that can then be
selected away from unfused lymphocytes and myeloma cells.
Hybridomas that produce monoclonal antibodies directed specifically
against a chosen antigen as determined by immunoprecipitation,
immunoblotting, or by an in vitro binding assay (e.g.,
radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))
can then be propagated either in vitro, for example, using known
methods (see, e.g., Goding, Monoclonal Antibodies: Principles and
Practice, Academic Press, 1986) or in vivo, for example, as ascites
tumors in an animal. The monoclonal antibodies can then be purified
from the culture medium or ascites fluid as described for
polyclonal antibodies above.
[0355] Alternatively monoclonal antibodies can also be made using
recombinant DNA methods, for example, as described in U.S. Pat. No.
4,816,567. For example, in one approach polynucleotides encoding a
monoclonal antibody are isolated from mature B-cells or hybridoma
cell, such as by RT-PCR using oligonucleotide primers that
specifically amplify the genes encoding the heavy and light chains
of the antibody, and their sequence is determined using
conventional procedures. The isolated polynucleotides encoding the
heavy and light chains are then cloned into suitable expression
vectors, which when transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
monoclonal antibodies are generated by the host cells. In other
approaches, recombinant monoclonal antibodies or antibody fragments
having the desired immunoreactivity can be isolated from phage
display libraries expressing CDRs of the desired species using
techniques known in the art (McCafferty et al., Nature 348:552-554
(1990); Clackson et al., Nature 352:624-628 (1991); and Marks et
al., J. Mol. Biol. 222:581-597 (1991)).
[0356] The polynucleotide(s) encoding a monoclonal antibody can
further be modified in a number of different ways, using
recombinant DNA technology to generate alternative antibodies. For
example, polynucleotide sequences that encode one or more MRDs and
optionally linkers, can be operably fused, for example, to the 5'
or 3' end of sequence encoding monoclonal antibody sequences. In
some embodiments, the constant domains of the light and heavy
chains of for example, a mouse monoclonal antibody can be
substituted (1) for those regions of, for example, a human antibody
to generate a chimeric antibody or (2) for a non-immunoglobulin
polypeptide to generate a fusion antibody. Techniques for
site-directed and high-density mutagenesis of the variable region
are known in the art and can be used to optimize specificity,
affinity, etc. of a monoclonal antibody.
[0357] In certain embodiments, the antibody of the MRD-containing
antibody is a human antibody. For example, human antibodies can be
directly prepared using various techniques known in the art.
Immortalized human B lymphocytes immunized in vitro or isolated
from an immunized individual that produce an antibody directed
against a target antigen can be generated (See, e.g., Cole et al.,
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77
(1985); Roemer et al., J. Immunol. 147 (1):86-95 (1991); and U.S.
Pat. Nos. 5,750,373 and 6,787,637). In one embodiment, the human
antibody can be derived from the "minilocus approach" in which an
exogenous Ig locus is mimicked through inclusion of individual
genes from the Ig locus (see e.g., U.S. Pat. No. 5,545,807).
Methods of preparing a human antibody from a phage library, and
optionally optimizing binding affinity are known in the art and
described, for example, in Vaughan et al., Nat. Biotech. 14:309-314
(1996); Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998);
Hoogenboom et al., Nat. Biotechnology 23:1105-1116 (2005);
Hoogenboom et al., J. Mol. Biol. 227:381 (1991); Persic et al.,
Gene 187:9-18 (1997); Jostock et al., J. Immunol. Methods 289:65-80
(2004); Marks et al., J. Mol. Biol., 222:581 (1991)); et al., Proc.
Natl. Acad. Sci. USA, 88:7978-7982 (1991); et al., Proc. Natl.
Acad. Sci. USA 91:3809-3813 (1994); Yang et al., J. Mol. Biol.
254:392-403 (1995); and Barbas et al., Proc. Natl. Acad. Sci. USA
89:4457-4461 (1992). Techniques for the generation and use of
antibody phage libraries are also described in: U.S. Pat. Nos.
5,545,807, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731,
6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484,
and 7,264,963; and Rothe et al., J. Mol. Bio. 130:448-54 (2007)
(each of which is herein incorporated by reference in its
entirety). Affinity maturation strategies and chain shuffling
strategies (Marks et al., Bio/Technology 10:779-783 (1992) (which
is herein incorporated by reference in its entirety) are known in
the art and can be employed to generate high affinity human
antibodies.
[0358] Antibodies can also be made in mice that are transgenic for
human immunoglobulin genes or fragments of these genes and that are
capable, upon immunization, of producing a broad repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. This approach is described in: Lonberg, Nat. Biotechnol
23:1117-1125 (2005), Green et al., Nature Genet. 7:13-21 (1994),
and Lonberg et al., Nature 368:856-859 (1994); U.S. Pat. Nos.
5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016,
6,596,541, 7,105,348, and 7,368,334 (each of which is herein
incorporated by reference in its entirety).
IV. LINKERS
[0359] Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) of the invention can contain a single
linker, multiple linkers, or no linker Thus, a MRD may be operably
attached (linked) to the antibody directly, or operably attached
through an optional linker peptide. Similarly, a MRD may be
operably attached to one or more MRD(s) directly, or operably
attached to one or more MRD(s) through one or more optional linker
peptide(s). Linkers can be of any size or composition so long as
they are able to operably attach an MRD and an antibody such that
the MRD enables the MRD containing antibody to bind the MRD
target.
[0360] In some embodiments, linkers have about 1 to 20 amino acids,
about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5
amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids,
about 2 to 10 amino acids, or about 2 to 5 amino acids. The linker
can also have about 4 to 15 amino acids. In certain embodiments,
the linker peptide contains a short linker peptide with the
sequence GGGS (SEQ ID NO:1), a medium linker peptide with the
sequence SSGGGGSGGGGGGSS (SEQ ID NO:2), or a long linker peptide
with the sequence SSGGGG SGGGGGGSSRSS (SEQ ID NO:19). In another
embodiment, the MRD is inserted into the fourth loop in the light
chain constant region. For example, the MRD can be inserted between
the underlined letters in the following amino acid sequence:
RTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDKLGTNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSLPVTKSFNRGEC (SEQ ID NO:102).
[0361] The linker can also be a non-peptide linker such as an alkyl
linker, or a PEG linker. For example, alkyl linkers such
as--NH--(CH.sub.2)s-C(O)--, wherein s=2-20 can be used. These alkyl
linkers may further be substituted by any non-sterically hindering
group such as lower alkyl (e.g., C.sub.1-C.sub.6) lower acyl,
halogen (e.g., Cl, Br), CN, NH.sub.2, phenyl, etc. An exemplary
non-peptide linker is a PEG linker. In certain embodiments, the PEG
linker has a molecular weight of about 100 to 5000 kDa, or about
100 to 500 kDa.
[0362] In some embodiments, the linker is a "cleavable linker"
facilitating release of an MRD or cytotoxic agent in the cell. For
example, an acid-labile linker (e.g., hydrazone),
protease-sensitive (e.g., peptidase-sensitive) linker, photolabile
linker, dimethyl linker or disulfide-containing linker (Chari et
al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020;
U.S. Appl. Pub. No. 20090110753) can be used wherein it is
desirable that the covalent attachment between an MRD or a cytoxic
agent and the multivalent and monovalent multispecific composition
(e.g., MRD-containing antibody) is intracellularly cleaved when the
composition is internalized into the cell. The terms
"intracellularly cleaved" and "intracellular cleavage" refer to a
metabolic process or reaction inside a cell on an antibody-drug
conjugate (ADC) whereby the covalent attachment, i.e., linked via a
linker between the MRD and cytotoxic agent, MRD and antibody,
antibody and cytotoxic agent, or between two MRDs is broken,
resulting in the free MRD and/or cytotoxic agent dissociated from
the antibody inside the cell. The cleaved moieties of the
zybody-ADC are thus intracellular metabolites.
[0363] Linker optimization can be evaluated using the techniques
described in Examples 1-18 and techniques otherwise known in the
art. Linkers preferably should not disrupt the ability of an MRD
and/or an antibody to bind target molecules.
V. ANTIBODIES CONTAINING MRDS
[0364] Using the methods described herein, multi-specie city and
greater multi-valency can be achieved through the fusion of MRDs to
antibodies.
[0365] MRDs of the multivalent and multispecific compositions
(e.g., MRD-containing antibodies) prepared according to the present
invention, may be operably linked to an antibody through the
peptide's N-terminus or C-terminus. The MRD may be operably linked
to the antibody at the C-terminal end of the heavy chain of the
antibody, the N-terminal end of the heavy chain of the antibody,
the C-terminal end of the light chain of the antibody, or the
N-terminal end of the light chain of the antibody. Optimization of
the MRD composition, MRD-antibody attachment location and linker
composition can be performed using the binding assays described in
Examples 1-18 and bioassays and other assays known in the art for
the appropriate target related biological activity.
[0366] In one embodiment, an MRD-containing antibody is an
MRD-containing antibody described in U.S. Application No.
61/489,249, filed May 24, 2011, which is herein incorporated by
reference in its entirety.
[0367] In one embodiment, multivalent and multispecific
compositions (e.g., MRD-containing antibodies) contain an MRD
operably linked to either the antibody heavy chain, the antibody
light chain, or both the heavy and the light chain. In one
embodiment, an MRD-containing antibody contains at least one MRD
linked to one of the antibody chain terminals. In another
embodiment, an MRD-containing antibody of the invention contains at
least one MRD operably linked to two of the antibody chain
terminals. In another embodiment, an MRD-containing antibody
contains at least one MRD operably linked to three of the antibody
chain terminals. In another embodiment, an MRD-containing antibody
contains at least one MRD operably attached to each of the four
antibody chain terminals (i.e., the N and C terminals of the light
chain and the N and C terminals of the heavy chain).
[0368] In certain specific embodiments, the MRD-containing antibody
has at least one MRD operably attached to the N-terminus of the
light chain. In another specific embodiment, the MRD-containing
antibody has at least one MRD operably attached to the N-terminus
of the heavy chain. In another specific embodiment, the
MRD-containing antibody has at least one MRD operably attached to
the C-terminus of the light chain. In another specific embodiment,
the MRD-containing antibody has at least one MRD operably attached
to the C-terminus of the heavy chain.
[0369] An MRD-containing antibody can be "multispecific" (e.g.,
bispecific, trispecific tetraspecific, pentaspecific or of greater
multispecificity), meaning that it recognizes and binds to two or
more different epitopes present on one or more different antigens
(e.g., proteins). Thus, whether an MRD-containing antibody is
"monospecific" or "multispecific," (e.g., bispecific, trispecific,
and tetraspecific) refers to the number of different epitopes that
the MRD-containing antibody binds. Multispecific antibodies may be
specific for different epitopes of a target polypeptide (e.g., as
described herein) or may be specific for a target polypeptide as
well as for a heterologous epitope, such as a heterologous
polypeptide target or solid support material. The present invention
contemplates the preparation of mono-, bi-, tri-, tetra-, and
penta-specific antibodies as well as antibodies of greater
multispecificity. In one embodiment, the MRD-containing antibody
binds two different epitopes. In an additional embodiment the
MRD-containing antibody binds two different epitopes
simultaneously. In another embodiment, the MRD-containing antibody
binds three different epitopes. In an additional embodiment the
MRD-containing antibody binds three different epitopes
simultaneously. In another embodiment, the MRD-containing antibody
binds four different epitopes. In an additional embodiment the
MRD-containing antibody binds four different epitopes
simultaneously. In another embodiment, the MRD-containing antibody
binds five different epitopes (see, e.g., FIG. 2D). In an
additional embodiment the MRD-containing antibody binds five
different epitopes simultaneously.
[0370] In other embodiments two MRDs of the MRD-containing antibody
bind the same antigen. In other embodiments three, four, five, six,
seven, eight, nine or ten MRDs of the MRD-containing antibody bind
the same antigen. In other embodiments at least two MRDs of the
MRD-containing antibody bind the same antigen. In other embodiments
at least three, four, five, six, seven, eight, nine or ten MRDs of
the MRD-containing antibody bind the same antigen. In other
embodiments two MRDs of the MRD-containing antibody bind the same
epitope. In other embodiments three, four, five, six, seven, eight,
nine or ten MRDs of the MRD containing antibody bind the same
epitope. In other embodiments at least two MRDs of the
MRD-containing antibody bind the same epitope. In other embodiments
at least three, four, five, six, seven, eight, nine or ten MRDs of
the MRD-containing antibody bind the same epitope.
[0371] In other embodiments, the antibody and one MRD of the
MRD-containing antibody bind the same antigen. In other embodiments
the antibody and two, three, four, five, six, seven, eight, nine or
ten MRDs of the MRD-containing antibody bind the same antigen. In
other embodiments, the antibody and at least one MRD of the
MRD-containing antibody bind the same antigen. In other embodiments
the antibody and at least two, three, four, five, six, seven,
eight, nine or ten MRDs of the MRD-containing antibody bind the
same antigen. In other embodiments, the antibody and one MRD of the
MRD-containing antibody bind the same epitope. In other embodiments
the antibody and two, three, four, five, six, seven, eight, nine or
ten MRDs of the MRD-containing antibody bind the same epitope. In
other embodiments, the antibody and at least one MRD of the
MRD-containing antibody bind the same epitope. In other embodiments
the antibody and at least two, three, four, five, six, seven,
eight, nine or ten MRDs of the MRD-containing antibody bind the
same epitope.
[0372] The present invention also provides for two or more MRDs
which are linked to any terminal end of the antibody. Thus, in one
non-exclusive embodiment, two, three, four, or more MRDs are
operably linked to the N-terminal of the heavy chain. In another
non-exclusive embodiment, two, three, four, or more MRDs are
operably linked to the N-terminal of the light chain. In another
non-exclusive embodiment, two, three, four, or more MRDs are
operably linked to the C-terminal of the heavy chain. In another
non-exclusive embodiment, two, three, four, or more MRDs are
operably linked to the C-terminal of the light chain. It is
envisioned that these MRDs can be the same or different. In
addition, any combination of MRD number and linkages can be used.
For example, two MRDs can be operably linked to the N-terminal of
the heavy chain of an antibody which contains one MRD linked to the
C-terminal of the light chain. Similarly, three MRDs can be
operably linked to the C-terminal of the light chain and two MRDs
can be operably linked to the N-terminal of the light chain.
[0373] Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) can contain one, two, three, four, five,
six, seven, eight, nine, ten or more than ten MRDs.
[0374] In one embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
one MRD (see, e.g., FIGS. 2B and 2C). In another embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains two MRDs. In another embodiment,
the multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains three MRDs. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains four MRDs
(see, e.g., FIGS. 2B and 2C). In another embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains five MRDs. In another embodiment,
the multivalent and monovalent multispecific composition
MRD-containing antibody) contains six MRDs. In an additional
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains between two
and ten MRDs.
[0375] In one embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least one MRD. In another embodiment, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) contains at least two MRDs. In another embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains at least three MRDs. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains at least four
MRDs. In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least five MRDs. In another embodiment, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) contains at least six MRDs.
[0376] In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
two different MRDs. In another embodiment, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) contains three different MRDs. In another embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains four different MRDs. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains five different
MRDs. In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
six different MRDs. In an additional embodiment, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) contains between two and ten different MRDs.
[0377] In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least two different MRDs. In another embodiment, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) contains at least three different MRDs. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains at least four
different MRDs. In another embodiment, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) contains at least live different MRDs. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains at least six
different MRDs.
[0378] Thus, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) can be MRD monomeric (i.e., containing
one MRD at the terminus of a peptide chain optionally connected by
a linker) or MRD multimeric (i.e., containing more than one MRD in
tandem optionally connected by a linker). The multimeric,
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) can be homo-multimeric (i.e., containing more than one
of the same MRD in tandem optionally connected by linker(s) (e.g.,
homodimers, homotrimers, homotetramer etc.)) or hetero-multimeric
(i.e., containing two or more MRDs in which there are at least two
different MRDs optionally connected by linker(s) where all or some
of the MRDs linked to a particular terminus are different (e.g.,
heterodimer, heterotrimer, heterotetramer etc.)). In one
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains two different
monomeric MRDs located at different immunoglobulin termini. In
another embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) contains three
different monomeric MRDs located at different immunoglobulin
termini. In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
four different monomeric MRDs located at different immunoglobulin
termini. In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
five different monomeric MRDs located at different immunoglobulin
termini. In another embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
six different monomeric MRDs located at different immunoglobulin
termini.
[0379] In an alternative embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least one dimeric and one monomeric MRD located at different
immunoglobulin termini. In another alternative embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains at least one homodimeric and one
monomeric MRD located at different immunoglobulin termini. In
another alternative embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least one heterodimeric and one monomeric MRD located at
different immunoglobulin termini.
[0380] In an alternative embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least one multimeric and one monomeric MRD located at different
immunoglobulin termini. In another alternative embodiment, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) contains at least one homomultimeric and
one monomeric MRD located at different immunoglobulin termini. In
another alternative embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
at least one heteromultimeric and one monomeric MRD located at
different immunoglobulin termini.
[0381] In an alternative embodiment, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) contains
MRDs operably linked to at least two different immunoglobulin
termini. In a specific embodiment, the MRDs fused to at least one
of the immunoglobulins are a multimer. In one embodiment, the MRDs
fused to a least one of the immunoglobulins are a homomultimeric
(i.e., more than one of the same MRD operably linked in tandem,
optionally linked via a linker). In another embodiment, the MRDs
fused to at least one of the immunoglobulins are a heteromultimeric
(i.e., two or more different MRDs operably linked in tandem,
optionally linked via a linker). In an additional embodiment, the
MRDs fused to at least one of the immunoglobulins are a dimer. In
another embodiment, the MRDs fused to a least one of the
immunoglobulins are a homodimer. In another embodiment, the MRDs
fused to at least one of the immunoglobulins are a heterodimer.
[0382] The multiple MRDs can target the same target binding site,
or two or more different target binding sites. Where the MRDs bind
to different target binding sites, the binding sites may be on the
same or different target molecules.
[0383] Similarly, the antibody and the MRD in a multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) may bind to the same target molecule or to different
target molecules.
[0384] In some embodiments, at least one MRD and the antibody in
the multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) can bind to their targets simultaneously.
In one embodiment, each MRD in the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) and the
antibody can bind to its target simultaneously. Therefore, in some
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) binds two, three, four,
five, six, seven, eight, nine, ten or more targets
simultaneously.
[0385] The ability of a multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) to bind to multiple
targets simultaneously can be assayed using methods known in the
art, including, for example, those methods described in the
examples below.
Multivalent and Multispecific Compositions having Monovalent
Specificity
[0386] In additional embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) of the invention
have a single binding site for (i.e., monovalently bind) a
target.
[0387] In some embodiments, the antigen binding domains of an
antibody component of a multivalent and monovalent multispecific
composition of the invention binds to different target epitopes
(i.e., the antibody is bispecific). The term "bispecific antibody"
is intended to include any antibody, which has two different
binding specificities, i.e. the antibody binds two different
epitopes, which may be located on the same target antigen or, more
commonly, on different target antigens. Methods for making
bispecific antibodies are known in the art. (See, for example,
Millstein et al., Nature, 305:537-539 (1983); Traunecker et al.,
EMBO J. 10:3655-3659 (1991); Suresh et al., Methods in Enzymology
121:210 (1986); Kostelny et al., J. Immunol. 148(5):1547-1553
(1992); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448
(1993); Gruber et al., J. Immunol. 152:5368 (1994); Tutt et al., J.
Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893, 4,676,980,
4,714,681, 4,925,648, 5,573,920, 5,601,819, 5,731,168, 5,807,706,
and 5,821,333; Intl. Appl. Publ. Nos. WO94/04690, WO91/00360,
WO92/05793, WO92/08802, WO92/200373, WO93/17715, WO00/44788, and
WO02/096948; EP 1870459A1 and EP 03089, the contents of each of
which is herein incorporated by reference in its entirety).
[0388] One method for generating bispecific antibodies has been
termed the "knobs-into-holes" strategy (see, e.g., Intl. Publ.
WO2006/028936). The mispairing of Ig heavy chains is reduced in
this technology by mutating selected amino acids forming the
interface of the CH3 domains in IgG. At positions within the CH3
domain at which the two heavy chains interact directly, an amino
acid with a small side chain (hole) is introduced into the sequence
of one heavy chain and an amino acid with a large side chain (knob)
into the counterpart interacting residue location on the other
heavy chain. In some embodiments, compositions of the invention
have immunoglobulin chains in which the CH3 domains have been
modified by mutating selected amino acids that interact at the
interface between two polypeptides so as to preferentially form a
bispecific antibody. The bispecific antibodies can be composed of
immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or
different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4)
[0389] In one embodiment, a bispecific antibody component of a
multispecific and multivalent composition (e.g., MRD-containing
antibody) comprises a T366W mutation in the "knobs chain" and
T366S, L368A, Y407V mutations in the "hole chain," and optionally
an additional interchain disulfide bridge between the CH3 domains
by, e.g., introducing a Y349C mutation into the "knobs chain" and a
E356C mutation or a S354C mutation into the "hole chain;" R409D,
K370E mutations in the "knobs chain" and D399K, E357K mutations in
the "hole chain;" R409D, K370E mutations in the "knobs chain" and
D399K, E357K mutations in the "hole chain;" a T366W mutation in the
"knobs chain" and T366S, L368A, Y407V mutations in the "hole
chain;" R409D, K370E mutations in the "knobs chain" and D399K,
E357K mutations in the "hole chain;" Y349C, T366W mutations in one
of the chains and E356C, T366S, L368A, Y407V mutations in the
counterpart chain; Y349C, T366W mutations in one chain and S354C,
T366S, L368A, Y407V mutations in the counterpart chain; Y349C,
T366W mutations in one chain and S354C, T366S, L368A, Y407V
mutations in the counterpart chain; and Y349C, T366W mutations in
one chain and S354C, T366S, L368A, Y407V mutations in the
counterpart chain (numbering according to the EU index of
Kabat).
[0390] In some embodiments, a bispecific antibody component of a
composition of the invention (e.g., MRD-containing antibody) is an
IgG4 antibody or a modified IgG4 antibody, or contains an IgG4
heavy chain or a modified IgG4 heavy chain. IgG4 antibodies are
dynamic molecules that undergo Fab arm exchange by swapping an IgG4
heavy chain and attached light chain for a heavy-light chain pair
from another IgG4 molecule, thus resulting in bispecific
antibodies. Accordingly, Fab arm exchange by swapping of
MRD-containing-IgG4 antibodies whether caused in vivo or in vitro
under physiologic conditions will lead to bispecific antibody
compositions. In particular embodiments, an IgG4 heavy chain of a
composition of the invention contains an S228P substitution. This
substitution has been shown to significantly inhibit Fab arm
exchange in the resulting mutant IgG4 antibodies, and to thereby
reduce the likelihood of Fab-arm-exchange between a recombinant
antibodies and endogenous IgG4. (See, e.g., Labrijn et al., Nat.
Biotechnol. 27(8):767-71 (2009)). In additional embodiments, an
IgG4 heavy chain of a composition of the invention contains a
substitution of the Arg at position 409 (e.g., with Lys, Ala, Thr,
Met or Leu), the Phe at position 405 (e.g., with Lys, Ala, Thr, Met
or Leu) or the Lys at position 370. In other embodiments, the CH3
region of an IgG4 heavy chain of a composition of the invention has
been replaced with the CHH3 region of IgG1, IgG2 or IgG3. In
additional embodiments, interactions between one or more MRDs
located at the C-termini of distinct heavy chains (e.g., IgG4 or
IgG4 and IgG3) favor and/or stabilize heterodimers between the
heavy chains, or otherwise reduces Fab arm exchange by the
heterodimer.
[0391] Exemplary bispecific antibody components of multivalent and
multispecific compositions of the invention include, IgG4 and IgG1,
IgG4 and IgG2, IgG4 and IgG2, IgG4 and IgG3, IgG1 and IgG3 chain
heterodimers. Such heterodimeric heavy chain antibodies, can
routinely be engineered by, for example, modifying selected amino
acids forming the interface of the CH3 domains in human IgG4 and
the IgG1 or IgG3 so as to favor heterodimeric heavy chain
formation. In additional embodiments, interactions between one or
more MRDs located at the C-termini of heteromeric heavy chains
favors or stabilizes heteromultimeric formation or structure,
respectively.
[0392] IgG4 antibodies are known to have decreased ADCC activity
and half-life compared to other immunoglobulins subclasses such as,
IgG1 and IgG3. Accordingly, IgG4 subclass-based formats provide an
attractive format for developing therapeutics that bind to and
block cell receptors, but do not deplete the target cell.
Alternatively, in those embodiments for which increased effector
activity is desired, an IgG4 heavy chain of a composition of the
invention can be modified as described herein or otherwise known in
the art, so as to increase effector function (e.g., modification of
the residues at positions 327, 330 and 331; numbering according to
EU index of Kabat). Similarly, where increased half-life is
desired, an IgG4 heavy chain of a composition of the invention can
be engineered as described herein, or otherwise known in the art to
more selectively bind the FcRn at pH 6.0, but not pH 7.4, by for
example, incorporating mutations located at the interface between
the CH2 and CH3 domains, such as substitutions at T250Q/M428L as
well as M252Y/S254T/T256E and H433K/N434F (numbering according to
the EU index of Kabat).
[0393] As exemplified above, it is envisioned that in some
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) of the invention have a single binding
site for (i.e., monovalently bind) a target. In some embodiments,
the single binding site (i.e., monovalent binding site) is an
antibody antigen binding domain. In other embodiments, the single
binding site is an MRD. Thus, the multivalent and multispecific
compositions of the invention encompass (and can be routinely
engineered to include) MRD-containing antibodies that that contain
1, 2, 3, 4 or more single binding sites for a target. The single
binding site(s) may be provided by one or more MRDs located at any
one or more of the 4 immunoglobulin heavy chain termini or 4
immunoglobulin light chain termini. Moreover, single binding site
may be provided by one of the antigen binding domains of the
antibody (wherein an MRD of the MRD-containing antibody binds the
same target epitope of the other antigen binding domain of the
antibody. Moreover, in a specific embodiment, the compositions of
the invention encompass (and can be routinely engineered to
include) MRD-containing antibodies that contain 1, 2, 3, 4 or more
single binding sites for a target and do not bivalenty bind another
target
[0394] In further embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibodies) has a
single binding site for (i.e., monovalently binds) a cell surface
target that forms multimers (e.g., homomers or heteromers). In some
embodiments, the single binding site binds a cell surface target
that requires multimerization for signaling. In some embodiments,
the multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) has a single binding site that binds a
cell surface target and inhibits binding of another molecule (such
as a ligand) to the cell surface target. In other embodiments,
binding of the single binding site inhibits multimerization of the
target (e.g., homomeric and heteromeric multimerization). In
additional embodiments, the composition has single binding sites
for different targets (i.e., monovalently binds more than one
different target). In some embodiments, the multiple single binding
sites of the composition bind targets on the same cell. In
additional embodiments, the multiple single binding sites of the
composition bind targets on different cells. Numerous receptors are
known in the art that require multimerization for affecting their
normal function. Such receptors are envisioned to be targets of
single binding sites in the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) of the invention. In
some, embodiments, the composition has a single binding site for a
receptor tyrosine kinase. In some embodiments, the composition has
a single binding site for a growth factor receptor. In additional
embodiments the composition has a single binding site for a G
protein coupled receptor. In additional embodiments the composition
has a single binding site for a chemokine receptor. In other
embodiments, the composition has a single binding site for a TNF
receptor superfamily member. In particular embodiments, the
composition has a single binding site for a receptor selected from:
RAGE, c-Met, ErbB2, VEGFR1, VEGFR2, VEGFR3, FGFR1 (e.g.,
FGFR1-1HC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, PDGFRA, PDGFRB, netrin, CD28, TNFRSF1A (TNFR1, p55, p60),
TNFRSF1B (TNFR2), TNFSF6 (Fas Ligand), TNFRSF6 (Fas, CD95),
TNFRSF21 or TNFRSF25, TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8
(CD30), TNFST11 (RANKL), TNFRSF11A (RANK), TNFRSF21 (DR6), TNFRSF25
(DR3), and LRP6.
[0395] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) has a
single binding site for (i.e., monovalently binds) a cell surface
target that forms a multimer and multiple sites (i.e.,
multivalently binds) for two or more different targets. In other
embodiments, the multivalent and monovalent multispecific
composition has a single binding site for a cell surface target and
multiple binding sites for 1, 2, 3, 4, 5 or more different targets.
In further embodiments, at least 1, 2, 3, 4, 5 or more of the
targets bound by the multivalent and monovalent multispecific
composition are located on a cell surface. In other embodiments, at
least 1, 2, 3, 4, 5 or more of the targets bound by the multivalent
and monovalent multispecific composition are soluble targets (e.g.,
chemokines, cytokines, and growth factors). In additional
embodiments, the composition binds 1, 2, 3, 4, 5 or more of the
targets described herein. In further embodiments, the targets bound
by the composition are tumor antigens (including tumor antigens and
tumor associated antigens). In additional embodiments, a target
bound by the composition is associated with a disease or disorder
of the immune system. In further embodiments, a targets bound by
the composition is associated with a disease or disorder of the
skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease.
[0396] In some embodiments, an MRD-containing antibody has a single
binding site for TNFRSF21 (DR6). In further embodiments, the
MRD-containing antibody has a single binding site for DR6 and binds
a target selected from: AGE (S100 A, amphoterin), IL1, IL6, IL18,
IL12, IL23, TNFSF12 (TWEAK), TNF alpha, VEGF, TNFRSF5 (CD40),
TNFSF5 (CD40 LIGAND), interferon gamma, GMCSF, an FGF, CXCL13, MCP
1, CCR2, NogoA, RGM A, OMgp MAG, a CPSG, LINGO, alpha-synuclein, a
semaphorin (e.g., Semaphorin 3A, Semaphorin 4), an ephrin, VLA4,
CD45, RB, C5, CD52 and CD200. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind DR6 and
also bind at least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. These compositions have applications
in treating diseases and disorders including neurological diseases
and disorders such as multiple sclerosis and other
neurodegenerative diseases. In specific embodiments, the antibody
component of the MRD-containing antibody binds DR6.
[0397] In some embodiments, an MRD-containing antibody has a single
binding site for TNFRSF25 (DR3). In further embodiments, the
MRD-containing antibody has a single binding site for DR3 and binds
a target selected from: TNF, IFN alpha, IFN gamma, IL1, IL1beta,
IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind DR3 and also bind at least 1, 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. These compositions
have applications in treating diseases and disorders including
inflammatory bowel disease and autoimmune diseases such as
rheumatoid arthritis. In specific embodiments, the antibody
component of the MRD-containing antibody binds DR3.
[0398] In further embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibodies) has
multiple binding site for (i.e., multivalently binds) a cell
surface target that forms multimers (e.g., homomers or heteromers).
In some embodiments, the multiple binding sites bind a cell surface
target that requires multimerization for signaling. In some
embodiments, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) has multiple binding
sites for a cell surface target. In further embodiments, binding of
the multiple binding sites result in multimerization of the target
(e.g., homomeric and heteromeric multimerization). In additional
embodiments, the composition has multiple binding sites for
different tai gets (i.e., multivalently binds more than one
different target). In some embodiments, the multiple single binding
sites of the composition bind targets on the same cell. In
additional embodiments, the multiple single binding sites of the
composition bind targets on different cells. Numerous receptors are
known in the art that require multimerization for affecting their
normal function. Such receptors are envisioned to be targets of the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies). In some embodiments, the composition has multiple
binding sites for a receptor tyrosine kinase. In some embodiments,
the composition has a multiple binding site for a growth factor
receptor. In additional embodiments the composition has multiple
binding sites for a G protein coupled receptor. In additional
embodiments the composition has multiple binding sites for a
chemokine receptor. In other embodiments, the composition has
multiple binding, sites for a TNF receptor superfamily member.
[0399] In some embodiments, an MRD-containing antibody binds
TNFRSF10A (DR4). In further embodiments, the MRD-containing
antibody binds DR4 and a target selected from: ErbB2, EGFR, IGF1R,
TNFRSF10b (DR5), CD19, CD20, CD22, CD30, CD33, TNFRSF5 (CD40),
TNFRSF9 (41BB), IL6, and IGF1,2. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind DR4 and
also bind at least 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. These compositions have applications
in treating diseases and disorders including cancers such as breast
cancer, colorectal cancer, head and neck cancer, B-cell lymphomas,
hairy cell leukemia, B-cell chronic lymphocytic leukemia and
melanoma. In specific embodiments, the antibody component of the
MRD-containing antibody binds DR4. In further embodiments, the
antibody component of the MRD-containing antibody is CS1008 or
mapatumumab.
[0400] In some embodiments, an MRD-containing antibody binds
TNFRSF10B (DR5). In some embodiments, an MRD-containing antibody
binds DR5 and a target selected from: ErbB2, EGFR, IGF1R, TNFRSF10A
(DR4), CD19, CD20, CD22, CD30, CD33, TNFRSF5 (CD40), TNFRSF9
(41BB), IL6, and IGF1,2. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind DR5 and also bind at
least 2, 3, 4, 5 or more of these targets are also encompassed by
the invention. These compositions have applications in treating
diseases and disorders including cancers such as breast cancer,
colorectal cancer, head and neck cancer, B-cell lymphomas, hairy
cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma.
In specific embodiments, the antibody component of the
MRD-containing antibody binds DR5. In further embodiments, the
antibody component of the MRD-containing antibody is LBY135, AMG66,
Apomab, PRO95780, lexatumumab, conatumumab or tigatuzumab.
Compositions that Redirect Effector Cell Function
[0401] The invention also encompasses multivalent and multispecific
compositions such as, multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that are capable of juxtaposing
host effector cells with cells that are desired to be eliminated
(e.g., immune cells, cancer cells, diseased cells, infectious
agents, and cells infected with infectious agents). The multivalent
and multispecific functionalities of the compositions of the
invention are particularly well suited for redirecting, host immune
responses and provide numerous advantages over alternative
multispecific composition platforms under development. In one
embodiment, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) binds (1) a target
on a cell, tissue, or infectious agent of interest (e.g., an immune
cell or a tumor antigen on a tumor cell) and (2) a target on an
effector cell so as to direct an immune response to the cell,
tissue, or infectious agent of interest. The target(s) to which the
multivalent, and monovalent multispecific composition binds can be
monomeric or multimeric. Moreover, the mulitimeric target to which
a multivalent and monovalent multispecific composition binds can be
homomultimeric or heteromultimeric. In additional embodiments, the
multivalent and monovalent multispecific composition binds at least
2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of
interest. In additional embodiments, one or more targets bound by
the multivalent and monovalent multispecific composition is a tumor
antigen (e.g., tumor antigens and tumor/cancer associated
antigens). The multivalent and multispecific compositions also have
applications in treating diseases and disorders including, but not
limited to, diseases of the immune system, skeletal system,
cardiovascular system, and nervous system, as well as infectious
disease. Thus, in some embodiments, 1, 2, 3, 4, 5 or more targets
bound by the multivalent and monovalent multispecific composition
is associated with a disease or disorder of the immune system (for
example, a disease or disorder of the immune system disclosed
herein, such as inflammation or an autoimmune disease (e.g.,
rheumatoid arthritis)). In additional embodiments, 1, 2, 3, 4, 5 or
more targets bound by the multivalent and monovalent multispecific
composition is associated with a disease or disorder of the
skeletal system (e.g., osteoporosis or another disease or disorder
of the skeletal system as disclosed herein). In additional
embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent
and monovalent multispecific composition is associated with a
disease or disorder of the cardiovascular system (e.g., a disease
or disorder of the cardiovascular system disclosed herein). In
additional embodiments, 1, 2, 3, 4, 5 or more targets bound by the
multivalent and monovalent multispecific composition is associated
with a disease or disorder of the nervous system (e.g., a disease
or disorder of the nervous system disclosed herein). In additional
embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent
and monovalent multispecific composition is associated with an
infectious agent or disease (e.g., an infectious disease or agent
disclosed herein).
[0402] Effector cells that can be bound by a multivalent and
monovalent multispecific composition (e.g., an MRD-containing
antibody) of the invention include, but are not limited to, T
cells, monocytes/macrophages, and natural killer cells.
[0403] In one embodiment, the target on a cell to which a
multivalent and monovalent multispecific composition (e.g., an
MRD-containing, antibody) directs an immune response is a tumor
antigen. The multivalent and multispecific compositions of the
invention (e.g., MRD-containing antibodies) are envisioned to be
capable of binding virtually any type of tumor and any type of
tumor antigen. Exemplary types of tumors that can be targeted
include, but are not limited to, one or more cancers selected from
the group: colorectal cancer, esophageal, gastric, head and neck
cancer, thyroid cancer, multiple myeloma, renal cancer, pancreatic
cancer, lung cancer, biliary cancer, glioma, melanoma, liver
cancer, prostate cancer, and urinary bladder cancer breast cancer,
ovarian cancer, cervical cancer, and endometrial cancer. Exemplary
types of tumors that may be targeted include hematological cancers.
Hematological cancers that may be targeted include, but are not
limited to, one or more cancers selected from the group Hodgkin's
lymphoma, medullary non-Hodgkin's lymphoma, acute lymphoblastic
leukemia, lymphocytic leukemia, and chronic myelogenous leukemia,
acute myelogenous leukemia.
[0404] Exemplary tumor antigens include ErbB1, ErbB2, ErbB3,
VEGFR1, VEGFR2, EGFRvIII, CD16, CD19, CD20, oncostatin M, PSA,
PSMA, integrin avb6, ADAM9, CD22, CD23, CD25, CD28, CD36, CD45,
CD46, CD56, CD79a/CD79b, CD103, JAM-3, gp100, ALCAM, PIPA, A33,
carboxypeptidease M, E-cadherin, CA125, CDK4, CEA, CTLA-4, RAAG10,
transferrin receptor, p-15, GD2, MUM-1, MAGE-1, MAGE-3, KSA, MOC31,
MIC-1, EphA2, GAGE-1, GAGE-2, MART, KID31, CD44v3, CD44v6, and
ROR1. Additional exemplary tumor antigens are described herein
and/or known in the art.
[0405] In one embodiment, the target on a cell to which a
multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) directs an immune response is an immune
cell or an inflammatory cell.
[0406] In some embodiments, the invention encompasses a multivalent
and monovalent multispecific composition that binds a tumor antigen
that is not expressed on tumor cells themselves, but rather on the
surrounding reactive and tumor supporting, non-malignant cells
comprising the tumor stroma (i.e., tumor associated antigens). The
tumor stroma comprises endothelial cells forming new blood vessels
and stromal fibroblasts surrounding the tumor vasculature. In one
embodiment, a multivalent and monovalent multispecific composition
binds a tumor associated antigen on an endothelial cell. In an
additional embodiment, a multivalent and monovalent multispecific
composition binds a tumor antigen and also binds a tumor associated
antigen on a fibroblast cell. In a further embodiment, a
multivalent and monovalent multispecific composition binds a tumor
antigen and also binds fibroblast activation protein (FAP).
[0407] Infectious agents to which a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) can
direct an immune response include, but are not limited to,
prokaryotic and eukaryotic cells, viruses (including
bacteriophage), foreign objects (e.g., toxins), and infectious
organisms such as funghi, and parasites (e.g., mammalian
parasites), as described herein and infectious agents associated
with infectious diseases described herein. The term infections
agents is also intended to encompass other prokaryotic and
eukaryotic cells, viruses (including bacteriophage), foreign
objects (e.g., toxins), and infectious organisms such as funghi,
and parasites otherwise known in the art.
[0408] In further embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
(1) a target on a cell, tissue, or infectious agent of interest
(e.g., a tumor antigen on a tumor cell) and (2) has a single
binding site for a target on an effector cell so as to direct an
immune response to the cell, tissue, or infectious agent of
interest. In some embodiments the single binding site is an MRD. In
other embodiments, the single binding site is an antibody antigen
binding domain. In further embodiments, binding, of the multivalent
and monovalent multispecific composition does not elicit a signal
when the composition binds a target on an effector cell. In
additional embodiments, the multivalent and monovalent
multispecific composition binds at least 2, 3, 4, or 5 targets on
the cell, tissue, or infectious agent of interest. According to
some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of
the multivalent and monovalent multispecific composition are
located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5
or more targets bound by the multivalent and monovalent
multispecific composition is a tumor antigen (e.g., tumor antigens
and tumor/cancer associated antigens). In additional embodiments,
one or more targets bound by the multivalent and monovalent
multispecific composition are associated with a disease or disorder
of the immune system. In additional embodiments, one or more
targets bound by the multivalent and monovalent multispecific
composition are associated with a disease or disorder of the
skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease.
[0409] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
(1) a target on a cell, tissue, or infectious agent of interest
(e.g., a tumor antigen on a tumor cell) and (2) a target on a
leukocyte so as to direct an immune response to the cell, tissue,
or infectious agent of interest. In additional embodiments, the
multivalent and monovalent multispecific composition binds at least
2, 3, 4, or 5 targets on the cell, tissue, or infectious agent of
interest. According to some embodiments, at least 1, 2, 3, 4, 5 or
more of the targets of the multivalent and monovalent multispecific
composition are located on a cell surface. In additional
embodiments the multivalent and monovalent multispecific
composition binds 1, 2, 3, 4, 5 or more targets described herein.
In additional embodiments, 1, 2, 3, 4, 5 or more targets bound by
the multivalent and monovalent multispecific composition are a
tumor antigen (e.g., tumor antigens and tumor/cancer associated
antigens). In additional embodiments, one or more targets bound by
the multivalent and monovalent multispecific composition are
associated with a disease or disorder of the immune system. In
additional embodiments, one or more targets bound by the
multivalent and monovalent multispecific composition are associated
with a disease or disorder of the skeletal system (e.g.,
osteoporosis), cardiovascular system, nervous system, or an
infectious disease.
[0410] The invention also encompasses multivalent and multispecific
compositions that bind a target expressed on a leukocyte. In some
embodiments, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) binds (1) a target
on a cell, tissue, or infectious agent of interest (e.g., a tumor
antigen on a tumor cell) and (2) has a single binding site for a
target on a leukocyte so as to direct an immune response to the
cell, tissue, or infectious agent of interest. In additional
embodiments, the multivalent and monovalent multispecific
composition binds at least 2, 3, 4, or 5 targets on the cell,
tissue, or infectious agent of interest. According to some
embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the
multivalent and monovalent multispecific composition are located on
a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more
antigens and tumor/cancer associated antigens). In additional
embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent
and monovalent multispecific composition are associated with a
disease or disorder of the immune system. In additional
embodiments, 1, 2, 3, 4, 5 or more targets bound by the multivalent
and monovalent multispecific composition are associated with a
disease or disorder of the skeletal system (e.g., osteoporosis),
cardiovascular system, nervous system, or an infectious
disease.
[0411] In one embodiment, the multivalent and monovalent
multispecific composition binds a target expressed on a T cell. In
some embodiments, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) binds (1) a target
on a cell, tissue, or infectious agent of interest (e.g., a tumor
antigen on a tumor cell) and (2) a target on a T cell so as to
juxtapose myeloid cells with the cell, tissue, or infectious agent
of interest. In some embodiments, the multivalent and monovalent
multispecific composition has multiple binding sites for (i.e.,
multivalently binds) a target on a T cell. In other embodiments,
the multivalent and monovalent multispecific composition has a
single binding site for (i.e., monovalently binds) a target on a T
cell. In some embodiments the single binding site is an MRD. In
other embodiments, the single binding site is an antibody antigen
binding domain. In further embodiments, binding of the multivalent
and monovalent multispecific composition does not elicit a signal
when the composition binds a target on a T cell. In other
embodiments, the binding of the multivalent and monovalent
multispecific composition does not result in lysis of the T cell
expressing the target. In some embodiments, the multivalent and
monovalent multispecific composition binds a target selected from:
CD2, CD3, CD4, CD8, CD161, a chemokine receptor. CD5, and CCR5. In
additional embodiments, the multivalent and monovalent
multispecific composition binds at least 2, 3, 4, or 5 targets on
the cell, tissue, or infectious agent of interest. According to
some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of
the multivalent and monovalent multispecific composition are
located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5
or more targets bound by the multivalent and monovalent
multispecific composition is a tumor antigen (e.g., tumor antigens
and tumor/cancer associated antigens). In additional embodiments,
1, 2, 3, 4, 5 or more targets bound by the multivalent and
monovalent multispecific composition are associated with a disease
or disorder of the immune system. In additional embodiments, 1, 2,
3, 4, 5 or more targets bound by the multivalent and monovalent
multispecific composition are associated with a disease or disorder
of the skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease.
[0412] In further embodiments, the multivalent and monovalent
multispecific composition contains a fusion protein containing one
or more peptides that bind to a protein on the surface of a cell,
such as a T cell. In additional embodiments, the multivalent and
monovalent multispecific composition bind target membrane proximal
protein sequences on a cell and inhibit the cross-linking (e.g.,
multimerization) of the target protein or its associated proteins.
In a particular embodiment, the multivalent and monovalent
multispecific composition binds to a T cell and inhibits the
cross-linking of the cell protein or its associated proteins. For
example, in one embodiment, the multivalent and multispecific
antibody comprises the amino terminal 27 amino acids of mature CD3
epsilon. In another embodiment, the multivalent and monovalent
multispecific composition comprises a fusion protein containing one
or more proteins corresponding to the G Domain of a CD3 protein
(e.g., CD3 epsilon, CD3 gamma, CD3 alpha (TCRA) or CD3 beta (TCRB).
Thus, in some embodiments, the fusion protein comprises a
polypeptide having an amino acid sequence selected from
GYYVCYPRGSKPEDANFYLYLR ARVC (SEQ ID NO:21), YLYLRAR (SEQ ID NO:22),
YRCNGTDIYKDKESTVQ VHYRMC (SEQ ID NO:23), and DKESTVQVH (SEQ ID
NO:24). In additional embodiments, the composition comprises a
fusion protein containing one or more proteins corresponding to a
portion of the extracellular domain of a CD3 protein (e.g., CD3
epsilon, CD3 gamma, CD3 alpha (TCRA) or CD3 beta (TCRB)) that is
able to bind CD3, or a CD3 multimer. Thus, in some embodiments, the
fusion protein comprises a portion of a CD3 protein that is able to
bind CD3 or a CD3 multimer wherein the portion comprises a CD3
binding fragment of a polypeptide having an amino acid sequence
selected from: KIPIEELEDRVFVNCNTSITWVEG
TVGTLLSDITRLDLGKRILDPRGIYRCNGTDIY KDKESTVQVHYRMCQSCVELD (human CD3
delta mature ECD, SEQ ID NO:25), QSIKGNHLVKVYDYQEDGSVLLTCDAEAK
NITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYRMCQNC IELN (human
CD3 gamma matte ECD, Ig-like domain highlighted; SEQ ID NO:26),
GNEEMGGITQTPYKVSTSGTTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGS DEDHL
SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVM (human CD3 epsilon
mature ECD, Ig-like domain highlighted, SEQ ID NO:27), and
QSFGLLDPK (human CD3 zeta mature ECD, SEQ ID NO:28), In alternative
embodiments, the fusion protein comprises a chemokine fragment that
binds a target on the cell surface. In some embodiments, the
chemokine fragment is a portion of a chemokine selected from: CCL20
(LARC/Ck.beta.4), CCL25 (TECK/Ck.beta.15), CXCL12 (SDF-1), CXCL13
(BCA-1), CXCL16 (SRPSOX), and CX3CL1 (Fractalkine). In some
embodiments, the chemokine fragment is a portion of a chemokine
selected from: CCL5 (RANTES), CCL8 (MCP-2), CXCL9 (MIG/CRG-10),
CXCL10 (IP-10/CRG-2) and CXCL11 (TAC/IP-9). In some embodiments,
the chemokine fragment is a portion of a chemokine selected from
CCL3 (MIP-1a) and CCL4 (MIP-1.beta.).
[0413] In specific embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
CD3. In particular embodiments, the composition binds a CD3 target
selected from CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, TCR
alpha, TCR beta, the TCR complex, or a heteromeric or
homomultimeric combination thereof. In a further embodiment, the
composition binds CD3 epsilon. In additional embodiments, the
multivalent and monovalent multispecific composition binds CD3 and
multiple binding sites for 1, 2, 3, 4, 5 or more different targets
(e.g., a tumor antigen as disclosed herein or otherwise known in
the art). In additional embodiments, the multivalent and monovalent
multispecific composition has a single binding site for (i.e.,
monovalently binds) CD3. In farther embodiments, the multivalent
and monovalent multispecific composition has a single MRD that
binds CD3 and multiple binding sites for 1, 2, 3, 4, 5 or more
different targets (e.g., a tumor antigen as disclosed herein or
otherwise known in the art). In further embodiments, the
multivalent and monovalent multispecific composition has a single
antibody antigen binding domain that binds CD3 and multiple binding
sites for 1, 2, 3, 4, 5 or more different targets (e.g., a tumor
antigen as disclosed herein or otherwise known in the art). In
particular embodiments, the CD3 binding compositions of the
invention are not single chain antibodies.
[0414] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD3 and a CD3 ortholog from another organism. In additional
embodiments, the multivalent and monovalent multispecific
composition binds human CD3 and a CD3 ortholog from another
primate. In further embodiments, the multivalent and monovalent
multispecific composition binds human CD3 and a CD3 ortholog from
cynomolgus Monkey or rhesus Monkey. In other embodiments, the
multivalent and monovalent multispecific composition binds human
CD3 and a CD3 ortholog from a primate selected from Saguinus
Oedipus and Callithrix jacchus). In an additional embodiment, the
multivalent and monovalent multispecific composition binds human
CD3 and a CD3 ortholog from cynomolgus monkey, and a CD3 ortholog
from mouse or rat. In particular embodiments, the human CD3 epsilon
binding compositions of the invention are not single chain
antibodies. In additional particular embodiments, the CD3 binding
compositions of the invention are not single chain antibodies.
[0415] According to one embodiment, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD3 epsilon. In a particular embodiment, the, multivalent and
monovalent multispecific composition binds human CD3 epsilon
protein having the sequence of amino acids 23-207 set forth in NCBI
Ref. Seq. No. NP.sub.--000724. In another embodiment, the
multivalent and monovalent multispecific composition binds a
polypeptide having the amino acid sequence of
QDGNEEMGGITQTPYKVSISGTT VILT (SEQ ID NO:29). In an additional
embodiment, the multivalent and monovalent multispecific
composition binds a polypeptide having the amino acid sequence of
QDGNEEMGGI (SEQ ID NO:30). In a further embodiment, the multivalent
and monovalent multispecific composition binds a polypeptide having
the amino acid sequence of QDGNEEMGG (SEQ ID NO:31). In particular
embodiments, the human CD3 epsilon binding compositions of the
invention are not single chain antibodies.
[0416] In some embodiments, a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) has a
single binding site for CD3 epsilon (i.e., monovalently binds CD3
epsilon) and multiple binding sites for 1, 2, 3, 4, 5 or more
different targets (e.g., a B cell or other target disclosed
herein). In further embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody)
competes for binding to CD3 with an antibody selected from: OKT-3,
otelixizumab, teplizumab, visilizumab, muromonab, X35-3, VIT3,
BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2,
TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, 12F6,
T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01. In
additional embodiments, an MRD of an MRD-containing antibody
competes for binding to CD3 with an antibody selected from OKT-3,
otelixizumab, teplizumab, visilizumab, muromonab X35-3, VIT3,
BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409. CLB-T3.4.2,
TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6,
T3/RW2-8C8, T3/RW24B6. OKT3D, M-T301, SMC2 and F101.01. In further
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) competes for binding to
CD3 with a CD3 binding composition disclosed in Int. Appt. Pub Nos.
WO2004/106380 and WO99/54440; funnacliffe et al., Int. Immunol.
1:546-550 (1989); Kjer-Nielsen, PNAS 101:7675-7680 (2004); or
Salmeron et al., J. Immunol. 147: 3047-3052 (1991).
[0417] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD3 epsilon and a CD3 epsilon ortholog from another organism.
In some embodiments, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) binds human CD3
epsilon and a CD3 epsilon ortholog from another primate. In
additional embodiments, the multivalent and monovalent
multispecific composition binds human CD3 epsilon and a CD3 epsilon
ortholog from cynomolgus monkey or rhesus monkey. In additional
embodiments, the multivalent and monovalent multispecific
composition binds human CD3 epsilon and a CD3 epsilon ortholog from
a primate selected from Saguinus Oedipus and Callithrix jacchus. In
an additional embodiment, the multivalent and monovalent
multispecific composition binds human CD3 epsilon and a CD3 epsilon
ortholog from cynomolgus monkey, and a CD3 epsilon ortholog from
mouse or rat. In particular embodiments, an MRD of the multivalent
and monovalent multispecific composition binds CD3 epsilon.
[0418] In another embodiment the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD3 delta. In a particular embodiment, the, multivalent and
monovalent multispecific composition binds human CD3 delta having
the sequence of amino acids 22-171 set forth in NCBI Ref Seq. No.
NP.sub.--000723. In particular embodiments, an MRD of the
multivalent and monovalent multispecific composition binds CD3
delta. In other embodiments, an antibody antigen binding domain of
the multivalent and monovalent multispecific composition binds CD3
delta. In particular embodiments, the human CD3 epsilon binding
compositions of the invention are not single chain antibodies.
[0419] In an additional embodiment, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing, antibody) binds
human CD3 gamma protein having the sequence of amino acids 23-182
set forth in NCBI Ref. Seq. No. NP.sub.--000064. In, particular
embodiments, an MRD of the multivalent and monovalent multispecific
composition binds gamma. In particular embodiments, an MRD of the
multivalent and monovalent multispecific composition binds CD3
gamma. In other embodiments, an antibody antigen binding domain of
the multivalent and monovalent multispecific composition binds CD3
gamma. In particular embodiments, the human CD3 gamma binding
compositions of the invention are not single chain antibodies.
[0420] In an additional embodiment, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD3 zeta protein having the sequence of amino acids 22-464
set forth in NCBI Ref. Seq. No. NP.sub.--932170. In particular
embodiments, an MRD of the multivalent and monovalent multispecific
composition binds CD3 zeta. In other embodiments, an antibody
antigen binding domain of the multivalent and monovalent
multispecific composition binds CD3 zeta. In particular
embodiments, the human CD3 zeta binding compositions of the
invention are not single chain antibodies.
[0421] The invention also encompasses multivalent and multispecific
compositions that bind a target expressed on a natural killer cell,
in some embodiments, the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) binds (1) a target
on a cell, tissue, or infectious agent of interest (e.g., a tumor
antigen on a tumor cell) and (2) a target on a natural killer cell.
In some embodiments, the multivalent and monovalent multispecific
composition has multiple binding sites for (i.e., monovalently
binds) a target on a natural killer cell. In other embodiments, the
multivalent and monovalent multispecific composition has a single
binding site for (i.e., monovalently hinds) a target on a natural
killer cell. In some embodiments the single binding site is an MRD.
In other embodiments, the single binding site is an antibody
antigen binding domain. In further embodiments, binding of the
multivalent and monovalent multispecific composition does not
elicit a signal when the composition binds a target on a natural
killer cell. In some embodiments, the multivalent and monovalent
multispecific composition binds a target selected from: KLRD1,
KLRK1, KLRB1, 2B4 (CD244), KIR2D4, KIR2D5, and KIR3DL1. In other
embodiments, the multivalent and monovalent multispecific
composition binds a target selected from: CD56, CD2, and CD161. In
additional embodiments, the multivalent and monovalent
multispecific composition binds at least 2, 3, 4, or 5 targets on
the cell, tissue, or infectious agent of interest. According to
some embodiments, at least 1, 2, 3, 4, 5 or more of the targets of
the multivalent and monovalent multispecific composition are
located on a cell surface. In additional embodiments, 1, 2, 3, 4, 5
or more targets bound by the multivalent and monovalent
multispecific composition are a tumor antigen (e.g., tumor antigens
and tumor/cancer associated antigens). In additional embodiments,
1, 2, 3, 4, 5 or more targets bound by the multivalent and
monovalent multispecific composition are associated with a disease
or disorder of the immune system. In additional embodiments, 1, 2,
3, 4, 5 or more targets bound by the multivalent and monovalent
multispecific composition are associated with a disease or disorder
of the skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease.
[0422] In specific embodiments, the multivalent and monovalent
multispecific composition binds CD2. According to one embodiment,
the multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) binds human CD2. In a particular
embodiment, the multivalent and monovalent multispecific
composition binds human CD2 protein having the sequence of amino
acids 25-209 set forth in NCBI Ref Seq. No. NP.sub.--001758. In
some embodiments, the multivalent and monovalent multispecific
composition has multiple binding sites for CD2. In some embodiments
the single binding site is an MRD. In other embodiments, the single
binding site is an antibody antigen binding domain. In other
embodiments, the multivalent and monovalent multispecific
composition has a single binding site for CD2. In further
embodiments, binding of the multivalent and monovalent
multispecific composition to CD2 does not elicit a signal by the
cell on which CD2 is expressed. In additional embodiments, the
multivalent and monovalent multispecific composition binds CD2 and
1, 2, 3, 4, 5 or more different targets (e.g., a tumor antigen as
disclosed herein or otherwise known in the art). In particular
embodiments, the CD2 binding compositions of the invention are not
single chain antibodies.
[0423] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
human CD2 at d a CD2 ortholog from another organism. In additional
embodiments, the multivalent and monovalent multispecific
composition binds human CD2 and a (D2 ortholog from another
primate. In further embodiments, the multivalent and monovalent
multispecific composition binds human CD2 and a CD2 ortholog from
cynomolgus monkey or rhesus monkey.
[0424] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
a target on a myeloid cell. In some, embodiments, the multivalent
and monovalent multispecific composition (e.g., an MRD-containing
antibody) binds (1) a target on a cell, tissue, or infectious agent
of interest (e.g., a tumor antigen on a tumor cell) and (2) a
target on an immune accessory cell (e.g., myeloid cell) so as to
juxtapose myeloid cells with the cell, tissue, or infectious agent
of interest. In some embodiments, the multivalent and monovalent
multispecific composition has multiple binding sites for (i.e.,
multivalently binds) a target on a myeloid cell. In other
embodiments, the multivalent and monovalent multispecific
composition has a single binding site for (i.e., monovalently
binds) a target on an accessory cell (e.g., myeloid cell). In some
embodiments the single binding site is an MRD. In other
embodiments, the single binding site is an antibody antigen binding
domain. In further embodiments, binding of the multivalent and
monovalent multispecific composition does not elicit a signal when
the composition binds a target on a myeloid cell. In some
embodiments, the multivalent and monovalent multispecific
composition binds an Fc gamma receptor selected from CD16 (i.e., Fc
gamma RIII), CD64 (i.e., Fc gamma RI), and CD32 (i.e., Fc gamma
RII). In particular embodiments, the multivalent and monovalent
multispecific composition binds CD64 (i.e., Fc gamma RI). In some
embodiments, the multivalent and monovalent multispecific
composition binds a target selected from, MHC class 2 and its
invariant chain, TLR1, TLR2, TLR4, TLR5 and TLR6. In additional
embodiments, the multivalent and monovalent multispecific
composition binds at least 2, 3, 4, or 5 targets on the cell,
tissue, or infectious agent of interest. According to some
embodiments, at least 1, 2, 3, 4, 5 or more of the targets of the
multivalent and monovalent multispecific composition are located on
a cell surface. In additional embodiments, 1, 2, 3, 4, 5 or more
targets bound by the multivalent and monovalent multispecific
composition are a tumor antigen (e.g., tumor antigens and
tumor/cancer associated antigens). In additional embodiments, 1, 2,
3, 4, 5 or more targets bound by the multivalent and monovalent
multispecific composition are associated with a disease or disorder
of the immune system. In additional embodiments, 1, 2, 3, 4, 5 or
more targets bound by the multivalent and monovalent multispecific
composition are associated with a disease or disorder of the
skeletal system (e.g., osteoporosis), cardiovascular system,
nervous system, or an infectious disease.
[0425] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
a target of interest on a cancer cell. In additional embodiments,
the multivalent and monovalent multispecific composition binds a
target of interest on an immune cell. In further embodiments, the
multivalent and monovalent multispecific composition binds a target
of interest on a diseased cell. In other embodiments, the
multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) binds a target of interest on an
infectious agent (e.g., a bacterial cell or a virus).
[0426] In further embodiments, the invention encompasses a method
of treating a disease or disorder by administering to a patient in
need thereof, a therapeutically effective amount of a multivalent
and monovalent multispecific composition of the invention.
Particular embodiments are directed to a method of treating a
disease or disorder by administering to a patient in need thereof,
a therapeutically effective amount a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) that
has a single binding site for a target (i.e., that monovalently
binds a target). In some embodiments, the administered multivalent
and monovalent multispecific composition has a single binding site
for a target on a leukocyte, such as a T-cell (e.g., CD3). In
additional embodiments, the administered multivalent and monovalent
multispecific composition has a single binding site for a target on
a leukocyte, such as a T-cell (e.g., CD3) and multiple binding
sites for (i.e., is capable of multivalently binding) a target
located on a cell or tissue of interest (e.g., a tumor antigen on a
tumor cell).
[0427] In further embodiments, the invention is directed to
treating a disease or disorder by administering to a patient a
therapeutically effective amount of a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) that
has a single binding site for a target (i.e., that monovalently
binds a target) and multiple binding sites for 1, 2, 3, 4, 5 or
more different targets.
[0428] In additional embodiments, the invention is directed to
treating a disease or disorder by administering to a patient in
need thereof, a therapeutically effective amount of a multivalent
and monovalent multispecific composition (e.g., an MRD-containing
antibody) that has a single binding site for CD3 (e.g., CD3
epsilon) that monovalently binds CD3 and multiple binding sites for
1, 2, 3, 4, 5 or more different targets.
[0429] According to some embodiments, the tumor cell is from a
cancer selected from breast cancer, colorectal cancer, endometrial
cancer, kidney (renal cell) cancer, lung cancer, melanoma,
Non-Hodgkin Lymphoma, leukemia, prostate cancer, bladder cancer,
pancreatic cancer, and thyroid cancer.
[0430] In some embodiments, the MRD(s) and the antibody in the
MRD-containing antibody are antagonists of their respective
targets. In other embodiments, the MRD(s) and the antibody in the
MRD-containing antibody are agonists of their respective target. In
yet other embodiments, at least one of the MRDs in the
MRD-containing antibody is an antagonist of its target molecule and
the antibody is an agonist of its target molecule. In yet another
embodiment, at least one of the MRDs in the MRD-containing antibody
is an agonist of its target molecule, and the antibody is an
antagonist of its target molecule.
[0431] In some embodiments, both the MRD(s) and the antibody in the
MRD-containing antibody bind to soluble factors. In some
embodiments, both the MRD(s) and the antibody in the MRD-containing
antibody bind to cell surface molecules. In some embodiments, at
least one MRD in the MRD-containing antibody binds to a cell
surface molecule and the antibody in the MRD-containing antibody
hinds to a soluble factor. In some embodiments, at least one MRD in
the MRD-containing antibody binds to a soluble factor and the
antibody in the MRD-containing antibody binds to a cell surface
molecule.
[0432] An improved multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) that specifically binds
a desired target or targets can also be prepared based on a
previously known MRD or multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody). For example, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50, 50-100, 100-150 or more
than 150 amino acid substitutions, deletions or insertions can be
introduced into an MRD or multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) sequence and the
resulting MRD or multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) can be screened for
binding to the desired target or targets, for antagonizing target
activity, or for agonizing target activity as described in the
examples or using techniques known in the art.
[0433] Additional peptide sequences may be added, for example, to
enhance the in vivo stability of the MRD or affinity of the MRD for
its target.
[0434] In certain embodiments, the binding of a multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) to its target (e.g., a cell) is enhanced compared to the
binding of the MRD alone, the antibody alone, and/or a combination
of the MRD and antibody. In some embodiments, the binding is at
least about 2-fold, at least about 5-fold, at least about 10-fold,
at least about 20-fold, at least about 50-fold, at least about
75-fold, at least about 100-fold, at least about 500-fold, or at
least about 1000-fold improved.
[0435] In addition, in some embodiments, the binding of a
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) to a target (e.g., a cell or a molecule
containing multiple epitopes) expressing both the MRD target and
the antibody target is enhanced compared to the binding of the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) to a target (e.g., a cell or a molecule
containing multiple epitopes) expressing only the MRD target or
only the antibody target. In some embodiments, the binding is at
least about 2-fold, at least about 5-fold, at least about 10-fold,
at least about 20-fold, at least about 50-fold, at least about
75-fold, at least about 100-fold, at least about 500-fold, or at
least about 1000-fold improved. This increased avidity can enable
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) to bind to targets that have previously been difficult
to target, e.g., G-protein coupled receptors and carbohydrate
molecules.
[0436] In addition, in some embodiments, the binding of a
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) to an MRD target is enhanced in a region
(e.g., of the body) where the antibody target is localized compared
to a region where the antibody target is not expressed or is
expressed at a lower level. In some embodiments, the binding of a
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) to an antibody target is enhanced in a
region (e.g., of the body) where the MRD target is localized
compared to a region where the MRD target is not expressed or is
expressed at a lower level. In some embodiments, the binding is at
least about 2-fold, at least about 5-fold, at least about 10-fold,
at least about 20-fold, at least about 50-fold, at least about
75-fold, at least about 100-fold, at least about 500-fold, or at
least about 1000-fold improved.
[0437] In preferred embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) retains
particular activities of the parent antibody. Thus, in certain
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) is capable of inducing
complement dependent cytotoxicity. In certain embodiments, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) is capable of inducing antibody dependent
cell mediated cytotoxicity (ADCC). In additional embodiments, the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) is capable of inducing apoptosis. In
additional embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) is
capable of reducing tumor volume. In additional embodiments, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) are capable of inhibiting tumor growth.
[0438] In some embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) shows
improved activity or pharmacodynamic properties compared to the
corresponding antibody without the attached MRD. Thus, in certain
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) has greater avidity
than the corresponding antibody without the attached MRD. In other
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) results in increased
receptor aggregation compared to the corresponding antibody without
the attached MRD. In another embodiment, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) antagonizes target activity to a greater extent than the
corresponding antibody without the attached MRD. In another
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) agonizes target
activity to a greater extent than the corresponding antibody
without the attached MRD. In another embodiment, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) has an improved pharmacodymamic profile than the
corresponding antibody without the attached MRD.
[0439] In another embodiment, the MRD-containing antibody has a
greater therapeutic efficacy than the corresponding antibody
without the attached MRD.
[0440] In other embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) have one or more of
the following effects: inhibit proliferation of tumor cells, reduce
the tumorigenicity of a tumor, inhibit tumor growth, increase
patient survival, trigger cell death of tumor cells, differentiate
tumorigenic cells to a non-tumorigenic state, or prevent metastasis
of tumor cells.
[0441] In certain embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) is at
least as stable as the corresponding antibody without the attached
MRD. In certain embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) is more
stable than the corresponding antibody without the attached MRD.
MRD-antibody stability can be measured using methods known to those
in the art, including, for example, ELISA techniques. In some
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) is stable in whole
blood at 37.degree. C. for at least about 10 hours, at least about
15 hours, at least about 20 hours, at least about 24 hours, at
least about 25 hours, at least about 30 hours, at least about 35
hours, at least about 40 hours, at least about 45 hours, at least
about 48 hours, at least about 50 hours, at least about 55 hours,
at least about 60 hours, at least about 65 hours, at least about 70
hours, at least about 72 hours, at least about 75 hours, at least
about 80 hours, at least about 85 hours, at least about 90 hours,
at least about 95 hours, or at least about 100 hours.
[0442] In certain embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) has at
least the same affinity for Fc receptors as the corresponding
parent antibody. In other nonexclusive embodiments, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) has at least the same affinity for complement receptors
as the corresponding parent antibody. In other nonexclusive
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) has at least the same
half-life as the corresponding parent antibody. In other
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) can be expressed at
levels commensurate with the corresponding parent antibody.
[0443] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) has an
increased affinity for Fc receptors compared to the corresponding
parent antibody. In other nonexclusive embodiments, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) has an increased affinity for complement receptors
compared to the corresponding parent antibody. In other
nonexclusive embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) has an
increased half-life compared to the corresponding parent antibody.
In other embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) can be expressed at
increased levels compared to that of the corresponding, parent
antibody.
Immunoconjugates (MRD-Containing Antibody Drug Conjugates)
[0444] The use of antibody-drug conjugates for the local delivery
of cytotoxic agents, allows targeted delivery of the drug to
tumors, and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., Lancet pages
603-05 (1986); Thorpe, "Antibody Carriers Of Cytotoxic Agents In
Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological
And Clinical Applications, A. Pinchera et al., (ed.s), pp. 475-506)
(1985)).
[0445] In additional embodiments, the invention encompasses a
multivalent and monovalent multispecific composition (e.g., an
MRD-containing antibody) that is covalently or otherwise associated
with a cytotoxic agent (payload) (i.e., as multivalent and
monovalent multispecific-cytoxic agent complexes (e.g.,
MRD-containing antibody-cytoxic agent complexes). According to some
embodiments, the cytoxic agent is covalently attached to a
multivalent and monovalent multispecific composition (e.g., MRD
containing antibody) by a linker. According to some embodiments,
the linker attaching the multivalent and monovalent multispecific
composition and the cytotoxic agent is cleavable by a protease. In
additional embodiments, the cytotoxic agent is a chemotherapeutic
agent, growth inhibitory agent, toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), a radioactive isotope (i.e., a radioconjugate)
or a prodrug. Methods of using immunoconjugates (MRD-containing
Antibody drug conjugates) are also encompassed by the
invention.
[0446] Cytotoxic agents that may be covalently or otherwise
associated with multivalent and multispecific compositions (e.g. an
MRD-containing antibody) include, but are not limited to any agent
that is detrimental to (e.g., kills) cells. Cytotoxins useful in
the compositions and methods of the invention include, inter alia,
alkylating agents intercalating agents, antiproliferative agents,
anti-mitototic agents, tubulin binding agents, vinca alkaloids,
enediynes, trichothecenes, podophyllotoxins or podophyllotoxin
derivatives, the pteridine family of drugs, taxanes, anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin, dolastatins
(e.g., dolastatin 10, dolastatin 11, and dolastatin 15)),
topoiosomerase inhibitors, and platinum complex chemotherapeutic
agents (e.g., cis-platinum).
[0447] In some embodiments, compositions of the invention include a
cytoxic agent that is a tubulin depolymerizing agent. Thus, in some
embodiments, compositions of the invention include an auristatin or
an auristatin derivative or analog. In one embodiment, compositions
of the invention contain monomethyl auristatin E (MMAE). In another
embodiment, compositions of the invention contain monomethyl
auristatin F (MMAF). In additional embodiments, an immunoconjugate
composition of the invention contains dolastatin or a dolastatin
peptidic analog or derivative, e.g., an auristatin (see, e.g., U.S.
Pat. Nos. 5,635,483, 5,780,588, and 5,663,149).
[0448] In additional embodiments, compositions of the invention
include a maytansinoid molecule. Maytansinoids are mitototic
inhibitors which act by inhibiting tubulin polymerization. Methods
of making maytansinoids and their therapeutic use are disclosed,
for example, in U.S. Pat. Nos. 5,208,020; 5,416,064, 6,441,163 and
European Pat. EP 0 425 235 B1; each of which is herein incorporated
by reference in its entirety.
[0449] Thus, in some embodiments, the cytotoxin is a maytansinoid
or a maytansinoid derivative or analog. Maytansinoid drug moieties
are attractive drug moieties in antibody-drug conjugates because
they are: (i) relatively accessible to prepare by fermentation or
chemical modification or derivatization of fermentation products,
(ii) amenable to derivatization with functional groups suitable for
conjugation through non-disulfide linkers to antibodies, (iii)
stable in plasma, and (iv) effective against a variety of tumor
cell lines. Maytansine compounds suitable for use as maytansinoid
drug moieties are well known in the art, and can be isolated from
natural sources according to known methods, produced using genetic
engineering techniques (see Yu et al PNAS 99:7968-7973 (2002)), or
maytansinol and maytansinol analogues can be prepared synthetically
according to known methods.
[0450] In particular embodiments compositions of the invention
include the maytansinoid DM1
(N(2')-deacetyl-N(2')-(3-mercapto-1-oxopropyl)-maytansine). In
other particular embodiments compositions of the invention include
the maytansinoid DM2. In additional embodiments, compositions of
the invention include the maytansinoid DM3
(N(2')-deacetyl-N-2-(4-mercapto-1-oxopentyl)-maytansine) or DM4
(N(2')-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine).
[0451] In some embodiments, compositions of the invention include a
cytoxic agent that is an alkylating agent. In particular
embodiments, the cytotoxic agent is selected from mechlorethamine,
thiotepa, thioepa chlorambucil, melphalan, carmustine (BSNU), BCNU
lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, and
streptozoicin.
[0452] In other embodiments, compositions of the invention include
a cytoxic agent that is an antimetabolite. In particular
embodiments, the cytotoxic agent is selected from methotrexate,
dichloromethotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil and 5-fluorouracil decarbazine.
[0453] In additional embodiments, the multivalent and multispecific
composition-drug conjugate (e.g., MRD-containing antibody-drug
conjugate) is capable of producing double-stranded DNA breaks. In
further embodiments, the MRD-containing antibody-drug conjugate
contains a member of the calicheamicin family of antibiotics
capable of producing double-stranded DNA breaks at sub-picomolar
concentrations. In further embodiments, a multivalent and
multispecific composition-drug conjugate (e.g., MRD-containing
antibody-drug conjugate) contains calicheamycin. For the
preparation of conjugates of the calicheamicin family, see e.g.,
U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American
Cyanamid Company). Structural analogues of calicheamicin which can
be contained in the multivalent and multispecific composition-drug
conjugate (e.g., MRD-containing antibody-drug conjugate) of the
invention include, but are not limited to, gamma.sub.1.sup.I,
alpha.sub.2.sup.I, alpha.sub.3.sup.I, N-acetylamma.sub.1.sup.I,
PSAG and theta.sub.1.sup.I (Hinman et al., Cancer Research
53:3336-3342 (1993), and Lode et al., Cancer Research 58:2925-2928
(1998).
[0454] In other embodiments, multivalent and multispecific
composition-drug conjugate (e.g., MRD-containing antibody-drug
conjugate) compositions of the invention include a cytoxic agent
selected from adriamicin, doxorubicin, mitomycin C, busulfan,
cytoxin, chlorambucil, etoposide, etoposide phosphate, CC-1065,
duocarmycin, KW-2189, CC1065, taxotere (docetaxel), methopterin,
aminopterin, topotecan, camptothecin, porfiromycin, bleomycin,
teniposide, esperamicins, mithramycin, anthramycin (AMC),
fludarabine, tamoxifen, taxotere (docetaxel), cytosine arabinoside
(Ara-C), adenosine arabinoside, cisplatin, carboplatin,
cis-dichlorodiamine platinum (II) (DDP) cisplatin, chloroquine,
cyclosporin A, docetaxel, paclitaxel, taxol, vinotelbine,
vindesine, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, ifosfamide, cyclophosphamide, tenoposide, caminomycin,
porfiromycin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
dactinomycin, actinomycin D, puromycin 1-dehydrotestosterone,
adriamycin, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, epithiolone, QFA, combretastatin, combretastatin A4
phosphate, vinblastine, vincristine, colchicine, geldanamycin,
doxorubicinchlorambucil, Auristatin F phenylene diamine (AFP)),
monomethylauristatin, the family of agents known collectively
LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710,
as well as esperamicins (U.S. Pat. No. 5,877,296) or a derivative
or analog thereof and derivatives and analog thereof.
[0455] Additional suitable toxins and chemotherapeutic agents are
described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and in Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan
Publishing Co. 1985). Moreover, for further discussion of types of
cytotoxins, linkers and other methods that can be use or routinely
adapted to conjugate therapeutic agents to the MRD-comprising
antibody complex, see e.g., Intl. Appl. Publ. WO2007/059404; Saito
et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail et al.,
Cancer Immunol Immunother. 52:328-337 (2003); Payne, Cancer Cell
3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan
et al., Curr. Opin. Investig. Drugs 3:1089-1091 (2002); and Senter
et al., Adv. Drug Deliv. Rev. 53:247-264 (2001), each of which is
hereby incorporated by reference in its entirety.
[0456] Cytotoxin chemotherapeutic agents that can be used in the
immunoconjugates of the invention (e.g., multivalent and
multispecific composition-drug conjugates such as MRD-containing
antibody-drug conjugates) include poisonous lectins and plant or
other toxins (e.g., ricin, abrin, modeccin, botulina, and
diphtheria toxins). It is envisioned that multiple copies of a
toxin or combinations of various toxins can optionally be coupled
to a multispecific and multivalent composition of the invention
(e.g., an MRD-containing antibody) thereby providing additional
cytotoxicity. Enzymatically active toxins and fragments thereof
that can be used in compositions of the invention include, but are
not limited to diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
Pseudomonas exotoxin, Pseudomonas endotoxin, ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
ribonuclease, DNase I, Staphylococcal enterotoxin-A, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. See, for example,
Pastan et al., Cell 47:641 (1986), Goldenberg et al., Cancer
Journal for Clinicians 44:43 (1994) and Intl Appl. Publ. Nos.
WO93/21232 and WO93/21232, each of which is herein incorporated by
reference in its entirety.
[0457] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp. 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. Nos. 5,635,483 and 5,780,588; Pettit et
al., J. Am. Chem. Soc. 111:5463-5465 (1989); Pettit et al.,
Anti-Cancer Drug Design 13:243-277 (1998); Pettit et al., Synthesis
719-725 (1996); Pettit et al., J. Chem. Soc. Perkin Trans.
15:859-863 (1996); and Doronina et al., Nat. Biotechnol
21(7):778-784 (2003).
[0458] According to some embodiments, the compositions of the
invention comprise a highly radioactive atom. A variety of
radioactive isotopes are available for the production of
radioconjugated multivalent and multispecific compositions (e.g.,
MRD-containing antibodies). Examples include At.sup.211, I.sup.131,
I.sup.125, Y..sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 to and radioactive isotopes of Lu.
When the conjugate is used for detection, it may comprise a
radioactive atom for scintiographic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0459] The radio- or other labels can be incorporated in the
conjugate using techniques known in the art. For example, the
peptide can be biosynthesized or can be synthesized by chemical
amino acid synthesis using suitable amino acid precursors
involving, for example, fluorine-19 in place of hydrogen. Labels
such as tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and
In.sup.111 can be attached via a cysteine residue in the peptide.
Yttrium-90 can be attached via a lysine residue. The IODOGEN method
(Fraker et al Biochem. Biophys. Res. Commun. 80: 49-57 (1978)) can
be used to incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes in detail
other methods that can be routinely applied to label the
compositions of the invention.
[0460] A linker can be a "cleavable linker," facilitating release
of a drug in the cell. For example, an acid-labile linker (e.g.,
hydrazone), protease-sensitive (e.g., peptidase-sensitive) linker,
photolabile linker, dimethyl linker or disulfide-containing linker
(Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.
5,208,020, U.S. Pat. Appl. Publ. No. 20110293513) can be used.
Thus, the invention encompasses multivalent and multispecific
compositions containing one or more linkers that can contain any of
a variety of groups as part of its chain that will cleave in vivo,
e.g., in a cell, at a rate which is enhanced relative to that of
constructs that lack such groups. Also provided are conjugates of
the linker arms with therapeutic and diagnostic agents. The linkers
are useful to form prodrug analogs of therapeutic agents and to
reversibly link a therapeutic or diagnostic agent (e.g., a
cytotoxin or MRD) to a targeting agent, a detectable label, or a
solid support. The linkers can be stable in plasma so as not to
release an MRD or cytotoxic agent. In the case of cytotoxins the
linkers can be stable in plasma and labile once internalized so as
to release the cytotoxin in an active form.
[0461] MRDs and/or cytotoxic agents are optionally attached to one
another or to the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) of the invention with a
linker as described herein or otherwise known in the art.
Conjugates of the MRD-containing antibody with an MRD or a
cytotoxic agent can be made using a variety of bifunctional protein
coupling agents known in the art, including, but not limited to,
coupling agents containing a group selected from 6-maleimidocaproyl
(MC), maleimidocaproyl-polyethylene glycol ("MC(PEG).sub.6-OH"
(amenable to attachment to antibody cysteines)), maleimidopropanoyl
(MP), MPBH, valine-citrulline (val-cit (exemplary dipeptide in a
protease cleavable linker)), methyl-valine-citrulline
("Me-Val-CitN," a linker in which a peptide bond has been modified
to prevent its cleavage by cathepsin B) alanine-phenylalanine
(ala-phe), p-aminobenzyloxycarbonyl (PAB (an example of a "self
immolative" linker component)),
valine-allin-p-aminobenzyloxycaronyl ("vc-PAB"), N-Succinimidyl
4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), LC-SMCC,
N-Succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), IT
(iminothiolane), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate),
6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl
(MC-vc-PAB), ethyleneoxy-CH.sub.2CH.sub.2O-- as one or more
repeating units ("EO" or "PEO"), BMPS, EMCS, GMBS, HBVS, MBS, SBAP,
SIA, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,
sulfo-SMCC, sulfo-SIAB, sulfo-SMPB, SVSG
(succinimidyl(4-vinylsulfone) benzoate), bifunctional derivatives
of imidoesters (such as dimethyl adipimidate HCl), active esters
(such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido compounds (such as
bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such
as toluene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene). Additional linker
components are known in the art and some are described herein.
[0462] In some embodiments, the multivalent and monovalent
multispecific composition is covalently attached to a cytotoxic
agent, via a linker at 1-5, 5-10, 1-10, or 1-20 sites on the
multivalent and multispecific composition. According to additional
embodiments, the multivalent and monovalent multispecific
composition is covalently attached to a cytotoxic agent via a
linker at more than 2, 5 or 10 sites on the multivalent and
multispecific composition.
[0463] In additional embodiments, the multivalent and monovalent
multispecific composition (e.g., MRD containing antibody) complex
is associated with a prodrug. Prodrug synthesis, chemical linkage
to antibodies, and pharmacodynamic properties are known in the art
and can routinely be applied to make and use multivalent and
multivalent compositions of the invention that contain prodrugs,
such as, MRD-containing antibody-prodrug compositions. See, e.g.,
Intl Publ. No. WO96/05863 and in U.S. Pat. No. 5,962,216, each of
which is herein incorporated by reference in its entirety.
[0464] Alternatively, a fusion protein comprising an antibody and a
cytotoxic agent can be made, e.g., by recombinant techniques or
peptide synthesis. A recombinant DNA molecule can comprise regions
encoding the antibody and cytotoxic portions of the conjugate
either adjacent to one another or separated by a region encoding a
linker peptide which does not destroy the desired properties of the
conjugate.
[0465] The multivalent and monovalent multispecific composition
(e.g., MRD-containing antibody) composition of the invention also
can be conjugated to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also referred to as radioimmunoconjugates.
Examples of radioactive isotopes that can be conjugated to
multivalent and monovalent multispecific compositions (e.g., MRD
containing antibodies) for use diagnostically or therapeutically
include, but are not limited to, iodine.sup.131, indium.sup.111,
yttrium.sup.90, and lutetium.sup.177. Methods for preparing
radioimmunconjugates are established in the art. Examples of
radioimmunoconjugates are commercially available, including
Zevalin.TM. (IDEC Pharmaceuticals) and Bexxar.TM. (Corixa
Pharmaceuticals), and similar methods can be used to prepare
radioimmunoconjugates using the MRD-containing antibodies of the
invention.
[0466] Methods for the conjugation of linker-drug moieties to
cell-targeted proteins such as antibodies are known in the art and
include those described for example, in U.S. Pat. Nos. 5,208,020
and 6,441,163; Intl. Appl. Publ. Nos. WO2005037992, WO2005081711,
and WO2006/034488, each of which is herein incorporated by
reference in its entirety. See, also e.g., Arnon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al., (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et
al., "Antibodies For Drug Delivery", in Controlled Drug Delivery
(2nd Ed.), Robinson et al., (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Saito et al., Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail
et al., Cancer Immunol. Immunother. 52:328-337 (2003); Payne,
Cancer Cell 3:207-212 (2003); Allen et al., Nat. Rev. Cancer
2:750-763 (2002); Pastan et al., Curr. Opin. Investig. Drugs
3:1089-1091 (2002); and Senter et al., Adv. Drug Deliv. Rev.
53:247-264 (2001), the contents of each of which, is herein
incorporated by reference in its entirety.
[0467] In some embodiments, a multivalent and monovalent
multispecific composition of the invention comprising a cytotoxic
agent (e.g., an MRD-containing antibody-cytotoxic agent conjugate)
and may generally be referred to herein as an immunoconjugate. In
some embodiments, an immunoconjugate of the invention binds a cell
surface target that is internalized into the cell. In farther
embodiments, the binding of an immunoconjugate of the invention
(e.g., an MRD-containing antibody-cytotoxic agent conjugate) to a
cell surface target results in the internalization of the
immunoconjugate into the cell in vitro. In further embodiments, the
binding of immunoconjugate to a cell surface target results in the
internalization of the composition into the cell in vivo. Methods
for treating a patient described herein can comprise: administering
to the patient a therapeutically effective amount of an
immunoconjugate (e.g., a multivalent and monovalent multispecific
composition of the invention comprising a cytotoxic agent, such as
an MRD-containing antibody-cytotoxic agent conjugate) that
comprises a cytotoxic agent and binds a target that is internalized
into a cell. In some embodiments, the immunoconjugate comprises a
cytotoxic agent disclosed herein. In additional embodiments, the
immunoconjugate comprises a cytotoxic agent selected from an
alkylating agent, antiproliferative agent, tubulin binding agent,
vinca alkaloid, enediyne, podophyllotoxin, podophyllotoxin
derivative, a member of the pteridine family of drugs, taxane, a
dolastatin, topoiosomerase inhibitor, or a platinum complex
chemotherapeutic agent. In further embodiments, the cytoxic agent
is a maytansinoid or a maytansinoid derivative or analog. In
specific embodiments the cytoxic agent is the maytansinoid DM1,
DM2, or DM3. In additional embodiments, the cytotoxic agent is
auristatin or an auristatin derivative or analog. In specific
embodiments the cytoxic agent is MMAE or MMAF. The cytotoxic agents
are optionally attached to the other components of the
immunoconjugate by a linker. In some embodiments the cytotoxic
agent is attached to the other components of the immunoconjugate by
an enzyme cleavable linker. In additional embodiments, the
cytotoxic agent is attached to the other components of the
immunoconjugate by an acid-labile linker.
[0468] In further embodiments, the cytoxic agent of an
immunoconjugate of the invention has a free drug potency of less
than 10.sup.-7M, 10.sup.-8M, or 10.sup.-9M. In additional
embodiments, the cytoxin has a free drug potency of 10.sup.-8 to
10.sup.-11M.
[0469] In some embodiments, a target bound by the immunoconjugate
is selected from CD19, CD22, CD30, CD33, CD56, CD70, CD79a, CD80,
CD83, CD95, CD126, CD133, CD138, PSMA, EphA2, ErbB2 (CD340),
SLC44A4, MN (carbonic anhydrase IX), GPNMB (glycoprotein
non-metastatic melanoma protein), Cripto, and .alpha.V integrin. In
additional embodiments, a target bound by the immunoconjugate is
selected from CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,
CD15, CD16, CL18, CD19, CD20, CD25, TNFRSF5 (CD40), CD64, CD74,
CD79, CD105, CD174, CD205, CD227, CD326, CD340, MUC16, EGP-1,
EGP-2, EGF receptor (ErbB1), ErbB2, ErbB3, Factor H, FHL-1, Flt-3,
folate receptor, Ga 733, GROB, HMGB-1, hypoxia inducible factor
(HIF), HM1.24, HER-2/neu, insulin-like growth factor (ILGF),
IFN-gamma, IFN-alpha, IFN-beta, IL2R, IL4R, IL6R, IL13R, IL15R,
IL17R, IL18R, IL2, IL6, IL8, IL12, IL15, IL17, IL18, IL25, IP-10,
IGF-1R, Ia, HM1.24, HCG, HLA-DR, ED-B, TMEFF2, EphB2, FAP
(fibroblast activation protein), mesothelin, EGFR, TAG-72, GD2
(encoded by the B4GALNT1 gene), and 5T4.
[0470] In additional embodiments, a target bound by the
immunoconjugate is a myeloid and hematopoietic target selected from
CD33, CD64, TNFRSF5 (CD40), CD56, and CD138. In further
embodiments, a target bound by the immunoconjugate is a carcinoma
target selected from EpCam, GD2, EGFR, CD74, CD227, CD340, MUC16,
GD2, GPNMB, PSMA, crypto, TMEFF2, EphB2, 5t4, mesothelin, TAG-72,
and MN.
[0471] In other embodiments, a target bound by the immunoconjugate
is a B cell target selected from CD19/CD21, CD20, CD22, TNFRSF5
(CD40), CD70, CD79a, CD79b, and CD205. In additional embodiments, a
target bound by the immunoconjugate is a T cell target selected
from CD25, CD30, TNFRSF5 (CD40), CD70, and CD205. In further
embodiments, a target bound by an endothelial cell target selected
from CD105, the stromal cell target FAP, and the vascular target
ED-B.
[0472] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0473] The following embodiments are further provided for any of
the above immunoconjugates. In one embodiment, an immunoconjugate
has in vitro or in vivo cell killing activity. In one embodiment,
the linker is attached to the antibody through a thiol group on the
antibody. In one embodiment, the linker is cleavable by a protease.
In one embodiment, the linker comprises a val-cit dipeptide. In one
embodiment, the linker comprises a p-aminobenzyl unit. In one
embodiment, the p-aminobenzyl unit is disposed between the drug and
a protease cleavage site in the linker. In one embodiment, the
p-aminobenzyl unit is p-aminobenzyloxycarbonyl (PAB). In one
embodiment, the linker comprises 6-maleimidocaproyl. In one
embodiment, the 6-maleimidocaproyl is disposed between the antibody
and a protease cleavage site in the linker. The above embodiments
may occur singly or in any combination with one another.
[0474] The MRD-containing antibody of the present invention may
also be conjugating to a prodrug-activating enzyme which converts a
prodrug (e.g., a peptidyl chemotherapeutic agent, see e.g.,
WO81/01145) to an active anti-cancer drug. See, for example,
WO88/07378 and U.S. Pat. No. 4,975,278 the contents of which are
herein incorporated by reference in its entirety. The enzyme
component of the immunoconjugate is preferably capable of acting on
a prodrug in such a way so as to convert it into its more active,
cytotoxic form. See, for example, Pastan et al., Cell, 47:641
(1986), and Goldenberg et al., Cancer Journal for Clinicians, 44:43
(1994). Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, non-binding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, eromycin and the
tricothecenes. See, for example, WO93/21232.
[0475] In some embodiments, the multivalent and multispecific
compositions of the invention (e.g., MRD-containing antibodies) are
conjugated to a radioisotope, such as, .sup.90Y, .sup.125I,
.sup.131I, .sup.123I, .sup.111In, .sup.105Rh, .sup.153Sm,
.sup.67Cu, .sup.67Ga, .sup.166Ho, .sup.177Lu, .sup.186Re and
.sup.188Re using anyone of a number of well-known chelators or
direct labeling. In other embodiments, the MRD-containing antibody
is coupled to drugs, prodrugs or lymphokines such as, interferon.
Compositions of the invention can be labeled with ligand reagents
that bind, chelate or otherwise complex a radioisotope metal where
the reagent is reactive with the engineered cysteine thiol of the
antibody, using techniques known in the art such as, those
described in Current Protocols in Immunology, Volumes 1 and 2,
Coligen et al, Ed. Wiley-Interscience, New York, N.Y. Pubs. (1991).
Chelating ligands which may complex a metal ion and that may have
use in the compositions and methods of the invention include DOTA,
DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).
Radionuclides can be targeted via complexation with the
antibody-drug conjugates of the invention (Wu et al Nature
Biotechnology 23(9): 1137-1146 (2005)). Linker reagents such as,
DOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared
by the reaction of aminobenzyl-DOTA with 4-maleimidobutyric acid
(Fluka) activated with isopropylchloroformate (Aldrich), following
the procedure of Axworthy et al., Proc. Natl. Acad. Sci. USA
97(4):1802-1807 (2000)). DOTA-maleimide reagents react with the
free cysteine amino acids of the cysteine engineered antibodies and
provide a metal complexing ligand on the antibody (Lewis et al.,
Bioconj. Chem. 9:72-86 (1998)). Chelating linker labeling reagents
such as, DOTA-NHS
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono
(N-hydroxysuccinimide ester) are commercially available
(Macrocyclics, Dallas, Tex.).
[0476] Conjugates of the multivalent and multispecific compositions
of the invention (e.g., MRD-containing antibodies) and cytotoxin
can routinely be made using a variety of bifunctional
protein-coupling agents such as,
N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as, dimethyl adipimidate HCL), active esters (such as,
disuccinimidyl suberate), aldehydes (such as, glutareldehyde),
bis-azido compounds (such as, bis(p-azidobenzoyl)hexanediamine),
bis-diazonium derivatives (such as,
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as,
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as, 1,5-difluoro-2,4-dinitrobenzene). In specific embodiments, the
toxin is conjugate to an MRD-containing antibody through an
enzyme-cleavable linker system (e.g., such as, that present in
SGN-35). Conjugates of an MRD-containing antibody and one or more
small molecule toxins, such as, a calicheamicin, maytansinoids, a
trichothene, and CC 1065, and the derivatives of these toxins that
have toxin activity, can also be used.
[0477] In some embodiments, the MRD-containing antibody can be
complexed, or have MRDs that bind with other immunologically active
ligands (e.g., chemokines, cytokines, and antibodies or fragments
thereof) wherein the resulting molecule binds to the neoplastic
cell or other target as well as the chemokine, cytokine, or an
effector cell such as, a T cell. In certain embodiments, these
conjugates can be generated as fusion proteins. The enzymes of this
invention can be covalently bound to the antibody by techniques
well-known in the art such as, the use of the heterobifunctional
crosslinking reagents discussed above. Alternatively, fusion
proteins comprising at least the antigen-binding region of an
antibody of the invention linked to at least a functionally active
portion of an enzyme of the invention can be constructed using
recombinant DNA techniques known in the art.
[0478] In some embodiments, the N-terminus or C-terminus of the
antibody to which an MRD is operably linked in the MRD-antibody
fusions is truncated. In preferred embodiments, this truncation
does not prevent or reduce the ability of the antibody to bind to
its target antigen via its antigen binding domain. In other
embodiments, the truncation does not prevent or reduce Fc effector
function, half-life and/or ADCC activity. In other embodiments,
MRDs are attached in the terminal region of the antibody chain.
More particularly, in certain embodiments, the MRD is attached
within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
residues of the C-terminal amino acid of the heavy chain. In other
embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20,
25, 30, 35, 40, 45, or 50 residues of the C-terminal amino acid of
the light chain. In additional embodiments, the MRD is attached
within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50
residues of the N-terminal amino acid of the heavy chain. In other
embodiments, the MRD is attached within 1, 2, 3, 4, 5, 10, 15, 20,
25, 30, 35, 40, 45, or 50 residues of the N-terminal amino acid of
the light chain. Thus, for example, a MRD that is linked to the
N-terminal end of the heavy chain can be linked to the first,
second, third, fourth, fifth, or tenth amino acid or the N-terminal
chain of the heavy chain. For example, an MRD-antibody fusion
containing an MRD linked to the N-terminal of the heavy chain may
contain amino acids 1-3 of the heavy chain sequence linked to the
MRD, which is linked to amino acid 4 of the heavy chain
sequence.
[0479] In certain embodiments, one or more MRDs are attached to an
antibody at locations other than the termini of the antibody light
and heavy chains. The MRD can be attached to any portion of the
antibody that does not prevent the ability of the antibody to bind
its target. Thus, in some embodiments, the MRD is located outside
the antibody combining site. For example, the MRD can be located
within a heavy chain sequence or within a light chain sequence. By
way of example only, the MRD can be located between the Fc domain
and the hinge region, between the hinge region and the CH1 domain
of the heavy chain, between the CH1 domain and the variable region
of the heavy chain, or between the constant region and the variable
region of the light chain.
[0480] Angiogenesis inhibitors targeting the vascular endothelial
growth factor (VEGF) signaling pathways have been observed to
provide at best transitory therapeutic benefits followed by
restoration of tumor growth and progression due to an apparent
ability of angiogenic tumors to adapt to the presence of these
inhibitors. Without being bound by theory, it is believed that the
multivalent and multispecific properties of multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind an angiogenesis target provide these compounds with an ability
to extend anti-angiogenic therapeutic benefits beyond those
observed from for example, conventional monoclonal antibody
therapies by binding multiple distinct angiogenesis related targets
and thereby disrupting resistance mechanisms available to the
angiogenic tumor.
[0481] In one embodiment, an MRD-containing antibody binds 2 or
more targets selected from: VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2,
FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g.,
FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc), FGFR3, TIE2, TNFSF2
(TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF,
PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1,
VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, .alpha.2.beta.1 integrin,
.alpha.4.beta.1 integrin, .alpha.5.beta.1 integrin, .alpha.v.beta.3
integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and
PDGFRB. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind VEGF and 2, 3, 4, 5 or more of
these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds VEGF. In further embodiments, the antibody component of the
MRD-containing antibody is bevacizumab. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind VEGF and 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds VEGF. In further
embodiments, the antibody component of the MRD-containing antibody
is bevacizumab.
[0482] In one embodiment, an MRD-containing antibody binds VEGF
(i.e., VEGFA) and additionally binds an angiogenic target selected
from: VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g.,
FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL-6, IL-8,
IL-18, HGF, TIE2, PDGFA, P1GF, PDGFB, CXCL12, KIT, GCSF, CXCR4,
PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7,
.alpha.2.beta.1 integrin, .alpha.4.beta.1 integrin, .alpha.5.beta.1
integrin, .alpha.v.beta.3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12,
PLAU, VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind VEGF and
2, 3, 4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds VEGF. In further embodiments, the
antibody component of the MRD-containing antibody is bevacizumab.
In additional embodiments, the antibody component of the
MRD-containing antibody competes for VEGF binding with
bevacizumab.
[0483] In one embodiment, an MRD-containing antibody binds TNF
alpha and additionally binds a target selected from: Te38, IL-12,
IL-12p40, IL-13, IL-15, IL-17, IL-18, IL-1beta, IL-23, MIF, PEG2,
PGE4, VEGF, TNFSF11 (RANKL), TNFSF13B (BLYS), GP130, CD-22, and
CTLA-4. In another embodiment, an MRD-containing antibody binds TNF
alpha, IL6, and TNFSF13B (BLYS). Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind TNF and 2,
3, 4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds TNF. In further embodiments, the
antibody component of the MRD-containing antibody is adalimumab,
certolizumab, golimumab or AME-527. In additional embodiments, the
antibody component of the MRD-containing antibody competes for TNF
binding with adalimumab, certolizumab, golimumab or AME-527.
[0484] In one embodiment, an MRD-containing antibody binds IL1
alpha and IL1 beta. In another embodiment, an MRD-containing
antibody binds IL1 beta and TNFSF11 (RANKL). In an additional
embodiment, an MRD-containing antibody binds IL1 beta and a target
selected from IL13, IL17A, TNF, VEGF, PGE2, VEGFR1, VEGFR2, TNFSF12
(TWEAK) and TNF. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind IL1 beta and at least 1, 2, 3,
4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds IL 1 beta. In further embodiments,
the antibody component of the MRD-containing antibody is
catumaxomab, Xoma052, canakinumab or ACZ885. In additional
embodiments, the antibody component of the MRD-containing antibody
competes for IL1 alpha or IL1 beta binding with catumaxomab,
Xoma052, canakinumab or ACZ885.
[0485] In another embodiment, an MRD-containing antibody binds
IL12. In a further embodiment, an MRD-containing antibody binds IL
12 and additionally binds IL18 or TNFSF12 (TWEAK). Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind CTLA-4 and at least 1, 2, 3, 4, 5 or more of these targets are
also encompassed by the invention. In specific embodiments, the
antibody component of the MRD-containing antibody binds CTLA-4. In
further embodiments, the antibody component of the MRD-containing
antibody is briakinumab or ustekinumab. In additional embodiments,
the antibody component of the MRD-containing antibody competes for
IL12 binding with briakinumab or ustekinumab.
[0486] In another embodiment, an MRD-containing antibody binds
CTLA-4. In a further embodiment, an MRD-containing antibody binds
CTLA4 and additionally binds PDL-1 or BTNO2. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind CTLA-4 and one or both of these targets are also encompassed
by the invention. In specific embodiments, the antibody component
of the MRD-containing antibody binds CTLA-4. In further
embodiments, the antibody component of the MRD-containing antibody
is tremelimumab or iplimumab. In additional embodiments, the
antibody component of the MRD-containing antibody competes for
CTLA-4 binding with tremelimumab or iplimumab.
[0487] In an additional embodiment, an MRD-containing binds IL13.
In a further embodiment, an MRD-containing antibody binds IL13 and
additionally binds a target selected from: IL1beta, IL4, IL9, IL13,
IL25, a LHR agonist, MDC, MIF, PED2, SPRR2a, SPRR2b; TARC, TGF-beta
and IL25. In another embodiment, an MRD-containing antibody binds
IL13 and a target selected from IL5, ADAM8, a LHR (agonist),
IL23p19 and IgE. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind IL13 and at least 1, 2, 3, 4,
5 or more of these targets are also encompassed by the invention.
In specific embodiments, the antibody component of the
MRD-containing antibody binds IL13. In further embodiments, the
antibody component of the MRD-containing antibody is TNX-650,
lebrikizumab or CAT354. In additional embodiments, the antibody
component of the MRD-containing antibody competes for IL13 binding
with TNX-650, lebrikizumab or CAT354.
[0488] In a further embodiment, an MRD-containing antibody binds
RGM A. In a further embodiment, an MRD-containing antibody binds
RGM A and additionally binds a target selected from RGM B, MAG,
NgR, NogoA, OMGp and CSPGs. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind RGM A and
at least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds RGM A.
[0489] In another embodiment, an MRD-containing antibody binds CD38
and additionally binds a target selected from CD20, TNFRSF5 (CD40)
ALK1, TNF, VEGF, VEGFA, VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b,
FGF19, (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa FGFR2-IIIb, and
FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, VEGFR1, VEGFR2 and CD138.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind CD38 and at least 1, 2 or all 3 of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds CD38. In further embodiments, the antibody component of the
MRD-containing antibody binds MOR202 or daratumumab. In additional
embodiments, the antibody component of the MRD-containing antibody
competes for CD38 binding with MOR202 or daratumumab.
[0490] In some embodiments an MRD-containing antibody binds ErbB1
(EGFR) and additionally binds ErbB3. In specific embodiments, the
antibody component of the MRD-containing antibody binds ErbB1. In
additional embodiments, the antibody component of the
MRD-containing antibody is ERBITUX.RTM.. In additional embodiments,
the antibody component, MRD component, and/or MRD-containing
antibody competes for ErbB1-binding with ERBITUX.RTM.. In another
embodiment, the antibody component of the MRD-containing antibody
is an ErbB1-binding antibody selected from: nimotuzumab,
zalutumumab, matuzumab, panitumumab, MEDX-214, and ABX-EGF. In
additional embodiments, the antibody component, MRD component,
and/or MRD-containing antibody competes for ErbB1-binding with an
antibody selected from: nimotuzumab, zalutumumab, matuzumab,
panitumumab, MEDX-214, and ABX-EGF.
[0491] In one embodiment, an MRD-containing antibody binds ErbB2
and IGF1R. In another embodiment, an MRD-containing antibody binds
ErbB2, Ang2, and IGF1R. In specific embodiments, the antibody
component of the MRD-containing antibody binds ErbB2. In additional
embodiments, the antibody component of the MRD-containing antibody
is HuMax-Her2.TM. or trastuzumab-DM1. In further embodiments, the
antibody component of the MRD-containing antibody is trastuzumab.
In additional embodiments, the antibody component, MRD component,
and/or MRD-containing antibody competes for ErbB2-binding with
trastuzumab.
[0492] In one embodiment, an MRD-containing antibody binds ErbB2
and additionally binds a target selected from: ErbB3, EGFR, IGF1R,
cMet, VEGF, RON (MST1R), DLL4, PLGF, CDCP1 (CD318), NRP1, TNFRSF10A
(DR4) and TNFRSF10B (DR5). In another embodiment, an MRD-containing
antibody binds ErbB2 and additionally binds, a target selected
from: CD2, CD3, CD4 and NKG2D. In an additional embodiment, an
MRD-containing antibody binds ErbB2 and IGF1, IGF2 or IGF1,2.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind ErbB2 and additionally bind 1, 2, 3, 4, 5 or
more of these targets are also encompassed by the invention. In
specific embodiments, the antibody component of the MRD-containing
antibody binds ErbB2. In additional embodiments, the antibody
component of the MRD-containing antibody is HuMax-Her2.TM. or
trastuzumab-DM1. In further embodiments, the antibody component of
the MRD-containing antibody is trastuzumab. In additional
embodiments, the antibody component, MRD component, and/or
MRI)-containing antibody competes for ErbB2-binding with
trastuzumab.
[0493] In some embodiments an MRD-containing antibody binds ErbB2
and additionally binds ErbB3. In specific embodiments, the antibody
component of the MRD-containing antibody binds ErbB2. In additional
embodiments, the antibody component of the MRD-containing antibody
is HuMax-Her2.TM. trastuzumab-DM1. In further embodiments, the
antibody component of the MRD-containing antibody is trastuzumab.
In additional embodiments, the antibody component, MRD component,
and/or MRD-containing antibody competes for ErbB2-binding with
trastuzumab. In another embodiment, the antibody component of the
MRD-containing antibody is an ErbB2-binding antibody selected from:
MDX-210 (Medarex), tgDCC-E1A (Targeted Genetics), MGAH22
(MacroGenics), and pertuzumab (OMNITARG.TM.). In additional
embodiments, the antibody component, MRD component, and/or
MRD-containing antibody competes for ErbB2-binding with an antibody
selected from: MDX-210, tgDCC-E1A, MGAH22, and pertuzumab.
[0494] In some embodiments, an MRD-containing antibody binds ErbB2
and HER2/3. In further embodiments, an MRD-containing antibody
binds ErbB2 and HER2/3 simultaneously.
[0495] Angiogenesis inhibitors targeting the vascular endothelial
growth factor (VEGF) signaling pathways have been observed to
provide at best transitory therapeutic benefits followed by
restoration of tumor growth and progression due to an apparent
ability of angiogenic tumors to adapt the presence of these
inhibitors. Without being bound by theory, it is believed that the
multivalent and multispecific properties of MRD-containing
antibodies that bind an angiogenesis target provide these compounds
with an ability to extend anti-angiogenic therapeutic benefits
beyond those observed from for example, conventional monoclonal
antibody therapies by binding multiple distinct angiogenesis
related targets and thereby disrupting resistance mechanisms
available to the angiogenic tumor.
[0496] In another embodiment, an MRD-containing antibody binds
PDGFRA and additionally binds an target selected from: VEGFA,
VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g.,
FGRF1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, 1L6, IL8,
IL18, IGF1, IGF2, IGF1,2, HGF, TIE2, PDGFA, PLGF, PDGFB, CXCL12,
KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, EGFR, cMET,
Notch 1, DLL4, EGFL7, .alpha.2.beta.1 integrin, .alpha.4.beta.1
integrin, .alpha.5.beta.1 integrin, .alpha.v.beta.3 integrin, TGFb,
MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, and PDGFRB. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind PDGFRA and binds at least 1, 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds PDGFRA. In further embodiments, the antibody component of the
MRD-containing antibody is olaratumab. In further embodiments, the
antibody component, MRD component, and/or MRD-containing antibody
competes for PDGFRA binding with olaratumab. In further
embodiments, the antibody component of the MRD-containing antibody
is MEDI-575. In further embodiments, the antibody component, MRD
component, and/or MRD-containing antibody competes for PDGFRA
binding with MEDI-575.
[0497] In another embodiment, an MRD-containing antibody binds
PDGFRB and additionally binds an target selected from: VEGFA,
VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g.,
FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2, IL6, IL8,
IL18, IGF1, IGF2, IGF1,2, HGF, TIE2, PDGFA, PLGF, PDGFB, CXCL12,
KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1, VEGFR2, VEGFR3, EGFR, cMET,
Notch 1, DLL4, EGFL7, .alpha.2.beta.1 integrin, .alpha.4.beta.1
integrin, .alpha.5.beta.1 integrin, .alpha.v.beta.3 integrin, TGFb,
MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, and PDGFRA. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind PDGFRB and also bind at least 1, 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds PDGFRB.
[0498] In another embodiment, an MRD-containing antibody binds
VEGFR1 and additionally binds an angiogenic target selected from:
VEGF (i.e., VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19,
FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and
FGFR2-IIIc), FGFR3, TNFSF2 (TNFa), FGFR3, EFNa1, EFNa2, ANG1, ANG2,
IL6, IL8, IL18, HGF, PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4,
PTPRC, TIE2, VEGFR2, VEGFR3, Notch 1, DLL4, EGFL7, .alpha.2.beta.1
integrin, .alpha.4.beta.1 integrin, .alpha.5.beta.1 integrin,
.alpha.v.beta.3 integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU,
VCAM1, PDGFRA, and PDGFRB. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind VEGFR1 and
additionally bind 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds VEGFR1. In further
embodiments, the antibody component of the MRD-containing antibody
is IMC-18F1. In additional embodiments, the antibody component, MRD
component, and/or MRD-containing antibody competes for VEGFR1
binding with IMC-18F1.
[0499] In another embodiment, an MRD-containing antibody hinds
VEGFR2 and additionally binds a target selected from: VEGF (i.e.,
VEGFA), VEGFB, FGF1, FGF2, FGF4, FGF7, FGF8b, FGF19, FGFR1 (e.g.,
FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIc),
FGFR3, TNFSF2 (TNFa), FGFR3, NRP1, ROBO4, CD30, CD33, CD55 CD80,
KIT, CXCL12, Notch1EFNa1, EFNa2, ANG1, ANG2, IL6, IL8, IL18, HGF,
PDGFA, PLGF, PDGFB, CXCL12, KIT, GCSF, CXCR4, PTPRC, TIE2, VEGFR1,
VEGFR3, Notch 1, DLL4, EGFL7, .alpha.2.beta.1 integrin,
.alpha.4.beta.1 integrin, .alpha.5.beta.1 integrin, .alpha.v.beta.3
integrin, TGFb, MMP2, MMP7, MMP9, MMP12, PLAU, VCAM1, PDGFRA, and
PDGFRB. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind VEGFR2 and additionally bind
1, 2, 3, 4, 5 or more of these targets are also encompassed by the
invention. In specific embodiments, the antibody component of the
MRD-containing antibody binds VEGFR2. In further embodiments, the
antibody component of the MRD-containing antibody is IMC-1C11 or
DC101. In additional embodiments, the antibody component, MRD
component, and/or MRD-containing antibody competes for VEGFR2
binding with IMC-1C11 or DC101.
[0500] In another embodiment, an MRD-containing antibody binds
VEGFR2 and additionally binds ANG2 or TIE2. In specific
embodiments, the antibody component of the MRD-containing antibody
binds VEGFR2. In further embodiments, the antibody component of the
MRD-containing antibody is IMC-1C11, DC101 or TTAC-0001. In
additional embodiments, the antibody component, MRD component,
and/or MRD-containing antibody competes for VEGFR2 binding with
IMC-1C11, DC101 or TTAC-0001. In further embodiments, the TIE2
binding component comprises a fragment of ANG2 that binds TIE2. In
particular embodiments, the TIE2 binding component comprises amino
acids 283-449 of the human ANG2 disclosed in NCBI Ref. Seq. No.
NP.sub.--001138.1.
[0501] In another embodiment, an MRD-containing antibody binds DLL4
and additionally binds a target selected from: EGFR, PLGF, VEGFR1,
VEGFR2 and VEGF. Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind DLL4 and at least 1, 2, 3, 4,
5 or more of these targets are also encompassed by the invention.
In further embodiments, the antibody component of the
MRD-containing antibody is REGN421. In additional embodiments, the
antibody component, MRD component, and/or MRD-containing antibody
competes for DLL4 binding with REGN421.
[0502] In additional embodiments, an MRD-containing antibody binds
to an anti-angiogenic and a metastatic or invasive cancer target.
In one embodiment, an MRD-containing antibody binds to an
angiogenic target and also binds a metastatic or invasive cancer
target selected from: CXCL12, CXCR4 (e.g., CXCR4b), CCR7 (e.g.,
CXCR7b), CD44 (e.g., CD44v3 and CD44v6), .alpha.2.beta.1 integrin,
.alpha.4.beta.1 integrin, .alpha.5.beta.31 integrin,
.alpha.v.beta.1 integrin, .alpha.v.beta.3 integrin, TGFb,
.alpha.v.beta.5 integrin, .alpha.9.beta.31 integrin,
.alpha.6.beta.4 integrin, .alpha.M.beta.2 integrin, PD-1, HGF,
cMET, MMP2, MMP-7, MMP-9, MMP-12, VEGFA, VEGFB, and IGF1.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind an angiogenic target and also bind 2, 3, 4, 5
or more of these metastatic or invasive cancer targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds VEGF. In further
embodiments, the antibody component of the MRD-containing antibody
is bevacizumab. In additional embodiments, the antibody component,
MRD component, and/or MRD-containing antibody competes for VEGF
binding with bevacizumab.
[0503] In one embodiment, an MRD-containing antibody binds to 2 or
more targets associated with distinct cell signaling pathways. In
additional embodiments, an MRD-containing antibody binds to 2 or
more targets associated with redundant, overlapping or
cross-talking signaling pathways. For example, in one embodiment,
an MRD-containing antibody binds to 2 or more targets associated
with PI3K/AKT/mTOR signaling (e.g., ErbB2, EGFR, IGF1R, Notch,
FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and
FGFR2-IIIb), FGFR3, FGFR4, GPCR, and/or c-MET). In some
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) binds 2, 3, 4, 5 or
more of these targets.
[0504] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with receptor tyrosine Raf/MEK/MAPK
signaling (e.g., VEGFR1, VEGFR2, VEGFR3, FGFR1 (e.g., FGFR1-IIIC),
FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, FGFR4,
CD28, RET, cMET, EGFR, ErbB2, Notch, Notch1, Notch3, Notch4, DLL1,
DLL4, Jagged, Jagged1, Jagged2, and Jagged3. In some embodiments,
the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) bind 1, 2, 3, 4, 5 or more of these
targets.
[0505] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with SMAD signaling (e.g., Notch,
TGF.beta., TGF.beta.R1, TGF.beta.R2, and a BMP). In some
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) bind 2, 3, 4, 5 or more of these
targets.
[0506] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with JAK/STAT signaling (e.g., IFNgR1,
IFNgR3, IFNG, IFN-AR2, IFN-AR1, IFN alpha, IFN beta, IL6a receptor
(GP130), IL6, IL12R131, IL12, and EGFR). Thus, the invention
encompasses an MRD-containing antibody that binds to 2 or more
targets selected from WNT1, WNT2, WNT2b, WNT3, WNT3A, WNT4, WNT5A,
WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A,
WNT1013, WNT11, WNT16, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8,
Notch, Notch1, Notch3, Notch4, DLL1, DLL4, Jagged, Jagged1,
Jagged2, and Jagged3. In some embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) bind
2, 3, 4, 5 or more of these targets.
[0507] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with NFkB signaling (e.g., BCR, TCR,
IL1R, IL1, FZD1, FZD2, FZD4, FZD5, FZD6, FZD7, FZD8, Notch, Notch1,
Notch3, Notch4, DLL4, Jagged, Jagged1, Jagged2, Jagged3, TNFSF1
(TNFb, LTa), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFSF6
(Fas LigaLd), TNFRSF6 (Fas, CD95), TNFRSF6B (DcR3), TNFSF7 (CD27
Ligand, CD70), TNFRSF7 (CD27), TNFSF8 (CD30 Ligand), TNFRSF8
(CD30), TNFSF11 (RANKL), TNFRSF11A (RANK), TNFSF12 (TWEAK),
TNFRSF12 (TWEAKR), INFSF13 (APRIL), TNFSF13B (BLYS), TNFRSF13B
(TACI), TNFRSF13C (BAFFR), TNFSF15 (TL1A), TNFRSF17 (BCMA),
TNFRSF19L (RELT), TNFRSF19 (TROY), TNFRSF21 (DR6), TNFRSF25 (DR3),
TNFSF5 (CD40 Ligand), TNFRSF5 (CD40), TNFSF2 (TNFa), TNFSF3 (LTb),
TNFRSF3 (LTBR), TNFSF14 (LIGHT, HVEM Ligand), TNFRSF14 (HVEM),
TNFSF18 (GITR Ligand), TNFRSF18 (GITR), TNFSF4 (OX40 Ligand),
TNFRSF4 (OX40), TNFSF9 (41BB Ligand), TNFRSF9 (41BB), a BMP, NGF,
and TGF alpha). In some embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) bind
2, 3, 4, 5 or more of these targets.
[0508] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with cell proliferation (e.g., FGF1,
FGF2, FGF7, FGF4, FGF10, FGF18b, FGF19, FGF23, FGFR1 (e.g.,
FGFR1-IIIC), FGFR2 (e.g., FGFRIIIB and FGFR-IIIC), FGFR3, FGFR4,
TCR, TNFRSF5 (CD40), TLR1, TLR2, TLR3, TLR 4, TLR5, and TLR6). In
some embodiments, the multivalent and multispecific compositions
(e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or more of these
targets.
[0509] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with toll-like receptor signaling (e.g.,
TLR1, TLR2, TLR3, TLR 4, TLR5, and TLR6).
[0510] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with B cell signaling (e.g., mIg,
Ig.alpha./Ig.beta. (CD79a/CD79b) heterodimers (.alpha./.beta.),
CD19, CD20, CD21, CD22, CD23, CD27, CD30, CD46, CD80, CD86, ICOSL
(B7-H2), HLA-DR (CD74), PD1, PDL1, TNFRSF1A (TNFR1, p55, p60),
TNFRSF1B (TNFR2), TNFRSF13B (TACI), TNFRSF13C (BAFFR), TNFRSF17
(BCMA), BTLA, TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), Fc gamma RIIB,
IL4R and CRAC. In a particular embodiment, the MRD-containing
antibody binds to CD19 and CD20. In an additional embodiment, the
MRD-containing antibody binds CD19, CD20, and CD22. In some
embodiments, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibodies) binds 2, 3, 4, 5 or
more of these targets.
[0511] In a further embodiment, an MRD-containing antibody binds to
1 or more B cell surface markers selected from: CD10, CD24, CD37,
CD53, CD72, CD75, CD77, CD79a, CD79b, CD81, CD82, CD83, CD84
(SLAMS) and CD85. In a further embodiment, an MRD-containing
antibody binds to 1 or more B cell surface markers selected from:
CD 10, CD24, CD37, CD53, CD72, CD75, CD77, CD79a, CD79b, CD81,
CD82, CD83, CD84 (SLAMS) and CD85. In some embodiments, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) bind 2, 3, 4, 5 or more of these B cell surface
markers.
[0512] In additional embodiments, an MRD-containing antibody binds
CD19 and a target
[0513] selected from: CD20, CD22, CD30, CD33, TNFRSF5 (CD40), CD52,
CD74, CD80, CD138, VEGFR1, VEGFR2, EGFR, TNFRSF10A (DR4), TNFRSF10B
(DR5), TNF, NGF, VEGF, IGF1,2, IGF2, IGF1 and TNFSF11 (RANKL). In
additional embodiments, an MRD-containing antibody binds CD20 and a
target selected from: CD3, CD4 and NKG2D. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind CD19 and also bind at least 2, 3, 4, 5 or more of these
targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds CD19. In further embodiments, the antibody component of the
MRD-containing antibody is MDX-1342, SGN-CD19A, XMAB.RTM.5574,
SGN-19A, ASG-5ME or MEDI-551. In additional embodiments, the
antibody component, MRD component, and/or MRD-containing antibody
competes for CD19 binding with MDX-1342, SGN-CD19A, XMAB.RTM.5574,
SGN-19A, ASG-5ME or MEDI-551.
[0514] In additional embodiments, an MRD-containing antibody binds
CD22 and a target selected from: CD19, CD20, CD23, CD30, CD33,
TNFRSF5 (CD40), CD52, CD74, CD80, TNFRSF10A (DR4), TNFRSF10B (DR5),
VEGF, TNF and NGF. In additional embodiments, an MRD-containing
antibody binds CD22 and a target selected from: CD3, CD4 and NKG2D.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind CD22 and also bind 2, 3, 4, 5 or more of
these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds CD22. In further embodiments, the antibody component of the
MRD-containing antibody is epratuzumab or inotuzumab. In additional
embodiments, the antibody component, MRD component, and/or
MRD-containing antibody competes for CD22 binding with epratuzumab
or inotuzumab.
[0515] In additional embodiments, the antibody component of the
MRD-containing antibody is moxetumomab (CAT-8015, Cambridge
Antibody Technologies). In additional embodiments, the antibody
component, MRD component, and/or MRD-containing antibody competes
for CD22 binding with moxetumomab.
[0516] In additional embodiments, an MRD-containing antibody binds
TNFRSF5 (CD40) and a target selected from: BCMA, TNFSF11 (RANKL),
VEGFR1, VEGFR2, TNFRSF10A (DR4), TNFRSF10B (DR5), CD22, CD30, CD38,
CD56 (NCAM), CD70, CD80, CD138, IL6, IGF1, IGF2, IGF1,2, BLyS,
APRIL and NGF. In additional embodiments, an MRD-containing
antibody binds CD40 and a target selected from: CD3, CD4 and NKG2D.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind CD40 and also bind 2, 3, 4, 5 or more of
these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds CD40. In further embodiments, the antibody component of the
MRD-containing antibody is CP870893, dacetuzumab, ANTOVA.RTM.,
lucatumumab, XMAB.RTM.5485 or teneliximab. In additional
embodiments, the antibody component, MRD component, and/or
MRD-containing antibody competes for CD40 binding with CP870893,
dacetuzumab, ANTOVA.RTM., lucatumumab, XMAB.RTM.5485 or
teneliximab.
[0517] In some embodiments, an MRD-containing antibody binds CD33
and a target selected from: FLT3, CD44, TNFRSF10A (DR4), TNFRSF10B
(DR5), CD80, MGC, VEGFR1, VEGFR2, IL1, IL6, TNF and VEGF.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind TNFRSF10B and also bind at least 2, 3, 4, 5
or more of these targets are also encompassed by the invention. In
specific embodiments, the antibody component of the MRD-containing
antibody binds CD33. In further embodiments, the antibody component
of the MRD-containing antibody is gemtuzumab or lintuzumab. In
additional embodiments the antibody component, MRD component,
and/or MRD-containing antibody competes for CD33 binding with
gemtuzumab or lintuzumab.
[0518] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with antigen presentation cell signaling
(e.g., mIg, Ig.alpha./Ig.beta. (CD79a/CD79b) heterodimers
(.alpha./.beta.), CD19, CD20, CD21, CD22, CD23, CD27, CD28, CD30,
CD30L, TNFSF14 (LIGHT, HVEM Ligand), CD70, ICOS, ICOSL (B7-H2),
CTLA4, PD-1, PDL1 (B7-H1), B7-H4, B7-H3, PDL2 (B7-DC), BTLA, CD46,
CD80 (B7-1), CD86 (B7-2), HLA-DR, CD74, PD1, TNFRSF4 (OX40),
TNFRSF9 (41BB), TNFSF4 (OX40 Ligand), TNFSF9 (41BB Ligand), TNFRSF9
(41BB), TNFRSF1A (TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF13B
(TACI), TNFRSF13C (BAFFR), TNFRSF17 (BCMA), BTLA, TNFRSF18 (GITR),
MHC-1, TNFRSF5 (CD40), TLR4, TNFRSF14 (HVEM), Fcgamma RIIB, IL4R
and CRAC). In some embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) bind 2, 3, 4, 5 or
more of these targets.
[0519] In another embodiment, an MRD-containing antibody binds to 2
or more targets associated with T cell receptor signaling (e.g.,
CD3, CD4, CD27, CD28, CD70, IL2R, LFA-1, C4, ICOS, CTLA-4, CD45,
CD80, CD86, PG-1, TIM1, TIM2, TIM3, TIM4, galectin 9, TNFRSF1A
(TNFR1, p55, p60), TNFRSF1B (TNFR2), TNFRSF21 (DR6), TNFRSF6 (Fas,
CD95), TNFRSF25 (DR3), TNFRSF14 (HVEM), TNFSF18, TNFRSF18 (GITR),
TNFRSF4 (OX40), TNFSF4 (OX40 Ligand), PD1, PDL1, CTLA4, TNFSF9
(41BB Ligand), TNFRSF9 (41BB), TNFSF14 (LIGHT, HVEM Ligand), TNFSF5
(CD40 Ligand), BTLA, and CRAC). In some embodiments, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) bind 2, 3, 4, 5 or more of these targets.
[0520] In additional embodiments an MRD-containing antibody binds
to a therapeutic target and a second target that is associated with
an escape pathway for resisting the therapeutic effect resulting
from targeting the therapeutic target. For example, in one
embodiment, an MRD-containing antibody binds to EGFR and a target
selected from MDR1, cMET, Notch, Notch1, Notch3, Notch4, DLL1,
DLL4, Jagged, Jagged1, Jagged2, and Jagged3. In some embodiments,
the multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibodies) binds 2, 3, 4, 5 or more of these
targets.
[0521] In specific embodiments, the MRD-containing antibody targets
ErbB2 and an angiogenic factor. In specific embodiments, the
MRD-containing antibody targets ErbB2 and IGR1R. In another
embodiment, the antibody targets ErbB2 and at least one MRD targets
an angiogenic factor and/or IGF1R. In one, embodiment, an antibody
that binds to the same ErbB2 epitope as trastuzumab is operably
linked to at least one MRD that targets an angiogenic factor and/or
IGF1R. In an additional embodiment, an antibody that competitively
inhibits trastuzumab binding is operably linked to at least one MRD
that targets an angiogenic factor and/or IGF1R. In additional
embodiments, an antibody that comprises the sequences of SEQ ID
NOS:59-64 is operably linked to at least one MRD that targets an
angiogenic factor and/or IGF1R. In additional embodiments, the
trastuzumab antibody is operably linked to at least one MRD that
targets an angiogenic factor and/or IGF1R.
[0522] In some embodiments, an antibody that binds to ErbB2 is
operably linked to an MRD that targets Ang2. In some embodiments,
the antibody that binds to ErbB2 is linked to an Ang2 binding MRD
that binds to, the same Ang2 epitope as an MRD comprising the
sequence of MGAQTNFMPMDNDELLLYEQFILQQGLE SEQ ID NO:8. In some
embodiments, the antibody that binds to ErbB2 is linked to an Ang2
binding MRD that competitively inhibits an MRD comprising the
sequence of SEQ ID NO:8. In some embodiments, the antibody that
binds to ErbB2 is linked to an MRD comprising the sequence of SEQ
ID NO:8.
[0523] In some embodiments, at least one Ang2 binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds to ErbB2. In some embodiments, at least one Ang2 binding
MRD is operably linked to the N-terminus of the heavy chain of an
antibody that binds to ErbB2. In some embodiments, at least one
Ang2 binding MRD is operably linked to the C-terminus of the light
chain of an antibody that binds to ErbB2. In some embodiments, at
least one Ang2 binding MRD is operably linked to the N-terminus of
the light chain of an antibody that binds to ErbB2.
[0524] In some embodiments, at least one Ang2 binding MRD is
operably linked directly to an antibody that binds to ErbB2. In
additional embodiments, at least one Ang2 binding MRD is operably
linked to an antibody that binds to ErbB2 via a linker.
[0525] In some embodiments, an antibody that binds to ErbB2 is
operably linked to an MRD that targets IGF1R. In some embodiments,
the antibody that binds to ErbB2 is linked to an IGF1R binding MRD
that binds to the same IGF1R epitope as an MRD comprising the
sequence of SEQ ID NO:14. In some embodiments, the antibody that
binds to ErbB2 is linked to an IGF1R binding MRD that competitively
inhibits an MRD comprising the sequence of SEQ ID NO:14. In some
embodiments, the antibody that binds to ErbB2 is linked to an MRD
comprising the sequence of SEQ ID NO:14. In some embodiments, the
antibody that binds ErbB2 is linked to an MRD encoding the sequence
SLFVPRPERK (SEQ ID NO:103). In some embodiments, the antibody that
binds ErbB2 is linked to an MRD encoding the sequence ESDVLHFTST
(SEQ ID NO:104). In some embodiments, the antibody that binds ErbB2
is linked to an MRD encoding the sequence LRKYADGTL (SEQ ID
NO:105).
[0526] In some embodiments, at least one IGF1R binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds to ErbB2. In some embodiments, at least one IGF1R
binding MRD is operably linked to the N-terminus of the heavy chain
of an antibody that binds to ErbB2. In some embodiments, at least
one IGF1R binding MRD is operably linked to the C-terminus of the
light chain of an antibody that binds to ErbB2. In some
embodiments, at least one IGF1R binding MRD is operably linked to
the N-terminus of the light chain of an antibody that binds to
ErbB2.
[0527] In some embodiments, at least one TGF binding MRD is
operably linked directly to an antibody that binds to ErbB2. In
additional embodiments, at least one IGF1R binding MRD is operably
linked to an antibody that binds to ErbB2 via a linker.
[0528] In some embodiments, an MRD-containing antibody targets
ErbB2 and HER2/3. In some embodiments, an MRD-containing antibody
can bind to ErbB2 and HER2/3 simultaneously. In some embodiments,
an antibody that binds to ErbB2 is operably linked to an MRD that
targets HER2/3. In additional embodiments, at least one
HER2/3-binding MRD is operably linked to the C-terminus of the
heavy chain of an antibody that binds to ErbB2. In further
embodiments, at least one HER2/3-binding MRD is operably linked to
the N-terminus of the heavy chain of an antibody that binds to
ErbB2. In additional embodiments, at least one HER2/3-binding MRD
is operably linked to the C-terminus of the light chain of an
antibody that binds to ErbB2. In additional embodiments, at least
one HER2/3-binding MRD is operably linked to the N-terminus of the
light chain of an antibody that binds to ErbB2.
[0529] In some embodiments, at least one HER2/3-binding MRD is
operably linked directly to an antibody that binds to ErbB2. In
additional embodiments, at least one HER2/3-binding MRD is operably
linked to an antibody that binds to ErbB2 via a linker.
[0530] In some embodiments, an MRD-containing antibody targets
ErbB2 and HER2/3. In some embodiments, an MRD-containing antibody
can bind to ErbB2 and HER2/3 simultaneously. In some embodiments,
an antibody that binds to HER2/3 is operably linked to an MRD that
targets ErbB2. In additional embodiments, at least one
ErbB2-binding MRD is operably linked to the C-terminus of the heavy
chain of an antibody that binds to HER2/3. In further embodiments,
at least one ErbB2-binding MRD is operably linked to the N-terminus
of the heavy chain of an antibody that binds to HER2/3. In
additional embodiments, at least one ErbB2-binding MRD is operably
linked to the C-terminus of the light chain of an antibody, that
binds to HER2/3. In additional embodiments, at least one
ErbB2-binding MRD is operably linked to the N-terminus of the light
chain of an antibody that binds to HER2/3.
[0531] In some embodiments, at least one ErbB2-binding MRD is
operably linked directly to an antibody that binds to HER2/3. In
additional embodiments, at least one ErbB2-binding MRD is operably
linked to an antibody that binds to HER2/3 via a linker.
[0532] In some embodiments, the MRD-containing antibody targets
ErbB2, Ang2, and IGF1R. In some embodiments, the MRD-containing
antibody comprises an antibody that targets ErbB2, an MRD that
targets Ang2, and an MRD that targets IGF1R. In some embodiments,
the Ang2 and IGF1R MRDs are attached to the same location on the
anti-ErbB2 antibody. In some embodiments, the Ang2 and IGF1R MRDs
are attached to different locations on the anti-ErbB2 antibody. In
some embodiments, the Ang2 and IGF1R MRDs are on the light chain of
the anti-ErbB2 antibody. In some embodiments, the Ang2 and IGF1R
MRDs are on the heavy chain of the anti-ErbB2 antibody. In some
embodiments, the Ang2 MRD is on the light chain of the ErbB2
antibody, and the IGF1R MRD is on the heavy chain of the anti-ErbB2
antibody. In some embodiments, the Ang2 MRD is on the heavy chain
of the ErbB2 antibody, and the IGF1R MRD is on the light chain of
the anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on
the N-terminus of the heavy chain of the ErbB2 antibody, and the
IGF1R MRD is on the C-terminus of the light chain of the anti-ErbB2
antibody. In some embodiments, the IGF1R MRD is on the N-terminus
of the heavy chain of the ErbB2 antibody, and the Ang2 MRD is on
the C-terminus of the light chain of the anti-ErbB2 antibody.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) comprising an antibody that targets Ang2, an MRD that
targets ErbB2, and an MRD that targets IGF1R; and multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
comprising an antibody that targets IGF1R, an MRD that targets
ErbB2, and an MRD that targets Ang2 are also encompassed by the
invention.
[0533] In some embodiments, the MRD-containing antibody targets
ErbB2, Ang2, and HER2/3. In some embodiments, the MRD-containing
antibody comprises an antibody that targets ErbB2, an MRD that
targets Ang2, and an MRD that targets HER2/3. In some embodiments,
the Ang2 and HER2/3 MRDs are attached to the same location on the
anti-ErbB2 antibody. In some embodiments, the Ang2 and HER2/3 MRDs
are attached to different locations on the anti-ErbB2 antibody. In
some embodiments, the Ang2 and HER2/3 MRDs are on the light chain
of the anti-ErbB2 antibody. In some embodiments, the Ang2 and
HER2/3 MRDs are on the heavy chain of the anti-ErbB2 antibody. In
some embodiments, the Ang2 MRD is on the light chain of the ErbB2
antibody, and the HER2/3 MRD is on the heavy chain of the
anti-ErbB2 antibody. In some embodiments, the Ang2 MRD is on the
heavy chain of the ErbB2 antibody, and the HER2/3 MRD is on the
light chain of the anti-ErbB2 antibody. In some embodiments, the
Ang2 MRD is on the N-terminus of the heavy chain of the ErbB2
antibody, and the HER2/3 MRD is on the C-terminus of the light
chain of the anti-ErbB2 antibody. In some embodiments, the HER2/3
MRD is on the N-terminus of the heavy chain of the ErbB2 antibody,
and the Ang2 MRD is on the C-terminus of the light chain of the
anti-ErbB2 antibody. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) comprising an antibody that
targets HER2/3, an MRD that targets ErbB2, and an MRD that targets
Ang2; and multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) comprising an antibody that targets
Ang2, an MRD that targets ErbB2, and an MRD that targets HER2/3 are
also encompassed by the invention.
[0534] In some embodiments, the MRD-containing antibody targets
ErbB2, HER2/3, and IGF1R. In some embodiments, the MRD-containing
antibody comprises an antibody that targets ErbB2, an MRD that
targets HER2/3, and an MRD that targets IGF1R. In some embodiments,
the HER2/3 and IGF1R MRDs are attached to the same location on the
anti-ErbB2 antibody. In some embodiments, the HER2/3 and IGF1R MRDs
are attached to different locations on the anti-ErbB2 antibody. In
some embodiments, the HER2/3 and IGF1R MRDs are, on the light chain
of the anti-ErbB2 antibody. In some embodiments, the HER2/3 and
IGF1R MRDs are on the heavy chain of the anti-ErbB2 antibody. In
some embodiments, the HER2/3 MRD is on the light chain of the ErbB2
antibody, and the IGF1R MRD is on the heavy chain of the anti-ErbB2
antibody. In some embodiments, the HER2/3 MRD is on the heavy chain
of the ErbB2 antibody, and the IGF1R MRD is on the light chain of
the anti-ErbB2 antibody. In some embodiments, the HER2/3 MRD is on
the N-terminus of the heavy chain of the ErbB2 antibody, and the
IGF1R MRD is on the C-terminus of the light chain of the anti-ErbB2
antibody. In some embodiments, the IGF1R MRD is on the N-terminus
of the heavy chain of the ErbB2 antibody, and the HER2/3 MRD is on
the C-terminus of the light chain of the anti-ErbB2 antibody.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) comprising an antibody that targets HER2/3, an MRD that
targets ErbB2, and an MRD that targets IGF1R; and multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
comprising an antibody that targets IGF1R, an MRD that targets
ErbB2, and an MRD that targets HER2/3 are also encompassed by the
invention.
[0535] In some embodiments, the MRD-containing antibody targets
ErbB2, Ang2, HER2/3, and IGF1R. In some embodiments, the
MRD-containing antibody comprises an antibody that targets ErbB2,
an MRD that targets Ang2, an MRD that targets HER2/3, and an MRD
that targets IGF1R. In some embodiments, the Ang2, HER2/3, and
IGF1R MRDs are attached to the same chain of the anti-ErbB2
antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are
attached to different chains of the anti-ErbB2 antibody. In some
embodiments, the Ang2, HER2/3, and IGF1R MRDs are on the light
chain of the anti-ErbB2 antibody. In some embodiments, the Ang2,
HER2/3, and IGF1R MRDs are on the heavy chain of the anti-ErbB2
antibody. In some embodiments, the Ang2, HER2/3, and IGF1R MRDs are
attached to the same terminus of the anti-ErbB2 antibody. In some
embodiments, the Ang2, HER2/3, and IGF1R MRDs are attached to
different termini of the anti-ErbB2 antibody. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
comprising: an antibody that targets HER2/3, an MRD that targets
ErbB2, an MRD that targets Ang2, and an MRD that targets IGF1R;
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) comprising an antibody that targets Ang2, an MRD that
targets ErbB2, an MRD that targets HER2/3, and an MRD that targets
IGF1R; and multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) comprising an antibody that targets
IGF1R, an MRD that targets ErbB2, an MRD that targets HER2/3, and
an MRD that targets Ang2 are also encompassed by the invention.
[0536] In some embodiments, the anti-ErbB2 antibody operably linked
to an Ang2 binding MRD binds to both ErbB2 and Ang2 simultaneously.
In some embodiments, the anti-ErbB2 antibody operably linked to an
IGF1R binding MRD binds to both ErbB2 and IGF1R simultaneously. In
some embodiments, the anti-ErbB2 antibody operably linked to a
HER2/3 binding MRD binds to both ErbB2 and HER2/3 simultaneously.
In some embodiments, the anti-ErbB2 antibody operably linked to an
Ang2 MRD, an IGF1R MRD, and/or a HER2/3 MRD binds to ErbB2, Ang2,
IGF1R, and/or HER2/3 simultaneously. In some embodiments, the
anti-ErbB2 antibody operably linked to an Ang2, IGF1R and/or HER2/3
binding MRD(s) exhibits ADCC activity. In additional embodiments,
the anti-ErbB2 antibody operably linked to an Ang2, IGF1R, and/or
HER2/3 binding MRD(s) down-regulates Akt signaling. In additional
embodiments, the anti-ErbB2 antibody operably linked to an Ang2
binding MRD inhibits Ang2 binding to TIE2. In additional
embodiments, the anti-ErbB2 antibody operably linked to an IGF1R
binding MRD(s) down-regulates IGF1R signaling. In additional
embodiments, the anti-ErbB2 antibody operably linked to an Ang2,
IGF1R and/or HER2/3 binding MRD(s) inhibits cell proliferation. In
additional embodiments, the anti-ErbB2 antibody operably linked to
an Ang2, IGF1R, and/or HER2/3 binding MRD(s) inhibits tumor
growth.
[0537] In specific embodiments, the MRD-containing antibody targets
VEGF and an angiogenic factor. In specific embodiments, the
MRD-containing antibody targets VEGF and IGF1R. In another
embodiment, the antibody targets VEGF and at least one MRD targets
an angiogenic factor and/or IGF1R. In one embodiment, an antibody
that binds to the same VEGF epitope as bevacizumab is operably
linked to at least one MRD that targets an angiogenic factor and/or
IGF1R. In an additional embodiment, an antibody that competitively
inhibits bevacizumab binding is operably linked to at least one MRD
that targets an angiogenic factor and/or IGF1R. In additional
embodiments, an antibody that comprises the sequences of SEQ ID
NOS:78-79 is operably linked to at least one MRD that targets an
angiogenic factor and/or IGF1R. In additional embodiments, the
bevacizumab antibody is operably linked to at least one MRD that
targets an angiogenic factor and/or IGF1R.
[0538] In some embodiments, an antibody that binds to VEGF is
operably linked to an MRD that targets Ang2. In some embodiments,
the antibody that binds to VEGF is linked to an Ang2 binding MRD
that binds to the same Ang2 epitope as an MRD comprising the
sequence of SEQ ID NO:8. In some embodiments, the antibody that
binds to VEGF is linked to an Ang2 binding MRD that competitively
inhibits an MRD comprising the sequence of SEQ ID NO:8. In some
embodiments, the antibody that binds to VEGF is linked to an MRD
comprising the sequence of SEQ ID NO:8.
[0539] In some embodiments, at least one Ang2 binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds to VEGF. In some embodiments, at least one Ang2 binding
MRD is operably linked to the N-terminus of the heavy chain of an
antibody that binds to VEGF. In some embodiments, at least one Ang2
binding MRD is operably linked to the C-terminus of the light chain
of an antibody that binds to VEGF. In some embodiments, at least
one Ang2 binding MRD is operably linked to the N-terminus of the
light chain of an antibody that binds to VEGF.
[0540] In some embodiments, at least one Ang2 binding MRD is
operably linked directly to an antibody that binds to VEGF. In
additional embodiments, at least one Ang2 binding MRD is operably
linked to an antibody that binds to VEGF via a linker.
[0541] In some embodiments, an antibody that binds to VEGF is
operably linked to an MRD that targets IGF1R. In some embodiments,
the antibody that binds to VEGF is linked to an IGF1R binding MRD
that binds to the same IGF1R epitope as an MRD comprising the
sequence of SEQ ID NO:14. In some embodiments, the antibody that
binds to VEGF is linked to an IGF1R binding MRD that competitively
inhibits an MRD comprising the sequence of SEQ ID NO:14. In some
embodiments, the antibody that binds to VEGF is linked to an MRD
comprising the sequence of SEQ ID NO:14. In some embodiments, the
antibody that binds ErbB2 is linked to an MRD encoding the sequence
SLFVPRPERK (SEQ ID NO:103). In some embodiments, the antibody that
binds ErbB2 is linked to an MRD encoding the sequence ESDVLHFTST
(SEQ ID NO:104). In some embodiments, the antibody that binds ErbB2
is linked to an MRD encoding the sequence LRKYADGTL (SEQ ID
NO:105).
[0542] In some embodiments, at least one IGF1R binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds to VEGF. In some embodiments, at least one IGF1R binding
MRD is operably linked to the N-terminus of the heavy chain of an
antibody that binds to VEGF. In some embodiments, at least one
IGF1R binding MRD is operably linked to the C-terminus of the light
chain of an antibody that binds to VEGF. In some embodiments, at
least one IGF1R binding MRD is operably linked to the N-terminus of
the light chain of an antibody that binds to VEGF.
[0543] In some embodiments, at least one IGF1R binding MRD is
operably linked directly to an antibody that binds to VEGF. In
additional embodiments, at least one IGF1R binding MRD is operably
linked to an antibody that binds to VEGF via a linker.
[0544] In some embodiments, the MRD-containing antibody targets
VEGF, Ang2, and IGF1R. In some embodiments, the MRD-containing
antibody comprises an antibody that targets VEGF, an MRD that
targets Ang2, and an MRD that targets IGF1R. In some embodiments,
the Ang2 and IGF1R MRDs are attached to the same location on the
anti-VEGF antibody. In some embodiments, the Ang2 and IGF1R MRDs
are attached to different locations on the anti-VEGF antibody. In
some embodiments, the Ang2 and IGF1R MRDs are on the light chain of
the anti-VEGF antibody. In some embodiments, the Ang2 and IGF1R
MRDs are on the heavy chain of the anti-VEGF antibody. In some
embodiments, the Ang2 MRD is on the light chain of the anti-VEGF
antibody, and the IGF1R MRD is on the heavy chain of the anti-VEGF
antibody. In some embodiments, the Ang2 MRD is on the heavy chain
of the anti-VEGF antibody, and the IGF1R MRD is on the light chain
of the anti-VEGF antibody. In some embodiments, the Ang2 MRD is on
the N-terminus of, the heavy chain of the anti-VEGF antibody, and
the IGF1R MRD is on the C-terminus of the light chain of the
anti-VEGF antibody. In some embodiments, the IGF1R MRD is on the
N-terminus of the heavy chain of the anti-VEGF antibody, and the
Ang2 MRD is on the C-terminus of the light chain of the anti-VEGF
antibody.
[0545] In some embodiments, the anti-VEGF antibody operably linked
to an Ang2 binding MRD binds to both anti-VEGF and Ang2
simultaneously. In some embodiments, the anti-VEGF antibody
operably linked to an IGF1R binding MRD binds to both anti-VEGF and
IGFR1 simultaneously. In some embodiments, the anti-VEGF antibody
operably linked to an Ang2 binding MRD and an IGF1R binding MRD
binds to VEGF, Ang2, and IGF1R simultaneously. In some embodiments,
the anti-VEGF antibody operably linked to an Ang2 and/or IGF
binding MRD(s) exhibits ADCC activity. In additional embodiments,
the anti-VEGF antibody operably linked to an Ang2 and/or IGF1R
binding MRD(s) down-regulates VEGF signaling. In additional
embodiments, the anti-VEGF antibody operably linked to an Ang2
binding MRD inhibits Ang2 binding to TIE2. In additional
embodiments, the anti-VEGF antibody operably linked to an IGF1R
binding MRD inhibits IGF1R signaling. In additional embodiments,
the anti-VEGF antibody operably linked to an Ang2 and/or IGF1R
binding MRD(s) inhibits cell proliferation. In additional
embodiments, the anti-VEGF antibody operably linked to an Ang2
and/or IGF1R binding MRD(s) inhibits tumor growth.
[0546] In some embodiments, the anti-ErbB2 antibody or the VEGF
antibody contains and MRD that inhibits the binding of pertuzumab
to ErbB2. In some embodiments, an anti-ErbB2 antibody contains at
least one MRD that binds to Ang2 or IGF1R and one MRD that inhibits
the binding of pertuzumab to ErbB2. In some embodiments, an
anti-VEGF antibody contains at least one MRD that binds to Ang2 or
IGF1R and one MRD that inhibits the binding of pertuzumab to ErbB2.
In some embodiments, an anti-ErbB2 antibody contains an MRD that
binds Ang2, an MRD that binds IGF1R, and an MRD that inhibits the
binding of pertuzumab to ErbB2. In some embodiments, an anti-VEGF
antibody contains an MRD that binds Ang2, an MRD that binds IGF1R,
and an MRD that inhibits the binding of pertuzumab to ErbB2.
[0547] In specific embodiments, the MRD-containing antibody targets
TNF and an angiogenic factor. In another embodiment, the antibody
targets TNF, and at least one MRD targets an angiogenic factor. In
one embodiment, an antibody that binds to the same TNF epitope as
adalimumab is operably linked to at least one MRD that targets an
angiogenic factor. In an additional embodiment, an antibody that
competitively inhibits adalimumab binding is operably linked to at
least one MRD that targets an angiogenic factor. In additional
embodiments, an antibody that comprises the sequences of SEQ ID
NOS:80-85 is operably linked to at least one MRD that targets an
angiogenic factor. In additional embodiments, the adalimumab
antibody is operably linked to at least one MRD that targets an
angiogenic factor. In one embodiment, an antibody that binds to the
same TNF epitope as golimumab is operably linked to at least one
MRD that targets an angiogenic factor. In an additional embodiment,
an antibody that competitively, inhibits golimumab binding is
operably linked to at least one MRD that targets an angiogenic
factor. In additional embodiments, the golimumab antibody is
operably linked to at least one MRD that targets an angiogenic
factor.
[0548] In some embodiments, an antibody that binds to TNF is
operably linked to an MRD that targets Ang2. In some embodiments,
the antibody that binds to TNF is linked to an Ang2 binding MRD
that binds to the same Ang2 epitope as an MRD comprising the
sequence of SEQ ID NO:8. In some embodiments, the antibody that
binds to TNF is linked to an Ang2 binding MRD that competitively
inhibits an MRD comprising the sequence of SEQ ID NO:8. In some
embodiments, the antibody that binds to TNF is linked to an MRD
comprising the sequence of SEQ ID NO:8.
[0549] In some embodiments, at least one Ang2 binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds to TNF. In some embodiments, at least one Ang2 binding
MRD is operably linked to the N-terminus of the heavy chain of an
antibody that binds to TNF. In some embodiments, at least one Ang2
binding MRD is operably linked to the C-terminus of the light chain
of an antibody that binds to TNF. In some embodiments, at least one
Ang2 binding MRD is operably linked to the N-terminus of the light
chain of an antibody that binds to TNF.
[0550] In some embodiments, at least one Ang2 binding MRD is
operably linked directly to an antibody that binds to TNF. In
additional embodiments, at least one Ang2 binding MRD is operably
linked to an antibody that binds to TNF via a linker.
[0551] In some embodiments, the anti-TNF antibody operably linked
to an Ang2 binding MRD binds to both TNF and Ang2 simultaneously.
In some embodiments, the anti-TNF antibody operably linked to an
Ang2 binding MRD exhibits ADCC activity. In additional embodiments,
the anti-TNF antibody operably linked to an Ang2 binding MRD
inhibits binding of TNF to the p55 and p75 cell surface TNF
receptors. In additional embodiments, the anti-TNF antibody
operably linked to an Ang2 binding MRD lyses surface TNF-expressing
cells in vitro in the presence of complement. In additional
embodiments, the anti-TNF antibody operably linked to an Ang2
binding MRD inhibits Ang2 binding to TIE2. In additional
embodiments, the anti-TNF antibody operably linked to an Ang2
binding MRD reduces the signs and symptoms of arthritis.
[0552] In some embodiments, the MRD-containing antibody targets TNF
and IL6. In some embodiments, the MRD-containing antibody is
capable of binding TNF and IL6 simultaneously. Thus, in some
embodiments, an antibody that binds to TNF is operably linked to an
MRD that targets IL6. In other embodiments, an antibody that binds
to IL6 is operably linked to an MRD that targets TNF.
[0553] In some embodiments, at least one IL6-binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds TNF. In some embodiments, at least one IL6-binding MRD
is operably linked to the N-terminus of the heavy chain of an
antibody that binds to TNF. In some embodiments, at least one
IL6-binding MRD is operably linked to the C-terminus of the light
chain of an antibody that binds to TNF. In some embodiments, at
least one IL6-binding MRD is operably linked to the N-terminus of
the light chain of an antibody that binds to TNF.
[0554] In some embodiments, at least one TNF-binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds IL6. In some embodiments, at least one TNF-binding MRD
is operably linked to the N-terminus of the heavy chain of an
antibody that binds to IL6. In some embodiments, at least one
TNF-binding MRD is operably linked to the C-terminus of the light
chain of an antibody that binds to IL6. In some embodiments, at
least one TNF-binding MRD is operably linked to the N-terminus of
the light chain of an antibody that binds to IL6.
[0555] In some embodiments, at least one IL6-binding MRD is
operably linked directly to an antibody that binds to TNF. In
additional embodiments, at least one IL6-binding MRD is operably
linked to an antibody that binds to TNF via a linker.
[0556] In some embodiments, at least one TNF-binding MRD is
operably linked directly to an antibody that binds to IL6. In
additional embodiments, at least one TNF-binding MRD is operably
linked to an antibody that binds to IL6 via a linker.
[0557] In some embodiments, the MRD-containing antibody targets TNF
and BLyS. In some embodiments, the MRD-containing antibody is
capable of binding TNF and BLyS simultaneously. In some
embodiments, an antibody that binds to TNF is operably linked to an
MRD that targets BLyS. In other embodiments, an antibody that binds
to BLyS is operably linked to an MRD that targets TNF.
[0558] In some embodiments, at least one BLyS-binding MRD is
operably linked to the C-terminus of the heavy chain of an antibody
that binds TNF. In some embodiments, at least one BLyS-binding MRD
is operably linked to the N-terminus of the heavy chain of an
antibody that binds to TNF. In some embodiments, at least one
BLyS-binding MRD is operably linked to the C-terminus of the light
chain of an antibody that binds to TNF. In some embodiments, at
least one BLyS-binding MRD is operably linked to the N-terminus of
the light chain of an antibody that binds to TNF.
[0559] In some embodiments at least one TNF-binding MRD is operably
linked to the C-terminus of the heavy chain of an antibody that
binds BLyS. In some embodiments, at least one TNF-binding MRD is
operably linked to the N-terminus of the heavy chain of an antibody
that binds to BLyS. In some embodiments, at least one TNF-binding
MRD is operably linked to the C-terminus of the light chain of an
antibody that binds to BLyS. In some embodiments, at least one
TNF-binding MRD is operably linked to the N-terminus of the light
chain of an antibody that binds to BLyS.
[0560] In some embodiments, at least one BLyS-binding MRD is
operably linked directly to an antibody that binds to TNF. In
additional embodiments, at least one BLyS-binding MRD is operably
linked to an antibody that binds to TNF via a linker.
[0561] In other embodiments, at least one TNF-binding MRD is
operably linked directly to an antibody that binds to BLyS. In
additional embodiments, at least one TNF-binding MRD is operably
linked to an antibody that binds to BLyS via a linker.
[0562] In some embodiments, the MRD-containing antibody targets
Ang2, TNF, and IL6. In some embodiments, the MRD-containing
antibody is capable of binding Ang2, TNF, and IL6 simultaneously.
In some embodiments, an antibody that binds to TNF is operably
linked to an MRD that targets Ang2 and an MRD that targets IL6. In
some embodiments, the Ang2 and IL6-binding MRDs are located on the
same antibody chain. In some embodiments, the Ang2 and IL6-binding
MRDs are located on the same antibody terminus. In some
embodiments, the Ang2 and IL6-binding MRDs are located on different
antibody chains. In some embodiments, the Ang2 and IL6-binding MRDs
are located on different antibody termini.
[0563] In some embodiments, an antibody that binds to Ang2 is
operably linked to an MRD that targets TNF and an MRD that targets
IL6. In some embodiments, the TNF and IL6-binding MRDs are located
on the same antibody chain. In some embodiments, the TNF and
IL6-binding MRDs are located on the same antibody terminus. In some
embodiments, the TNF and IL6-binding MRDs are located on different
antibody chains. In some embodiments, the TNF and IL6-binding MRDs
are located on different antibody termini.
[0564] In some embodiments, an antibody that binds to IL6 is
operably linked to an MRD that targets Ang2 and an MRD that targets
TNF. In some embodiments, the Ang2 and TNF-binding MRDs are located
on the same antibody chain. In some embodiments, the Ang2 and
TNF-binding MRDs are located on the same antibody terminus. In some
embodiments, the Ang2 and TNF-binding MRDs are located on different
antibody chains. In some embodiments, the Ang2 and TNF-binding MRDs
are located on different antibody termini.
[0565] In some embodiments, the MRD-containing antibody targets
Ang2, TNF, and BLyS. In some embodiments, the MRD-containing
antibody is capable of binding Ang2, TNF, and BLyS simultaneously.
In some embodiments, an antibody that binds to TNF is operably
linked to an MRD that targets Ang2 and an MRD that targets BLyS. In
other embodiments, an antibody that binds to BLyS is operably
linked to an MRD that targets TNF and an MRD that targets Ang2. In
other embodiments, an antibody that binds to Ang2 is operably
linked to an MRD that targets TNF and an MRD that targets BLyS. In
some embodiments, the Ang2, BLyS, and/or TNF-binding MRDs are
located on the same antibody chain. In some embodiments, Ang2,
BLyS, and/or TNF-binding MRDs are located on the same antibody
terminus. In some embodiments, the Ang2, BLyS, and/or TNF-binding
MRDs are located on different antibody chains. In some embodiments,
the Ang2, BLyS, and/or TNF-binding MRDs are located on different
antibody termini.
[0566] In some embodiments, the MRD-containing antibody targets
Ang2, TNF, IL6, and BLyS. In some embodiments, the MRD-containing
antibody is capable of binding Ang2, TNF, IL6 and BLyS
simultaneously. In some embodiments, an antibody that binds to TNF
is operably linked to an MRD that targets Ang2, an MRD that,
targets IL6, and an MRD that targets BLyS. In some embodiments, an
antibody that binds to Ang2 is operably linked to an MRD that
targets TNF, an MRD that targets IL6, and an MRD that targets BLyS.
In some embodiments, an antibody that binds to IL6 is operably
linked to an MRD that targets Ang2, an MRD that targets TNF, and an
MRD that targets BLyS. In some embodiments, an antibody that binds
to BLyS is operably linked to an MRD that targets Ang2, an MRD that
targets IL6, and an MRD that targets TNF. In some embodiments, the
TNF, Ang2, IL6, and/or BLyS-binding MRDs are located on the same
antibody chain. In some embodiments, the TNF, Ang2, IL6 and/or
BLyS-binding MRDs are located on the same antibody terminus. In
some embodiments, the TNF, Ang2, IL6, and/or BLyS-binding MRDs are
located on different antibody chains. In some embodiments, the TNF,
Ang2, IL6 and/or BLyS-binding MRDs are located on different
antibody termini.
VI. METHODS OF MAKING ANTIBODY-MRD FUSIONS
[0567] The multivalent and multispecific compositions of the
invention (e.g., MRD-containing antibodies) and MRDs can be
produced by any method known in the art for the synthesis of
antibodies, polypeptides, immunoconjugates, and cytotoxins, in
particular, by chemical synthesis or by recombinant expression
techniques. An advantage of multivalent and multispecific
compositions (e.g., MRD-containing antibodies) is that they can be
produced using protocols that are known in the art for producing
antibodies. The antibody-MRD fusion molecules can be encoded by a
polynucleotide comprising a nucleotide sequence. Thus, the
polynucleotides described herein can encode an MRD, an antibody
heavy chain, an antibody light chain, a fusion protein comprising
an antibody heavy chain and at least one MRD, and/or a fusion
protein comprising an antibody light chain and at least one
MRD.
[0568] Accordingly, the invention provides vector constructs
comprising a polynucleotide sequence(s) encoding multivalent and
multispecific compositions (e.g., MRD-containing antibodies) and a
host cell comprising these rector constructs. Standard techniques
for cloning and transformation may be used in the preparation of
cell lines expressing the multivalent and multispecific
compositions (e.g. MRD-containing antibodies) of the invention.
[0569] Recombinant expression vectors containing a polynucleotide
sequence(s) encoding multivalent and multispecific compositions
(e.g., MRD-containing antibodies) of the invention can be prepared
using well known techniques. The expression vectors include a
polynucleotide coding sequence operably linked to suitable
transcriptional or translational regulatory nucleotide sequences
such as, those derived from mammalian, microbial, viral, or insect
genes. Exemplary regulatory sequences present in the expression
vector constructs include transcriptional promoters, operators,
enhancers, mRNA ribosomal binding sites, and/or other appropriate
sequences which control transcription and translation initiation
and termination. Nucleotide sequences are "operably linked" when
the regulatory sequence functionally relates to the nucleotide
sequence for the appropriate polypeptide. Thus, a promoter sequence
is operably linked to, for example, an antibody heavy chain-MRD
sequence if the promoter nucleotide sequence controls the
transcription of the appropriate nucleotide sequence.
[0570] The polynucleotide coding sequence in the expression vector
can include additional heterologous sequences encoding polypeptides
such as, signal peptides that are not naturally associated with
antibody heavy and/or light chain sequences. For example, a
nucleotide sequence for a signal peptide (secretory leader) can be
fused in-frame to the polypeptide sequence so that the
MRD-containing antibody is secreted to the periplasmic space or
into the medium. A signal peptide that is functional in the
intended host cells enhances extracellular secretion of the
appropriate antibody. The signal peptide can be cleaved from the
polypeptide upon secretion of antibody from the cell. Examples of
sequences encoding secretory signals that can be included in the
expression vectors include those described in for example, U.S.
Pat. Nos. 5,698,435, 5,698,417, and 6,204,023.
[0571] A variety of host-expression vector systems can be utilized
to express the coding sequence an MRD-containing antibody.
[0572] Host cells useful in the present invention include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing, antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., Baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing MRD-containing
antibody coding sequences. In particular embodiments, the mammalian
cell systems are used to produce the multivalent and multispecific
compositions of the invention (e.g., MRD-containing antibodies).
Mammalian cell systems typically utilize recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter). Examples of mammalian host cells useful for
producing the multivalent and multispecific compositions of the
invention include, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO
myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells,
COS cells, 293 cells, 3T3 cells and hybridoma cells.
[0573] Vectors containing the polynucleotides encoding the
multivalent and multispecific compositions of the invention (e.g.,
MRD containing antibodies) or portions or fragments thereof,
include plasmid vectors, a single and double-stranded phage
vectors, as well as single and double-stranded RNA or DNA viral
vectors. The vectors can be routinely introduced into host cells
using known techniques for introducing DNA and RNA into cells.
Phage and viral, vectors may also be introduced into host cells in
the form of packaged or encapsulated virus using known techniques
for infection and transduction. Moreover, viral vectors may be
replication competent or alternatively, replication defective.
Alternatively, cell-free translation systems may also be used to
produce the protein using RNAs derived from the DNA expression
constructs of the invention (see, e.g., Intl. Appl. Publ.
WO86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464).
[0574] Also provided herein, are methods of producing an
MRD-containing antibody, the method comprising: culturing a host
cell comprising one or more polynucleotides or an expression vector
comprising one or more isolated polynucleotides in a medium under
conditions allowing the expression of said one or more
polynucleotide, wherein said one or more polynucleotides encodes
one or more polypeptides that form part of MRD-containing antibody;
and recovering said MRD-containing antibody.
[0575] Prokaryotes useful as host cells in producing the
compositions of the invention (e.g., MRDs) include gram negative or
gram positive organisms such as, E. coli and B. subtilis.
Expression vectors for use in prokaryotic host cells generally
contain one or more phenotypic selectable marker genes (e.g., genes
encoding proteins that confer antibiotic resistance or that supply
an autotrophic requirement). Examples of useful prokaryotic host
expression vectors include the pKK223-3 (Pharmacia, Uppsala,
Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA) and
pRSET (Invitrogen, Calif., USA) series of vectors (see, e.g.,
Studier, J. Mol. Biol, 219:37 (1991) and Schoepfer, Gene 124:83
(1993)). Exemplary promoter sequences frequently used in
prokaryotic host cell expression vectors include T7, (Rosenberg et
al., Gene 56: 125-135 (1987)), beta-lactamase (penicillinase),
lactose promoter system (Chang et al., Nature 275:615 (1978)); and
Goeddel et al., Nature 281:544 (1979)), tryptophan (tip) promoter
system (Goeddel et al., Nucl. Acids Res. 8:4057, (1980)), and tac
promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0576] In alternative embodiments, eukaryotic host cell systems can
be used, including yeast cells transformed with recombinant yeast
expression vectors containing the coding sequence of an
MRD-containing antibody of the present invention, such as, the
expression systems taught in U.S. Pat. Appl. No. 60/344,169 and
WO03/056914 (methods for producing human-like glycoprotein in a
non-human eukaryotic host cell) (the contents of each of which are
incorporated by reference in their entirety). Exemplary yeast that
can be used to produce compositions of the invention, such as,
MRDs, include yeast from the genus Saccharotnyces, Pichia,
Actinomycetes and Kluyveromyces. Yeast vectors typically contain an
origin of replication sequence from a 2 mu yeast plasmid, an
autonomously replicating sequence (ARS), a promoter region,
sequences for polyadenylation, sequences for transcription
termination, and a selectable marker gene. Examples of promoter
sequences in yeast expression constructs include promoters from
metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.
Biol. Chem. 255:2073, (1980)) and other glycolytic enzymes, such
as, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. Additional suitable vectors and promoters for use in
yeast expression as well as yeast transformation protocols are
known in the art. See, e.g., Fleer et al., Gene, 107:285-195 (1991)
and Hinnen et al., Proc. Natl. Acad. Sci., 75:1929 (1978).
[0577] Insect and plant host cell culture systems are also useful
for producing the compositions of the invention. Such host cell
systems include for example, insect cell systems infected with
recombinant virus expression vectors (e.g., baculovirus) containing
the coding sequence of an MRD-containing antibody; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing the coding sequence of an MRD-containing
antibody, including, but not limited to, the expression systems
taught in U.S. Pat. No. 6,815,184, WO2004/057002, WO2004/024927,
U.S. Pat. Appl. Nos. 60/365,769, 60/368,047, and WO2003/078614, the
contents of each of which is herein incorporated by reference in
its entirety.
[0578] In alternate embodiments, other eukaryotic host cell systems
may be used, including animal cell systems infected with
recombinant virus expression vectors (e.g., adenovirus, vaccinia
virus) including cell lines engineered to contain multiple copies
of the DNA encoding an MRD-containing antibody either stably
amplified (CHO/dhfr) or unstably amplified in double-minute
chromosomes (e.g., murine cell lines). In one embodiment, the
vector comprising the polynucleotide(s) encoding the MRD-containing
antibody of the invention is polycistronic.
[0579] Exemplary mammalian cells useful for producing these
compositions include 293 cells (e.g., 293T and 293F), CHO cells,
BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse
myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells or
hybridoma cells, other mammalian cells. Additional exemplary
mammalian host cells that are useful in practicing the invention
include but are not limited, to VERY, Hela, COS, MDCK, 3T3, W138,
BT483, Hs578T, HTB2, BT20 and T47D, CRL7O3O and HsS78Bst cells.
Some examples of expression systems and selection methods are
described in the following references and references cited therein:
Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000-2001), in
Werner et al., Arzneimittelforschung/Drug Res. 48(8):870-80 (1998),
in Andersen and Krummen, Curr. Op. Biotechnol. 13:117-123 (2002),
in Chadd and Chamow, Curr. Op. Biotechnol. 12:188-194 (2001), and
in Giddings, Curr. Op. Biotechnol. 12: 450-454 (2001). Additional
examples of expression systems and selection methods are described
in Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359
(1984), Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
Transcriptional and translational control sequences for mammalian
host cell expression vectors are frequently derived from viral
genomes. Commonly used promoter sequences and enhancer sequences in
mammalian expression vectors include, sequences derived from
Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human
cytomegalovirus (CMV). Exemplary commercially available expression
vectors for use in mammalian host cells include pCEP4
(Invitrogen.RTM.) and pcDNA3 (Invitrogen.RTM.).
[0580] A number of selection systems can be used in, mammalian
host-vector expression systems, including, but not limited to, the
herpes simplex virus thymidine kinase, hypoxanthine-guanine
phosphoribosyltransferase and adenine phosphoribosyltransferase
(Lowy et al., Cell 22:817 (1980)) genes, which can be employed in
tk, hgprt.sup.- or aprt.sup.- cells, respectively. Additionally,
antimetabolite resistance can be used as the basis of selection for
e.g., dhfr, gpt, neo, hygro, trpB, hisD, ODC (ornithine
decarboxylase), and the glutamine synthase system.
[0581] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the coding
sequence of an MRD-containing antibody along with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Maniatis et al., MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989) and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene
Publishing Associates and Wiley Interscience, N.Y. (1989).
[0582] A variety of host-expression vector systems may be utilized
to express the coding sequence an MRD-containing antibody. A host
cell strain can be chosen which modulates the expression of
inserted antibody sequences, or modifies and processes the antibody
gene product in the specific fashion desired. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of protein
products can be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
antibody or portion thereof expressed. To this end, eukaryotic host
cells which possess the cellular machinery for proper processing of
the primary transcript, glycosylation, and phosphorylation of the
gene product may be used.
[0583] Stable expression typically achieves more reproducible
results than transient expression and also is more amenable to
large-scale production; however, it is within the skill of one in
the art to determine whether transient expression is better for a
particular situation. Rather than using expression vectors which
contain viral origins of replication, host cells can be transformed
with the respective coding nucleic acids controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows selection of cells which have stably
integrated the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines.
[0584] In some embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are expressed at
levels (titers) comparable to those of antibodies. In some
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are expressed at least about 10 .mu.g/ml
at least about 20 .mu.g/ml, or at least about 30 .mu.g/ml. In some
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are expressed at least about 40 .mu.g/ml
or at least about 50 .mu.g/ml. In some embodiments, the multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
are expressed at least about 60 .mu.g/ml, at least about 70
.mu.g/ml, at least about 80 .mu.g/ml, at least about 90 .mu.g/ml,
at least about 95 .mu.g/ml, at least about 100 .mu.g/ml, at least
about 110 .mu.g/ml, at least about 120 .mu.g/ml, at least about 130
.mu.g/ml, at least about 140 .mu.g/ml, at, least about 150
.mu.g/ml, at least about 160 .mu.g/ml, at least about 170 .mu.g/ml,
at least about 180 .mu.g/ml, at least about 190 .mu.g/ml, or at
least about 200 .mu.g/ml. The expression levels of an antibody
molecule can be increased by vector amplification and the use
recombinant methods and tools known in the art, including chromatin
remodeling strategies to, enhance transgene, expression.
[0585] The present invention is further directed to a method for
modifying the glycosylation profile of an MRD-containing antibody
that is produced by a host cell, comprising expressing in said host
cell a nucleic acid encoding an MRD-containing antibody and a
nucleic acid encoding a polypeptide with a glycosyltransferase
activity, or a vector comprising such nucleic acids. Genes with
glycosyltransferase activity include
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTII),
.alpha.-mannosidase II (ManII), .beta.(1,4)-galactosyltransferase
(GalT), .beta.(1,2)-N-acetylglucos aminyltransferase I (GnTI), and
.beta.(1,2)-N-acetylglucosaminyltransferase II (GnTII). In one
embodiment, a combination of genes with glycosyltransferase
activity are expressed in the host cell (e.g., GnTIII and Man II).
Likewise, the method also encompasses expression of one or more
polynucleotide(s) encoding the MRD-containing antibody in a host
cell in which a glycosyltransferase gene has been disrupted or
otherwise deactivated (e.g., a host cell in which the activity of
the gene encoding .alpha.1-6 core fucosyltransferase has been
knocked out). In another embodiment, the MRD-containing antibody
can be produced in a host cell that further expresses a
polynucleotide encoding a polypeptide having GnTIII activity to
modify the glycosylation pattern. In a specific embodiment, the
polypeptide having GnTIII activity is a fusion polypeptide
comprising the Golgi localization domain of a Golgi resident
polypeptide. In another embodiment, the expression of the
MRD-containing antibody in a host cell that expresses a
polynucleotide encoding a polypeptide having GnTIII activity
results in an MRD-containing antibody with increased Fc receptor
binding affinity and increased effector function. Accordingly, in
one embodiment, the present invention is directed to a host cell
comprising (a) an isolated nucleic acid comprising a sequence
encoding a polypeptide having GnTIII activity; and (b) an isolated
polynucleotide encoding an MRD-containing antibody of the present
invention, such as, a chimeric, primatized or humanized antibody.
In another embodiment, the polypeptide having GnTIII activity is a
fusion polypeptide comprising the catalytic domain of GnTIII and
the Golgi localization domain is the localization domain of
mannosidase II. Methods for generating such fusion polypeptides and
using them to produce antibodies with increased effector functions
are disclosed in U.S. Provisional Pat. Appl. No. 60/495,142 and
U.S. Pat. Appl. Publ. No. 2004/0241817, each of which is herein
incorporated by reference.
[0586] The multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) with altered glycosylation produced by
the host cells of the invention typically exhibit increased Fc
receptor binding, affinity and/or increased effector function as a
result of the modification of the host cell (e.g., by expression of
a glycosyltransferase gene). The increased Fc receptor binding
affinity can be increased binding to an Fey activating receptor,
such as, the Fc.gamma.RIIIa receptor. The increased effector
function can be an increase in one or more of the following:
increased antibody-dependent cellular cytotoxicity, increased
antibody-dependent cellular phagocytosis (ADCP), increased cytokine
secretion, increased immune-complex-mediated antigen uptake by
antigen-presenting cells, increased Fc-mediated cellular
cytotoxicity, increased binding to NK cells, increased binding to
macrophages, increased binding to polymorphonuclear cells (PMNs),
increased binding to monocytes, increased crosslinking of
target-bound antibodies, increased direct signaling inducing
apoptosis, increased dendritic cell maturation, and increased T
cell, priming.
[0587] Once a multivalent and monovalent multispecific composition
(e.g., MRD-containing antibody) of the invention has been produced
by recombinant expression, it can be purified by any method known
in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. In additional embodiments, the multivalent and
multispecific compositions of the present invention or fragments
thereof are optionally fused to heterologous polypeptide sequences
described herein or otherwise known in the art to facilitate
purification. In additional embodiments, the multivalent and
multispecific compositions or fragments thereof are optionally
fused to heterologous polypeptide sequences described herein or
otherwise known in the art to facilitate purification. More
particularly, it is envisioned that ligands (e.g., antibodies and
other affinity matrices) for MRDs or other components of the
multivalent and multispecific compositions can be used in affinity
columns for affinity purification and that optionally, the MRDs or
other components of the multivalent and monovalent multispecific
composition that are bound by these ligands are removed from the
composition prior to final preparation of the multivalent and
multispecific compositions using techniques known in the art.
VII. USES OF ANTIBODY-MRD FUSIONS
[0588] The multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) described herein are useful in a variety
of applications including, but not limited to, therapeutic
treatment methods, such as, the treatment of cancer. In certain
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are useful for inhibiting tumor growth,
reducing neovascularization, reducing angiogenesis, inducing
differentiation, reducing tumor volume, and/or reducing the
tumorigenicity of a tumor. The methods of use may be in vitro, ex
vivo, or in vivo methods. Cancer therapies and their dosages,
routes of administration and recommended usage are known in the art
and have been described in such literature as the
[0589] Physician's Desk Reference (PDR). The PDR discloses dosages
of the agents that have been used in treatment of various cancers.
The dosing regimen and dosages of these aforementioned
chemotherapeutic drugs that are therapeutically effective will
depend on the particular cancer being treated, the extent of the
disease and other factors familiar to the physician of skill in the
art and can be determined by the physician. The contents of the PDR
are expressly incorporated herein in its entirety by reference. The
2006 edition of the Physician's Desk Reference (PDR) discloses the
mechanism of action and preferred doses of treatment and dosing
schedules for thalidomide (p 979-983), VELCADE.RTM. (p 2102-2106)
and melphalan (p 976-979).
[0590] The multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are formulated, dosed, and administered
in a fashion consistent with good medical practice. Factors for
consideration in this context include the particular disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the agent, the method of administration, the
scheduling of administration, and other factors known to medical
practitioners. The dosage ranges for the administration of the
multivalent and multispecific compositions of the invention are
those large enough to produce the desired effect in which the
disease symptoms mediated by the target molecule are ameliorated.
The dosage should not be so large as to cause adverse side effects,
such as, hyperviscosity syndromes, pulmonary edema, congestive
heart failure, and the like. Generally, the dosage will vary with
the age, condition, sex and extent of the disease in the patient
and can be determined by one of skill in the art. The dosage can be
adjusted by the individual physician in the event of any
complication.
[0591] The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein is well
understood in the art. Typically such compositions are prepared as
sterile injectables either as liquid solutions or suspensions,
aqueous or nonaqueous. However, solid forms suitable for solution,
or suspensions, in liquid prior to use can also be prepared. The
preparation can also be emulsified. Thus, an antibody-MRD
containing composition can take the form of solutions, suspensions,
tablets, capsules, sustained release formulations or powders, or
other compositional forms.
[0592] In some embodiments, the compositions of the invention
(e.g., multivalent and multispecific compositions (e.g.,
MRD-containing antibodies)) are formulated to ensure or optimize
distribution in vivo. For example, the blood-brain barrier (BBB)
excludes many highly hydrophilic compounds and if so desired, the
compositions are prepared so as to increase transfer across the
BBB, by for example, formulation in liposomes. For methods of
manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;
5,374,548; and 5,399,331. The liposomes may comprise one or more
moieties which are selectively transported into specific cells or
organs, thus enhance targeted drug delivery (see, e.g., Ranade
Clin. Pharmacol. 29:685 (1989)).
[0593] The active ingredient can be mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient and in amounts suitable for use in the therapeutic
methods described herein. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol or the like and
combinations thereof. Physiologically tolerable carriers are well
known in the art. Likewise, composition of the present invention
can include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
tartaric, mandelic and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases for
example, sodium, potassium, ammonium, calcium or ferric hydroxides,
and such organic bases as isopropylamine, trimethylamine,
2-ethylamino ethanol, histidine, procaine and the like
[0594] Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as,
cottonseed oil, organic esters such as, ethyl oleate, and water-oil
emulsions.
[0595] In one embodiment, a therapeutic composition contains a
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) of the present invention, typically in an
amount of at least 0.1 weight percent of MRD-containing antibody
fusion per weight of total therapeutic composition. A weight
percent is a ratio by weight of MRD-containing antibody per total
composition. Thus, for example, 0.1 weight percent is 0.1 grams of
MRD-containing antibody per 100 grams of total composition.
[0596] The MRD-containing antibody are formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners.
[0597] The dosage schedule and amounts effective for therapeutic
and prophylactic uses, i.e., the "dosing regimen", will depend upon
a variety of factors, including the cause, stage and severity of
the disease or disorder, the health, physical status, age of the
mammal being treated, and the site and mode of the delivery of the
MRD-containing antibody. Therapeutic efficacy and toxicity of the
complex and formation can be determined by standard pharmaceutical,
pharmacological, and toxicological procedures in cell cultures or
experimental animals. Data obtained from these procedures can
likewise be used in formulating a range of dosages for human use.
Moreover, therapeutic index (i.e., the dose therapeutically
effective in 50 percent of the population divided by the dose
lethal to 50 percent of the population (ED.sub.50/LD.sub.50)) can
readily be determined using known procedures. The dosage is
preferably within a range of concentrations that includes the
ED.sub.50 with little or no toxicity, and may vary within this
range depending on the dosage form employed, sensitivity of the
patient, and the route of administration.
[0598] The dosage regimen also takes into consideration
pharmacokinetics parameters known in the art, such as, drug
absorption rate, bioavailability, metabolism and clearance (see,
e.g., Hidalgo-Aragones, J. Steloid Biochem. Mol. Biol. 58:611-617
(1996); Groning et al., Pharmazie 51:337-341 (1996); Fotherby
Contraception 54:59-69 (1996); and Johnson et al., J. Pharm. Sci.
84:1144-1146 (1995)). It is well within the state of the art for
the clinician to determine the dosage regimen for each subject
being treated. Moreover, single or multiple administrations of a
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) containing compositions can be
administered depending on the dosage and frequency as required and
tolerated by the subject. The duration of prophylactic and
therapeutic treatment will vary depending on the particular disease
or condition being treated. Some diseases are amenable to acute
treatment whereas others require long-term, chronic therapy. When
treating with an additional therapeutic agent, MRD-containing
antibody) can be administered serially, or simultaneously with the
additional therapeutic agent.
[0599] Therapeutically effective amounts of MRD-containing antibody
of the invention vary according to, for example, the targets of the
MRD-containing antibody and the potency of conjugated cytotoxic
agents encompassed by various embodiments of the invention Thus,
for example therapeutically effective dose of an a multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) that "mops up" a soluble ligand, such as, TNF alpha, is
expected to be higher than that for an a multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) that
redirects T cell effector function to a target on a hematological
malignancy. Likewise, therapeutically effective amounts of a
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) comprising a maytansinoid cytotoxic agent are likely to
be lower than the dosage of an a multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody)
comprising a less potent chemotherapeutic, such as, taxol, or the
counterpart a multivalent and monovalent multispecific composition
does not contain a cytotoxic agent.
[0600] According to one embodiment, a therapeutically effective
dose of an a multivalent and monovalent multispecific composition
(e.g., MRD-containing antibody) is an amount selected from about
0.00001 mg/kg to about 20 mg/kg, from about 0.00001 mg/kg to about
10 mg/kg, from about 0.00001 mg/kg to about 5 mg/kg, from about
0.0001 mg/kg to about 20 mg/kg, from about 0.0001 mg/kg to about 10
mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about 0.001
mg/kg to about 20 mg/kg, from about 0.001 mg/kg to about 10 mg/kg,
and from about 0.001 mg/kg to about 5 mg/kg of the patient's body
weight, in one or more dose administrations daily, for one or
several days.
[0601] According to another embodiment, a therapeutically effective
amount of an a multivalent and monovalent multispecific composition
(e.g., MRD-containing antibody) is an amount such that when
administered in a physiologically tolerable composition is
sufficient to achieve a plasma concentration of from about 0.1
microgram (.mu.g) per milliliter (ml) to about 100 .mu.g/ml, from
about 1 .mu.g/ml to about 5 .mu.g/ml, and usually about 5 .mu.g/ml.
Stated differently, in another embodiment, the dosage can vary from
about 0.1 mg/kg to about 300 mg/kg, from about 0.2 mg/kg to about
200 mg/kg, from about 0.5 mg/kg to about 20 mg/kg, in one or more
dose administrations daily, for one or several days.
[0602] In some embodiments, the a multivalent and monovalent
multispecific composition (e.g., MRD-containing antibody) is
administered at about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to
about 25 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to
about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg
to about 5 mg/kg.
[0603] In additional embodiments, the interval between dose
administration of the multivalent and monovalent multispecific
composition (e.g., an MRD-containing antibody) is about daily,
about twice a week, about every week, about every other week, or
about every three weeks. In some embodiments, the multivalent and
monovalent multispecific composition is administered first at a
higher loading dose and subsequently at a lower maintenance
dose.
[0604] In further embodiments, therapeutic composition comprise
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) in an amount of at least 0.1 weight percent of antibody
per weight of total therapeutic composition. A weight percent is a
ratio by weight of antibody/total composition. Thus, for example,
0.1 weight percent is 0.1 grams of antibody-MRD per 100 grams of
total composition. According to some embodiments, a therapeutic
composition comprising a multivalent and monovalent multispecific
composition contains about 10 micrograms (.mu.g) per milliliter
(ml) to about 100 milligrams (mg) per ml of antibody as active
ingredient per volume of composition. In additional embodiments, a
therapeutic composition comprising a multivalent and monovalent
multispecific composition contains about 1 mg/ml to about 10 mg/ml
(i.e., about 0.1 to 1 weight percent) of antibody as active
ingredient per volume of composition.
[0605] As shown in the examples herein, a multivalent and
multispecific composition (e.g., an MRD containing antibody) can
have a similar PK profile to a corresponding antibody. Thus, in
some embodiments, an antibody-MRD is administered in a dosing
concentration and regimen that is the same as the antibody
component of the antibody-MRD molecule alone (e.g., a commercial
antibody, or a so-called "biosimilar" or a "biobetter" thereof).
Likewise, the multivalent and multispecific composition can have a
different PK profile from a corresponding antibody. For example, in
embodiments where the multivalent and multispecific compositions
redirect a T cell response and/or include a cytotoxic agent, the
dosing concentration is expected to be less than that of the
corresponding antibody. In these instances, therapeutically
effective dosing concentrations and regimens for these compositions
can routinely be determined using factors and criteria known in the
art.
[0606] The multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) need not be, but optionally are,
formulated with one or more agents currently used to prevent or
treat the disorder in question. The effective amount of such other
agents depends on the amount of multivalent and monovalent
multispecific composition present in the formulation, the type of
disorder or treatment, and other factors discussed above.
[0607] As discussed above, the appropriate dosage of the
multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
previous therapy, the patient's clinical history, and the
discretion of the attending physician. The multivalent and
monovalent multispecific composition is suitably administered to
the patient at one time or over a series of treatments. Preferably,
the multivalent and monovalent multispecific composition is
administered by intravenous infusion or by subcutaneous injections.
According to some embodiments, the multivalent and monovalent
multispecific composition is administered parenterally by injection
or by gradual infusion over time. Although the target molecule can
typically be accessed in the body by systemic administration and
therefore most often treated by intravenous administration of
therapeutic compositions, other tissues and delivery means are
contemplated where there is likelihood that the tissue targeted
contains the target molecule. Thus, the multivalent and monovalent
multispecific composition can be administered intravenously,
intraperitoneally, intramuscularly, subcutaneously, intracavity,
transdermally, and can be delivered by peristaltic means.
Multivalent and multispecific compositions can also be delivered by
aerosol to airways and lungs. In some embodiments, the antibody-MRD
molecule is administered by intravenous infusion. In some
embodiments, the antibody-MRD molecule is administered by
subcutaneous injection.
[0608] The therapeutic compositions containing a multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) can conventionally be administered intravenously, as by
injection of a unit dose, for example. The term "unit dose" when
used in reference to a therapeutic composition of the present
invention refers to physically discrete units suitable as unitary
dosage for the patient, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect in association with the required diluent; i.e.,
carrier, or vehicle. In a specific embodiment, the therapeutic
compositions containing a human monoclonal antibody or a
polypeptide are administered subcutaneously.
[0609] The compositions of the invention are administered in a
manner compatible with the dosage formulation, and in a
therapeutically effective amount. The quantity to be administered
depends on the patient to be treated, capacity of the patient's
system to utilize the active ingredient, and degree of therapeutic
effect desired. Precise amounts of active ingredient required to be
administered depend on the judgment of the practitioner and are
peculiar to each individual. However, suitable dosage ranges for
systemic application are disclosed herein and depend on the route
of administration. Suitable regimes for administration are also
variable but are typified by an initial administration followed by
repeated doses at one or more hour intervals by a subsequent
injection or other administration. Alternatively, continuous
intravenous infusion sufficient to maintain concentrations in the
blood in the ranges specified for in vivo therapies are
contemplated.
[0610] In other embodiments, the invention provides a method for
treating or preventing a disease, disorder, or injury comprising
administering a therapeutically effective amount or
prophylactically effective amount of antibody-MRD molecule to a
patient in need thereof. In some embodiments, the disease, disorder
or injury is cancer. In other embodiments, the disease, disorder or
injury is a disease or disorder of the immune system, such as,
inflammation or an autoimmune disease.
[0611] Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are expected to have at least the same
therapeutic efficacy as the antibody contained in the MRD antibody
containing antibody when administered alone. Accordingly, it is
envisioned that the multivalent and multispecific compositions
(e.g., MRD-containing antibodies) can be administered to a patient
to treat or prevent a disease, disorder, or injury for which the
antibody contained in the MRD-containing antibody, or an antibody
that functions in the same way as the antibody contained in the
MRD-containing antibody, demonstrates a reasonably correlated
beneficial activity in treating or preventing such disease,
disorder or injury. This beneficial activity can be demonstrated in
vitro, in an in vivo animal model, or in human clinical trials. In
one embodiment, an MRD-containing antibody is administered to a
patient to treat or prevent a disease, disorder or injury for which
the antibody component of the MRD-containing antibody, or an
antibody that functions in the same way as the antibody contained
in the MRD-containing antibody, demonstrates therapeutic or
prophylactic efficacy in vitro or in an animal model. In another
embodiment, an MRD-containing antibody is administered to a patient
to treat or prevent a disease, disorder or injury for which the
antibody component of the MRD-containing antibody, or an antibody
that functions in the same way as the antibody contained in the
MRD-containing antibody, demonstrates therapeutic or prophylactic
efficacy in humans. In another embodiment, an MRD-containing
antibody is administered to a patient to treat or prevent a
disease, disorder or injury for which the antibody component of the
MRD-containing antibody, or an antibody that functions in the same
way as the antibody contained in the MRD-containing antibody, has
been approved by a regulatory authority for use in such treatment
or prevention.
[0612] In another embodiment, an MRD-containing antibody is
administered in combination with another therapeutic to treat or
prevent a disease, disorder or injury for which the antibody
component of the MRD-containing antibody, or an antibody that
functions in the same way as the antibody contained in the MRD
antibody, in combination with the therapeutic, or a different
therapeutic that functions in the same way as the therapeutic in
the combination, demonstrates therapeutic or prophylactic efficacy
in vitro or in an animal model. In another embodiment, an
MRD-containing antibody is administered in combination with another
therapeutic to treat or prevent a disease, disorder or injury for
which the antibody component of the MRD-containing antibody, or an
antibody that functions in the same way as the antibody contained
in the MRD antibody, in combination with the therapeutic, or a
different therapeutic that functions in the same way as the
therapeutic in the combination, demonstrates therapeutic or
prophylactic efficacy in humans. In another embodiment, an
MRD-containing antibody, is administered in combination with
another therapeutic to treat or prevent a disease, disorder or
injury for which the antibody component of the MRD-containing
antibody, or an antibody that functions in the same way as the
antibody contained in the MRD antibody, in combination with the
therapeutic, or a different therapeutic that functions in the same
way as the therapeutic in the combination, has been approved by a
regulatory authority for use in such treatment or prevention. The
administration of an MRD-containing antibody in combination with
more than one therapeutic as described above is also encompassed by
the invention.
[0613] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that promotes
apoptosis, inhibits apoptosis, promotes cell survival, inhibits
cell survival, promotes senescence of diseased or aberrant cells,
inhibits cell senescence, promotes cell proliferation, inhibits
cell proliferation, promotes cell differentiation, inhibits cell
differentiation, promotes cell activation, inhibits cell
activation, promotes cell metabolism, inhibits cell metabolism,
promotes cell adhesion, inhibits cell adhesion, promotes cell
cycling or cell division, inhibits cell cycling or cell division,
promotes DNA replication or repair, inhibits DNA replication or
repair, promotes transcription or translation, or inhibits
transcription or translation.
[0614] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that promotes apoptosis
or senescence of diseased or aberrant cells. In some embodiments,
the MRD-containing antibody is administered in combination with a
compound that agonizes, antagonizes or reduces the activity of:
EGFR, ErbB2, cMET, TNFa, TGFb, integrin .alpha.v.beta.3, TLR2,
TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TNFR1, TNFRSF10A (TRAIL R1
DR4), TNFRSF10B (TRAIL R2 DR5), TNF, TRAIL, IFN beta, MYC, Ras,
BCR, ABL, JNK, CKH2, CHK1, CDK1, RAC1, MEK, MOS, mTOR, AKT, NFkB,
Ikk, IAP1, IAP2, XIAP, b-catenin, survivin, HDAC, HSP70, HSP90,
proteasome 20S, topoisomerase 1, MDM2, E2F, or E2F1.
[0615] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that inhibits cell
survival. In some embodiments, the MRD-containing antibody is
administered in combination with a compound that antagonizes or
reduces the activity of: VEGF, VEGFR1, VEGFR2, IGF1R, IGF1, IGF2,
PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D, PDGFRA, PDGFRB, TFGa,
TGFB3, PI3K, TNFSF13B (BLYS), TNFRSF13C (BAFFR), INK, NFKB, SIP,
integrin .alpha.v.beta.3, or survivin.
[0616] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates cell
proliferation. In some embodiments, the MRD-containing antibody is
administered in combination with a compound that antagonizes or
reduces the activity of: VEGF, VEGFR, EGFR, ErbB2, NFKB, HIF, MUC1,
MUC2, or HDAC.
[0617] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates cell
adhesion. In some embodiments, the MRD-containing antibody is
administered in combination with a compound that inhibits or
reduces the activity of: MMP1, MMP2, MMP7, MMP9, MMP12, PLAU,
.alpha.v.beta.1 integrin, .alpha.v.beta.3 integrin, .alpha.v.beta.5
integrin, TGFb, EPCAM, .alpha.1.beta.1 integrin, .alpha.2.beta.1
integrin, .alpha.4.beta.1 integrin, .alpha.2.beta.1 integrin,
.alpha.5.beta.1 integrin, .alpha.9.beta.1 integrin, .alpha.6.beta.4
integrin, .alpha.M.beta.2 integrin, CEA, L1, Mel-CAM, or HIF1. In
one embodiment the MRD-containing antibody is administered in
combination with a compound that inhibits or reduces the activity
of .alpha.v.beta.3 integrin, .alpha.v.beta.5 integrin, or
.alpha.5.beta.1 integrin. In specific embodiments the
MRD-containing antibody is administered in combination with:
MEDI-522 (VITAXIN, Abegrin; MedImmune), ATN-161 (Attenuon), EMD
121974 (Merck KGaA), CNTO 95 (Cenotocor), or velociximab (M200,
Protein Design Labs).
[0618] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates cell
activation. In some embodiments, the MRD-containing antibody is
administered in combination with a compound that promotes, inhibits
or reduces the activity of: CD80, CD86, MHC, PDL2 (B7-DC), B7-H1,
B7-H2 (ICOSL), B7-H3, B7-H4, CD28, CTLA4, TCR, PD1, CD80, or
ICOS.
[0619] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates cell
cycling, cell division or mitosis. In some embodiments, the
MRD-containing antibody is administered in combination with a
compound that antagonizes or reduces the activity of PI3K, SMO,
Ptch, HH, SHH, plk1, plk2, plk3, plk4, aurora A, aurora B, aurora
C, CDK1, CDK2, CDK4, CHK1, CHK2, GSK3B, PAK, NEK2A, ROCK 2, MDM2
EGF (KSP), proteasome 20S, HDAC, or survivin.
[0620] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates DNA
replication or repair. In some embodiments, the MRD-containing
antibody is administered in combination with a compound that
antagonizes or reduces the activity of: BRCA1, CHK1, CHK2, E2F,
E2FL, MDM2, MDM4, or PARP1.
[0621] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates
transcription or translation. In some embodiments, the
MRD-containing antibody is administered in combination with a
compound that antagonizes or reduces the activity of IGF1R, IGF1,
IGF2, PDGFRA, PDGFRB, PDGF-A, PDGF-B, PDGF-CC, PDGF-C, PDGF-D, KIT,
MYC, CD28, CDK4, CDK6, mTOR, MDM2, HDAC, E2F, E2F1, or HIF1.
[0622] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates
migration, invasion or metastasis. In some embodiments, the
MRD-containing antibody is administered in combination with a
compound that inhibits or reduces the activity of: c-MET, RON,
CXCR4, PI3K, AKT, MMP2, FN1, CATHD, AMF, .alpha.v.beta.1 integrin,
.alpha.v.beta.3 integrin, .alpha.v.beta.5 integrin, TGFb,
.alpha.1.beta.1 integrin, .alpha.2.beta.1 integrin, .alpha.4.beta.1
integrin, .alpha.2.beta.1 integrin, .alpha.5.beta.1 integrin,
.alpha.9.beta.1 integrin, .alpha.6.beta.4 integrin, .alpha.M.beta.2
integrin, or HIF1.
[0623] According to one embodiment, an MRD-containing antibody is
administered in combination with a compound that regulates cell
metabolism. In some embodiments, the MRD-containing antibody is
administered in combination with a compound that inhibits or
reduces the activity of: Erb132, EGFR, IGF1R, IGF1, IGF2, TGFa,
ICOS, PI3K, VEGFR1, VEGFR2, mTOR, HIF1, or HDAC.
[0624] According to one embodiment, an MRD-containing antibody is
administered in combination with an inhibitor of one or more
protein kinases. In one embodiment, the protein kinase inhibitor
inhibits a target of the MRD containing antibody (e.g., by either
one or more MRDs or the antibody of the MRD containing antibody).
In an alternative embodiment, the protein kinase inhibitor inhibits
a protein kinase that is not a target of the MRD containing
antibody. In some embodiments, the protein kinase inhibitor
inhibits one protein kinase. In other embodiments, the protein
kinase inhibitor inhibits more than one protein kinase.
[0625] In some embodiments, an MRD containing antibody is
administered in combination with an inhibitor (e.g., small
molecule, antibody, etc.,) of a protein kinase selected from: EGFR,
FGFR1 (e.g., FGFR1-IIIC), FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and
FGFR2-IIIb), FGFR3, ErbB2, VEGFR2, VEGFR3, Tie-2, PDGFR, PDGFRB,
RON, and c-Met. In other embodiments, the inhibitor inhibits a
protein kinase that is not targeted by the MRD containing antibody.
In an additional embodiment, an MRD-containing antibody is
administered in combination with an inhibitor of one or more
protein kinases selected from: EGFR, FGFR1 (e.g., FGFR1-IIIC),
FGFR2 (e.g., FGFR2-IIIa, FGFR2-IIIb, and FGFR2-IIIb), FGFR3, ErbB2,
VEGFR1, VFGFR2, VEGFR3, Tie-2, PDGFRA, PDGFRB, FIT3, ALK, RET, Kit,
raf, p38, RON, c-Met, PI3K, ERK, FAK, AKT, SYK, JAK1, JAK2, JAK3,
TYK2, SIP, FAK, PTK7, PKD1, PKA, PKC, PKG, PRKDC, Pim, CDK, plk,
p38MAPK, SRC, ABL, FGR, FYN, HCK, LCK, LYN, YES, EPH4, BMK1, ERK5,
mTOR, CHK1, CHK2, CSNK1G1, CSNK1G2, CSNK1G3, GSK3, BTK, JNK, Aurora
Kinase, Aurora Kinase A, Aurora Kinase B, and Aurora Kinase C.
[0626] In an additional embodiment, an MRD-containing antibody is
administered in combination with a protein kinase inhibitor
selected from: imatinib mesylate (e.g., GLEEVEC.TM.), gefitinib
(e.g., IRESSA.TM., Astra Zeneca), vandetanib (e.g., ZACTIMA.TM.,
Astra Zeneca), erlotinib (e.g., TARCEVA.TM., Genentech/OSI),
sunitinib (e.g., SUTENT.TM., Pfizer), lapatanib (GSK), and
sorafenib (e.g., NEXAVAR.TM., Bayer).
[0627] In a further embodiment, an MRD-containing antibody is
administered in combination with a protein kinase inhibitor
selected from nilotinib (e.g., AMN107, Novartis), dasatinib (e.g.,
BMS 354825, BMS), ABT-869, botsutinib (e.g., SKI-606, Wyeth),
cediranib, recentib, captastatin, AEE788 (Novartis), AZD0530
(AstraZeneca) Exel 7646/Exel 0999 Exelixis), cabozantinib (e.g.,
XL184; Exelixis), XL880/GSK1363089 (Exelixis/GSK), ARQ-197 (Arqule
and Daiichi Sankyo), Inno-406 (Innovive), SGS523 (SGX), PF-2341066
(Pfizer), CI-1033 (Pfizer), motesanib (e.g., AMG-706, Amgen),
AG-013736 (Axitinib), AMG-705 (Amgen), pegaptanib (OSI/Pfizer),
lestaurtinib, SB1518, CYT387, LY3009104, TG101348 JANEX-1,
tofacitinib (Pfizer), INCB18424, LFM-A13, pazopanib (e.g.,
GW786034B, Glaxo SmithKline), GW-572016, EKB-569 (Wyeth-Ayerst),
vatalanib (e.g., PTK787/ZK), AZD2171, MK-0457 (VX-680, Merck), PHA
739358 (Nerviano), mubritinib (Takeda), E7080 (Eisai), fostamatinib
(Rigel/AstraZeneca), SGX523, SNS-032 (Sunesis), XL143, SNS-314
(Sunesis), SU6668 (Pfizer), AV-951 (AVEO), AV-412 (AVEO), tivizanib
(AVEO), PX-866 (Oncothyreon), canertinib (CI-1033), NSC 109555,
VRX0466617, UCN-01, CHK2 inhibitor II, EXEL-9844, XL844, CBP501,
PF-004777736, debromohymerialdisine, Go6976, AEG3482, cediranib
(e.g., RECENTIN.TM., AstraZeneca), semaxanib (SU5416), SU5616, CGP,
53716, mastinib, and ZD6474 (AstraZeneca).
[0628] In a further embodiment, an MRD-containing antibody is
administered in combination with a FGFR protein kinase inhibitor
selected from: sunitinib, SU5402, PD173074, TKI258 (Novartis), BIBF
1120 (Boehringer Ingelheim), brivanib (BMS-582,664), E7080 (Eisai),
and TSU-68 (Taiho).
[0629] In an additional embodiment, an MRD-containing antibody is
administered in combination with a protein kinase inhibitor of
JAK1, JAK2, JAK3, or SYK. In a further embodiment the protein
kinase inhibitor is selected from: lestaurtinib, tofacitinib,
SB1518, CYT387, LY3009104, TG101348, fostamatinib, BAY 61-3606, and
sunitinib.
[0630] In one embodiment, an ErbB2 (HER2) binding MRD-containing
antibody (e.g., an MRD-binding antibody that binds ErbB2 by either
one or more MRDs or the antibody of the MRD containing antibody) is
administered in combination with a protein kinase inhibitor of
ErbB2. In another specific embodiment a trastuzumab antibody-based
MRD-containing antibody is administered in combination with a
protein kinase inhibitor of ErbB2. In one embodiment, an
ErbB2-binding MRD-containing antibody is administered in
combination with lapatinib. In a specific embodiment a trastuzumab
antibody-based MRD-containing antibody is administered in
combination with lapatinib. In one embodiment, an ErbB2-binding
MRD-containing antibody is administered in combination with,
sunitinib. In a specific embodiment a trastuzumab antibody-based
MRD-containing antibody is administered in combination with
sunitinib. In one embodiment, an ErbB2-binding MRD-containing
antibody is administered in combination with neratinib. In a
specific embodiment a trastuzumab antibody-based MRD-containing
antibody is administered in combination with neratanib. In one
embodiment, an ErbB2-binding MRD-containing antibody is
administered in combination with iapatinib. In a specific
embodiment a trastuzumab antibody-based MRD-containing antibody is
administered in combination with iapatinib. In an additional
embodiment, an ErbB2 (HER2) binding MRD-containing antibody is
administered in combination with a protein kinase inhibitor
selected from canertinib (GW-572016), AV-412 (AVEO), tivozanib
(AVEO), vandetanib (e.g., ZACTIMA.TM., AstraZeneca), AEE788
(Novartis), Exel 7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), and
EKB-569 (Wyeth-Ayerst). In a specific embodiment a trastuzumab
antibody-based MRD-containing antibody is administered in
combination with a protein kinase inhibitor selected from:
canertinib (GW-572016), AV-412 (AVEO), tivozanib (AVEO), vandetanib
(e.g., ZACTIMA.TM., AstraZeneca), AEE788 (Novartis), Exel 7646/Exel
0999 (Exelixis), CI-1033 (Pfizer), PX-866 (Oncothyreon), and
EKB-569 (Wyeth-Ayerst).
[0631] In another embodiment, an EGFR binding MRD-containing
antibody (e.g., an MRD-binding antibody that binds EGFR by either
one or more MRDs or the antibody of the MRD containing antibody) is
administered in combination with a protein kinase inhibitor of
EGFR. In a specific embodiment a cetuximab antibody-based
MRD-containing antibody is administered in combination with a
protein kinase inhibitor of EGFR. In one embodiment, an EGFR
binding MRD-containing antibody is administered in combination with
gefitinib (e.g., IRESSA.TM., AstraZeneca). In a specific embodiment
a cetuximab antibody-based MRD-containing antibody is administered
in combination with gefitinib (e.g., IRESSA.TM., AstraZeneca). In
one embodiment, an EGFR binding MRD-containing antibody is
administered in combination with erlotinib (e.g., TARCEVA.TM.,
Genentech/OSI). In a specific embodiment a cetuximab antibody-based
MRD-containing antibody is administered in combination with
erlotinib (e.g., TARCEVA.TM., Genentech/OSI). In one embodiment, an
EGFR binding MRD-containing antibody is administered in combination
with lapatinib. In a specific embodiment a cetuximab antibody-based
MRD-containing antibody is administered in combination with
lapatinib. In one embodiment, an EGFR binding MRD-containing
antibody is administered in combination with sorafenib (e.g.,
NEXAVAR.TM., Bayer). In a specific embodiment a cetuximab
antibody-based MRD-containing antibody is administered in
combination with sorafenib (e.g., NEXAVAR.TM., Bayer). In another
embodiment, an EGFR binding MRD-containing antibody is administered
in combination with a protein kinase inhibitor selected from:
canertinib (GW-572016), ZD6474, AV-412 (AVEO), tivozanib (AVEO),
vandetanib (ZACTIMA, AstraZeneca), AEE788 (Novartis), Exel
7646/Exel 0999 (Exelixis), CI-1033 (Pfizer), and EKB-569
(Wyeth-Ayerst). In a specific embodiment a cetuximab antibody-based
MRD-containing antibody is administered in combination with a
protein kinase inhibitor selected from: canertinib (GW-572016),
ZD6474, AV-412 (AVEO), tivozanib (AVEO), vandetanib (ZACTIMA,
AstraZeneca), AEE788 (Novartis), Exel 7646/Exel 0999 (Exelixis),
CI-1033 (Pfizer), PX-866 (Oncothyreon), and EKB-569
(Wyeth-Ayerst).
[0632] In one embodiment, a VEGFA, VEGFR1, or VEGFR2 binding
MRD-containing antibody (e.g., an MRD-binding antibody that binds
VEGFR1 by either one or more MRDs or the antibody of the MRD
containing antibody) is administered in combination with a protein
kinase inhibitor of VEGR1, VEGFR2, or VEGFR3. In one embodiment,
the VEGFA, VEGFR1 or VEGFRr2 binding MRD-containing antibody is
administered in combination with: sunitinib, sorafenib, pazopanib
(e.g., GW786034B), AZD2171, vatalanib, ZD6474, AMG-706, or
AC013736.
[0633] In a further embodiment, an MRD-containing antibody is
administered in combination with a proteasome inhibitor. In a
specific embodiment, the inhibitor is bortezomib (e.g.,
VELCADE.TM.). In another specific embodiment, the inhibitor is
PR-171 (Proteolix).
[0634] In a farther embodiment, an MRD-containing antibody is
administered in combination with a HDAC inhibitor.
[0635] In a further embodiment, an MRD-containing antibody is
administered in combination with a mTOR inhibitor.
[0636] In a further embodiment, an MRD-containing antibody is
administered in combination with a NFKB inhibitor.
[0637] In one embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of a VEGFA or VEGFR binding MRD-containing
antibody to a patient in need thereof. In a specific embodiment,
the invention provides a method of treating cancer comprising
administering a therapeutically effective amount of bevacizumab
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating colorectal
cancer by administering a therapeutically effective amount of
bevacizumab comprising at least one MRD to a patient having
colorectal cancer. In another embodiment, the invention provides a
method of treating breast cancer by administering a therapeutically
effective amount of bevacizumab comprising at least one MRD to a
patient having breast cancer. In another embodiment, the invention
provides a method of treating non-small cell lung carcinoma by
administering a therapeutically effective amount of bevacizumab
comprising at least one MRD to a patient having non-small cell lung
carcinoma. In other embodiments, therapeutic effective amounts of
bevacizumab comprising at least one MRD are administered to a
patient to treat metastatic colorectal cancer, metastatic breast
cancer, metastatic pancreatic cancer, or metastatic non-small cell
lung carcinoma. In another embodiment, the invention provides a
method of treating cancer by administering to a patient a
therapeutically effective amount of bevacizumab comprising at least
one MRD to a patient having renal cell carcinoma, glioblastoma
multiforme, ovarian cancer, prostate cancer, liver cancer or
pancreatic cancer.
[0638] Combination therapy and compositions including multivalent
and multispecific compositions (e.g., MRD-containing antibodies) of
the invention and another therapeutic are also encompassed by the
invention, as are methods of treatment using these compositions. In
other embodiments, compositions of the invention are administered
alone or in combination with one or more additional therapeutic
agents. Combinations may be administered either concomitantly,
e.g., as an admixture, separately but simultaneously or
concurrently; or sequentially. This includes presentations in which
the combined agents are administered together as a therapeutic
mixture, and also procedures in which the combined agents are
administered separately but simultaneously, e.g., as through
separate intravenous lines into the same individual. Administration
"in combination" further includes the separate administration of
one of the therapeutic compounds or agents given first, followed by
the second. Accordingly, in one embodiment, a VEGFA or VEGFR
binding MRD-containing antibody is administered in combination with
5-fluorouracil, carboplatin, paclitaxel, or interferon alpha. In
another embodiment, bevacizumab comprising at least one MRD is
administered in combination with 5-fluorouracil, carboplatin,
paclitaxel, or interferon alpha.
[0639] In another embodiment, the invention provides a method of
treating macular degeneration comprising administering a
therapeutically effective amount of a VEGFA or VEGFR binding
MRD-containing antibody to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating macular
degeneration comprising administering a therapeutically effective
amount of bevacizumab comprising at least one MRD to a patient in
need thereof. In a specific embodiment, the invention provides a
method of treating macular degeneration comprising administering a
therapeutically effective amount of ranibizumab comprising at least
one MRD to a patient in need thereof.
[0640] In some embodiments, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of an ErbB2 (HER2) binding MRD-containing antibody
to a patient in need thereof. In various embodiments, the
ErbB2-binding multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are administered to patients who have
been previously shown to respond to another ErbB2-based therapy
(e.g., HERCEPTIN, chemotherapy and/or radiation) or are predicted
to respond to another ErbB2-based therapy. In other embodiments,
the ErbB2-binding multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are administered to patients who have
previously failed to respond to another ErbB2-based therapy or are
predicted to fail to respond to another ErbB2-based therapy.
[0641] In a specific embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of trastuzumab comprising at least one MRD to a
patient in need thereof. In one embodiment, the invention provides
a method of treating breast cancer by administering a
therapeutically effective amount of trastuzumab comprising at least
one MRD to a patient having breast cancer. In other embodiments,
therapeutic effective amounts of trastuzumab comprising at least
one MRD are administered to a patient to treat metastatic breast
cancer.
[0642] In another embodiment, an ErbB2 (HER2) binding
MRD-containing antibody is administered in combination with
cyclophosphamide, paclitaxel, docetaxel, carboplatin,
anthracycline, or a maytansinoid. In a specific embodiment,
trastuzumab comprising at least one MRD is administered in
combination with cyclophosphamide, paclitaxel, docetaxel,
carboplatin, anthracycline, or a maytansinoid.
[0643] In another embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of a CD20-binding MRD-containing antibody to a
patient in need thereof. In a specific embodiment, the invention
provides a method of treating a hematologic cancer comprising
administering a therapeutically effective amount of rituximab
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating CD20
positive NHL by administering a therapeutically effective amount of
bevacizumab comprising at least one MRD to a patient having CD20
positive NHL. In one embodiment, the invention provides a method of
treating CD20 positive CLL by administering a therapeutically
effective amount of bevacizumab comprising at least one MRD to a
patient having CD20 positive CLL.
[0644] In another embodiments, a therapeutically effective amount
of a CD20-binding MRD-containing antibody is administered in
combination with: ludarabine, cyclophosphamide, FC (fludarabine and
cyclophosphamide), anthracycline based chemotherapy regimen (e.g.,
CHOP (cyclophosphamide, adriamycin, vincristine and prednisone)),
or CVP (cyclophosphamide, prednisone, and vincristine)
chemotherapy. In a specific embodiment, a therapeutically effective
amount of bevacizumab comprising at least one MRD is administered
in combination with: ludarabine, cyclophosphamide, FC (fludarabine
and cyclophosphamide), anthracycline based chemotherapy regimen
(e.g., CHOP (cyclophosphamide, adriamycin, vincristine and
prednisone)), or CVP (cyclophosphamide, prednisone, and
vincristine) chemotherapy.
[0645] Any of the antibody-MRD fusions containing antibodies and/or
MRDs that bind CD20 can be used according to the methods of
treating a disorder associated with CD20, or that can be treated by
targeting cells that express CD20 (e.g., hematological cancers and
autoimmune disease). In some embodiments, the antibody component of
the antibody-MRD-fusion is selected from rituximab, ocrelizumab,
GA101, and PF-5,230,895.
[0646] The invention also provides a method of treating a disorder
of the immune system comprising administering a therapeutically
effective amount of an MRD-containing antibody. In some
embodiments, the administered MRD-containing antibody binds a
target selected from: CD20, TNFRSF5 (CD40), CD45RB, CD52, CD200,
CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5 (CD40 LIGAND), TLR2,
TLR4, GPR44, FASL, TREM1, IL1, IL1 beta, IL1RN, tissue factor, MIF,
MIP2, IL6, IL8, IL10, IL12, IL13, IL15, IL17, IL18, IL23, TNF,
TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN alpha, IFN gamma, GMCSF,
FGF, TGFb, C5, and CCR3. Multivalent and multispecific compositions
(e.g., MRD-containing antibodies) that bind 2, 3, 4, 5 or more of
these targets are also encompassed by the invention.
[0647] In particular embodiments, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of an
MRD-containing antibody that binds TNF and ANG2.
[0648] In additional embodiments, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of an
MRD-containing antibody that binds IL1, IL12, and TNF. In further
embodiments, the MRD-containing antibody binds IL1, IL12, TNF and
ANG2.
[0649] In additional embodiments, the administered MRD-containing
antibody binds IL1, IL6 and TNF. In further embodiments, the
MRD-containing antibody binds ILL IL6 TNF and ANG2.
[0650] target selected from: CD20, TNFRSF5 (CD40), CD45RB, CD52,
CD200, CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5 (CD40
LIGAND), TLR2, TLR4, GPR44, FASL, TREM1, IL1, IL1 beta, IL1RN,
tissue factor, MIF, MIP2, IL6, IL8, IL10, IL12, IL13, IL15, IL17,
IL18, IL23, TNF, TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN alpha, IFN
gamma, GMCSF, FGF, TGFb, C5, and CCR3. Multivalent and
multispecific compositions (e.g., MRD-containing antibodies) that
bind 2, 3, 4, 5 or more of these targets are also encompassed by
the invention.
[0651] In additional embodiments, the invention provides a method
of treating an autoimmune disease comprising administering a
therapeutically effective amount of an MRD-containing antibody. In
a specific embodiment, the administered MRD-containing antibody
binds a target selected from: CD1C, CD3, CD4, CD19, CD20, CD21,
CD22, CD23, CD24, CD28, CD37, CD38, CD45RB, CD52, CD69, CD72, CD74,
CD75, CD79A, CD79B, CD80, CD81, CD83, CD86, CD200, IL2RA, IL1R2,
IL6R, VLA4, HLA-DRA, HLA-A, ITGA2, ITGA3, CSF2, TLR2, TLR4, GPR44,
TREM1, TIE2, TNF, FASL, tissue factor, MIF, MIP2, IL1, IL1 beta,
IL1RN, IL2, IL4, IL6, IL8, IL10, IL11, IL12, IL13, IL15, IL17,
IL18, IL23, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7
(CD27), TNFRSF8 (CD30), TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand),
TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFSF12 (TWEAK),
TNFSF13B (BLyS), ANG2, ICOSL (B7-H2), MS4A1, IFN alpha, IFN beta1,
IFN gamma, TNFSF7 (CD27 Ligand, CD70), PAFR, INHA, INHBA, DPP4,
NT5E, CTLA4, B7.1/B7.2, LPS, VEGF, GMCSF, FGF, C5, CXCL13, CXCR4,
CCR2 and CCR3. In further embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) are
administered to treat rheumatoid arthritis and the multivalent and
multispecific compositions bind a target selected from: CD19, CD20,
CD45RB, CD52CD200, IL1, IL6, IL12, IL15, IL17, IL18, IL23, TNF,
TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 Ligand), TNFSF13B
(BLyS), VEGF, VLA4, IFN gamma, IFN alpha, GMCSF, FGF, C5, CXCL13
and CCR2. In additional embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) are
administered to treat systemic lupus erythematous and the
multivalent and multispecific compositions bind IFN alpha and
TNFSF13B (BLyS). In further embodiments, the multivalent and
multispecific compositions (e.g., MRD-containing antibodies) are
administered to treat multiple sclerosis and the multivalent and
multispecific compositions bind a target selected from: ANG2, IL1,
IL12, IL18, IL23, CXCL13, TNF, TNFRSF5 (CD40), TNFSF5 (CD40
Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma, GM-CSF, FGF,
C5, CD52, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6 (Fas, CD95), TNFRSF7
(CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK), TNFRSF13C (BAFFR), TNFSF5
(CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand), TNFRSF21
(DR6), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, AGE (S100 A,
amphoterin), ICOSL (B7-H2), MS4A 1, IFN alpha, IFN beta1, IFN
gamma, TNFSF7 (CD27 Ligand, CD70), MCP1, CCR2 and CXCL13.
Multivalent and multispecific compositions that bind at least 2, 3,
4, 5 or more of these targets are also encompassed by the
invention.
[0652] In a further embodiment, the invention provides a method of
treating, a disorder of the immune system comprising administering
a therapeutically effective amount of a CD20-binding MRD-containing
antibody to a patient in need thereof. In a specific embodiment,
the invention provides a method of treating an autoimmune disease
comprising administering a therapeutically effective amount of a
CD20-binding MRD-containing antibody to a patient in need thereof.
In one embodiment, the invention provides a method of treating an
autoimmune disease comprising administering a therapeutically
effective amount of a ritaximab-MRD-containing antibody to a
patient in need thereof. In another embodiment, the invention
provides a method of treating rheumatoid arthritis comprising
administering a therapeutically effective amount of a
rituximab-MRD-containing antibody to a patient in need thereof. In
another embodiment, the invention provides a method of treating
systemic lupus erythematous comprising administering a
therapeutically effective amount of a rituximab-MRD-containing
antibody to a patient in need thereof. In another embodiment, the
invention provides a method of treating multiple sclerosis
comprising administering a therapeutically effective amount of a
rituximab-MRD-containing antibody to a patient in need thereof.
[0653] In an additional embodiment, the invention provides a method
of treating an autoimmune disease comprising administering a
therapeutically effective amount of an ocrelizumab-MRD-containing
antibody to a patient in need thereof. In one embodiment, the
invention provides a method of treating rheumatoid arthritis
comprising administering a therapeutically effective amount of an
ocrelizumab-MRD-containing antibody to a patient in need thereof.
In a further embodiment, the invention provides a method of
treating systemic lupus erythematous comprising administering a
therapeutically effective amount of a ocrelizumab-MRD-containing
antibody to a patient in need thereof. In another embodiment, the
invention provides a method of treating multiple sclerosis
comprising administering a therapeutically effective amount of an
ocrelizumab-MRD-containing antibody to a patient in need
thereof.
[0654] In another embodiment, the invention provides a method of
treating an autoimmune disease comprising administering a
therapeutically effective amount of a PF5,230,895-MRD-containing
antibody to a patient in need thereof. In one embodiment, the
invention provides a method of treating rheumatoid arthritis
comprising administering a therapeutically effective amount of a
PF5,230,895-MRD-containing antibody to a patient in need thereof.
In a further embodiment, the invention provides a method of
treating systemic lupus erythematous comprising administering a
therapeutically effective amount of a PF5,230,895-MRD-containing
antibody to a patient in need thereof. In another embodiment, the
invention provides a method of treating multiple sclerosis
comprising administering a therapeutically effective amount of an
PF5,230,895-MRD-containing antibody to a patient in need
thereof.
[0655] In some embodiments, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of an MRD-containing antibody that
binds CD20. In further embodiments, the administered MRD-containing
antibody binds CD20 and a target selected from: TNF, TNFRSF5
(CD40), TNFSF5 (CD40 LIGAND), TNFSF12 (TWEAK), TNFRSF1A, CD45, RB,
CD52, CD200, CCR2, PAFR, IL6R, VLA4, CSF2, RAGE, TLR2, TLR4, GPR44,
FASL, TREM1, TIE2, tissue factor, MIF, MIP2, LPS, IL1, IL1 beta,
IL1RN, IL6, IL6R, IL8, IL10, IL12, IL13, IL15, IL17, IL18, IL23,
CXCL13, VEGF, IFN alpha, IFN gamma, GMCSF, FGF, C5, and CCR3.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind CD20 and also bind at least 1, 2, 3, 4, 5 or
more of these targets are also encompassed by the invention. In
specific embodiments, the antibody component of the MRD-containing
antibody binds CD20. In further embodiments, the antibody component
of the MRD-containing antibody is a rituximab, ocrelizumab, GA101
or PF-5,230,895.
[0656] In some embodiments, the invention provides a method of
treating an autoimmune disease comprising administering a
therapeutically effective amount of an MRD-containing antibody that
binds CD20. In a specific embodiment, the administered
MRD-containing antibody binds CD20 and a target selected from:
CD1C, CD3, CD4, CD19, CD21, CD22, CD23, CD24, CD28, CD37, CD38,
CD45RB, CD52, CD69, CD72, CD74, CD75, CD79A, CD79B, CD80, CD81,
CD83, CD86, CD200, IL2RA, IL1R2, IL6R, VLA4, HLA-DRA, HLA-A, ITGA2,
ITGA3, CSF2, TLR2, TLR4, GPR44, TREM1, TIE2, TNF, FASL, tissue
factor, MIF, MIP2, IL1, IL1 beta, IL1RN, IL2, IL4, IL6, IL8, IL10,
IL11, IL12, IL13, IL15, IL17, IL18, IL23, TIE2, TNFRSF1A, TNFRSF5
(CD40), TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30),
TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand),
TNFSF8 (CD30 Ligand), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, ICOSL
(B7-H2), MS4A1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27
Ligand, CD70), PAFR, INHA, INHBA, DPP4, NT5E, CTLA4, B7.1/B7.2,
LPS. VEGF, GMCSF, FGF, C5, CXCL13, CXCR4, CCR2 and CCR3. In further
embodiments, the multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are administered to treat rheumatoid
arthritis and the multivalent and multispecific compositions bind
CD20 and a target selected from: CD19, CD45RB, CD52, CD200, IL12,
IL15, IL17, IL18, IL23, TNF, TNFSF12 (TWEAK). TNFRSF5 (CD40),
TNFSF5 (CD40 Ligand), VEGF, VLA4, IFN gamma, interferon alpha,
GMCSF, FGF, C5, CXCL13 and CCR2. In further embodiments, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) are administered to treat multiple sclerosis and the
multivalent and multispecific compositions bind CD20 and a target
selected from: ANG2, IL12, IL18, IL23, CXCL13, TNFRSF5 (CD40),
TNFSF5 (CD40 Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma,
GM-CSF, FGF, C5, CD52, TIE2, TNFRSF1A, TNFRSF5 (CD40), TNFRSF6
(Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK),
TNFRSF13C (BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand),
TNFSF8 (CD30 Ligand), TNFRSF21 (DR6), TNFSF12 (TWEAK), TNFSF13B
(BLyS), ICOSL (B7-H2), MS4A 1, IFN alpha, IFN beta1, IFN gamma,
TNFSF7 (CD27 Ligand, CD70), CCR2 and CXCL13. Multivalent and
multispecific compositions that bind a least 1, 2, 3, 4, 5 or more
of these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds TNF. In further embodiments, the antibody component of the
MRD-containing antibody is selected from rituximab, ocrelizumab,
GA101 and PF-5,230,895.
[0657] In some embodiments, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of a TNF-binding MRD-containing
antibody to a patient in need thereof. In various embodiments, the
TNF-binding multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are administered to patients who have
been previously shown to respond to another TNF-based therapy or
are predicted to respond to another TNF-based therapy (e.g., TNF
antagonists such as, anti-TNFs (e.g., HUMIRA), EMBREL, CD28
antagonists, CD20 antagonists, and IL6/IL6R antagonists). In other
embodiments, the TNF-binding multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are administered to
patients who have previously failed to respond to another TNF-based
therapy or are predicted to fail to respond to another TNF-based
therapy.
[0658] In some embodiments, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of an MRD-containing antibody that
binds TNF.
[0659] In further embodiments, the administered MRD-containing
antibody binds TNF and a target selected from CD20, TNFRSF5 (CD40),
CD45RB, CD52, CD200, CCR2, PAFR, IL6R, TNFRSF1A, VLA4, CSF2, TNFSF5
(CD40 LIGAND), TLR2, TLR4, GPR44, FASL, TREM1, IL1, IL1 beta,
IL1RN, tissue factor, MIF, MIP2, IL6, IL8, IL10, IL12, IL13, IL15,
IL17, IL18, IL23, TNFSF12 (TWEAK), LPS, CXCL13, VEGF, IFN gamma,
GMCSF, FGF, C5, and CCR3. Multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that bind TNF and at
least 1, 2, 3, 4, 5 or more of these targets are also encompassed
by the invention. In specific embodiments, the antibody component
of the MRD-containing antibody binds TNF. In further embodiments,
the antibody component of the MRD-containing antibody is selected
from adalimumab, certolizumab, golimumab and AME-527.
[0660] In some embodiments, the invention provides a method of
treating an autoimmune disease comprising administering a
therapeutically effective amount of an MRD-containing antibody that
binds TNF. In a specific embodiment, the administered
MRD-containing antibody binds TNF and a target selected from: CD1C,
CD3, CD4, CD19, CD20, CD21, CD22, CD23, CD24, CD28, CD37, CD38,
CD45RB, CD52, CD69, CD72, CD74, CD75, CD79A, CD79B, CD80, CD81,
CD83, CD86, CD200, IL2RA, IL1R2, IL6R, VLA4, HLA-DRA, HLA-A, ITGA2,
ITGA3, CSF2, TLR2, TLR4, GPR44, TREM1, TIE2, FASL, tissue factor,
MIF, MIP2, ILL IL1 beta, IL1RN, IL2, IL4, IL6, IL8, IL10, IL11,
IL12, IL13, IL15, IL17, IL18, IL23, TIE2, TNFRSF1A, TNFRSF5 (CD40),
TNFRSF6 (Fas, CD95), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF13C
(BAFFR), TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30
Ligand), TNFSF12 (TWEAK), TNFSF13B (BLyS), ANG2, ICOSL (B7-H2),
MS4A1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand, CD70),
PAFR, INHA, INHBA, DPP4, NT5E, CTLA4, B7.1/B7.2, LPS, VEGF, GMCSF,
FGF, C5, CXCL13, CXCR4, CCR2 and CCR3. In further embodiments, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) are administered to treat rheumatoid arthritis and the
multivalent and multispecific compositions bind TNF and a target
selected from CD19, CD20, CD45RB, CD52CD200, IL12, IL15, IL17,
IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40 Ligand),
TNFSF13B (BLyS), VEGF, VLA4, IFN gamma, interferon alpha, GMCSF,
FGF, C5, CXCL13 and CCR2. In further embodiments, the multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
are administered to treat multiple sclerosis and the multivalent
and multispecific compositions bind TNF and a target selected from:
ANG2, IL12, IL18, IL23, CXCL13, TNFRSF5 (CD40), TNFSF5 (CD40
Ligand), VEGF, VLA4, TNF, CD45RB, CD200, IFN gamma, GM-CSF, FGF,
C5, CD52, TNFRSF1A, TNFRSF5 (CD40), TIE2, TNFRSF6 (Fas, CD95),
TNFRSF7 (CD27), TNFRSF8 (CD30), TNFSF12 (TWEAK), TNFRSF13C (BAFFR),
TNFSF5 (CD40 Ligand), TNFSF6 (Fas Ligand), TNFSF8 (CD30 Ligand),
TNFRSF21 (DR6), TNFSF12 (TWEAK), TNFSF13B (BLyS), ICOSL (B7-H2),
MS4A 1, IFN alpha, IFN beta1, IFN gamma, TNFSF7 (CD27 Ligand,
CD70), CCR2 and CXCL13. Multivalent and multispecific compositions
that bind a least 1, 2, 3, 4, 5 or more of these targets are also
encompassed by the invention. In specific embodiments, the antibody
component of the MRD-containing antibody binds TNF. In further
embodiments, the antibody component of the MRD-containing antibody
selected from adalimumab, certolizumab, golimumab and AME-527.
[0661] In other embodiments, the TNF-binding multivalent and
multispecific compositions (e.g., MRD-containing antibodies) are
administered to patients who have been previously shown to respond
to an autoimmune disease based therapy or are predicted to respond
to other autoimmune disease based therapies (e.g., TNF antagonists
such as, Anti-TNFs (e.g., HUMIRA.RTM.), ENBREL.RTM., CD28
antagonists, CD20 antagonists, BLyS antagonists, and IL6/IL6R
antagonists). In other embodiments, the TNF-binding multivalent and
multispecific compositions (e.g., MRD-containing antibodies) are
administered to patients who have previously failed to respond to
another autoimmune disease based therapy or are predicted to fail
to respond to another autoimmune disease based therapy.
[0662] In a specific embodiment, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of adalimumab comprising at least
one MRD to a patient in need thereof. In one embodiment, the
invention provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of adalimumab
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating rheumatoid
arthritis, by administering a therapeutically effective amount of
adalimumab comprising at least one MRD to a patient in need
thereof. In one embodiment, the invention provides a method of
treating an inflammatory disorder, by administering a
therapeutically effective amount of adalimumab comprising at least
one MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating Crohn's disease, by
administering a therapeutically effective amount of adalimumab
comprising at least one MRD to a patient in need thereof. In
another embodiment, the invention provides a method of treating
ulcerative colitis, by administering a therapeutically effective
amount of adalimumab comprising at least one MRD to a patient in
need thereof. In another embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering a
therapeutically effective amount of adalimumab comprising at least
one MRI) to a patient in need thereof.
[0663] In an additional embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of ATN-103
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating an
inflammatory disorder, by administering a therapeutically effective
amount of ATN-103 comprising at least one MRD to a patient in need
thereof. In another embodiment, the invention provides a method of
treating an autoimmune disease, by administering a therapeutically
effective amount of ATN-103 comprising at least one MRD to a
patient in need thereof. In a further embodiment, the invention
provides a method of treating rheumatoid arthritis, by
administering a therapeutically effective amount of ATN-103
comprising at least one MRD to a patient in need thereof. In
another embodiment, the invention provides a method of treating
Crohn's disease, by administering a therapeutically effective
amount of ATN-103 comprising at least one MRD to a patient in need
thereof. In an additional embodiment, the invention provides a
method of treating ulcerative colitis, by administering a
therapeutically effective amount of ATN-103 comprising at least one
MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating psoriatic arthritis,
ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis
by administering a therapeutically effective amount of ATN-103
comprising at least one MRD to a patient in need thereof.
[0664] In a specific embodiment, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of infliximab comprising at least
one MRD to a patient in need thereof. In one embodiment, the
invention provides a method of treating an inflammatory disorder,
by administering a therapeutically effective amount of infliximab
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating an
autoimmune disease, by administering a therapeutically effective
amount of infliximab comprising at least one MRD to a patient in
need thereof. In one embodiment, the invention provides as method
of treating rheumatoid arthritis, by administering a
therapeutically effective amount of infliximab comprising at least
one MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating Crohn's disease, by
administering a therapeutically effective amount of infliximab
comprising at least one MRD to a patient in need thereof. In
another embodiment, the invention provides a method of treating
ulcerative colitis, by administering a therapeutically effective
amount of infliximab comprising at least one MRD to a patient in
need thereof. In another embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering a
therapeutically effective amount of infliximab comprising at least
one MRD to a patient in need thereof.
[0665] In some embodiments, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of an MRD-containing antibody that
binds TNFSF15 (TL1A).
[0666] In further embodiments, the administered MRD-containing
antibody binds TL1A and a target selected from: TNF, IFN gamma,
IL1, IL1beta, IL6, IL8, IL12, IL15, IL17, IL18, IL23 and IL32.
Multivalent and multispecific compositions (e.g., MRD-containing
antibodies) that bind TL1A and at least 1, 2, 3, 4, 5 or more of
these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds TL 1A.
[0667] In an additional embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of a IL22-binding
MRD-containing antibody to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating a disorder
of the immune system comprising administering a therapeutically
effective amount of PF5,212,367 (ILV-094) comprising at least one
MRD to a patient in need thereof. In one embodiment, the invention
provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of PF5,212,367
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating rheumatoid
arthritis, by administering a therapeutically effective amount of
PF5,212,367 comprising at least one MRD to a patient in need
thereof. In one embodiment, the invention provides a method of
treating an inflammatory disorder, by administering a
therapeutically effective amount of PF5,212,367 comprising at least
one MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating Crohn's disease, by
administering a therapeutically effective amount of PF5,212,367
comprising at least one MRD to a patient in need thereof. In a
further embodiment, the invention provides a method of treating,
ulcerative colitis, by administering a therapeutically effective
amount of PF5,212,367 comprising at least one MRD to a patient in
need thereof. In another embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering a
therapeutically effective amount of PF5,212,367 comprising at least
one MRD to a patient in need thereof.
[0668] In an additional embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of a alpha4
integrin-binding MRD-containing antibody to a patient in need
thereof. In a specific embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of natalizumab
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating an
autoimmune disease by administering a therapeutically effective
amount of natalizumab comprising at least one MRD to a patient in
need thereof. In another embodiment, the invention provides a
method of treating rheumatoid arthritis, by administering a
therapeutically effective amount of natalizumab comprising at
least, one MRD to a patient in need thereof. In a further
embodiment, the invention provides a method of treating systemic
lupus erythematous comprising administering a therapeutically
effective amount of a natalizumab-MRD-containing antibody to a
patient in need thereof. In another embodiment, the invention
provides a method of treating multiple sclerosis comprising
administering a therapeutically effective amount of a
natalizumab-MRD-containing antibody to a patient in need thereof.
In a further embodiment, the invention provides a method of
treating an inflammatory disorder, by administering a
therapeutically effective amount of natalizumab comprising at least
one MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating Crohn's disease, by
administering a therapeutically effective amount of natalizumab
comprising at least one MRD to a patient in need thereof. In an
additional embodiment, the invention provides a method of treating
ulcerative colitis, by administering a therapeutically effective
amount of natalizumab comprising at least one MRD to a patient in
need thereof. In another embodiment, the invention provides a
method of treating multiple sclerosis, by administering a
therapeutically effective amount of natalizumab comprising at least
one MRD to a patient in need thereof. In an additional embodiment,
the invention provides a method of treating psoriatic arthritis,
ankylosing spondylitis, psoriasis, or juvenile idiopathic arthritis
by administering a therapeutically effective amount of natalizumab
comprising at least one MRD to a patient in need thereof
[0669] In an additional embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of a TNFSF5 (CD40
LIGAND)-binding MRD-containing antibody to a patient in need
thereof. In a specific embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of CDP7657
comprising at least one MRD to a patient in need thereof. In one
embodiment, the invention provides a method of treating an
autoimmune disease by administering a therapeutically effective
amount of CDP7657 comprising at least one MRD to a patient in need
thereof. In another embodiment, the invention provides a method of
treating rheumatoid arthritis, by administering a therapeutically
effective amount of CDP7657 comprising at least one MRD to a
patient in need thereof. In a further embodiment, the invention
provides a method of treating systemic lupus erythematous
comprising administering a therapeutically effective amount of a
CDP7657-MRD-containing antibody to a patient in need thereof. In
another embodiment, the invention provides a method of treating
multiple sclerosis comprising administering a therapeutically
effective amount of a CDP7657-MRD-containing antibody to a patient
in need thereof. In one embodiment, the invention provides a method
of treating an inflammatory disorder, by administering a
therapeutically effective amount of CDP7657 comprising at least one
MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating Crohn's disease, by
administering a therapeutically effective amount of CDP7657
comprising at least one MRD to a patient in need thereof. In a
further embodiment, the invention provides a method of treating
ulcerative colitis, by administering a therapeutically effective
amount of CDP7657 comprising at least one MRD to a patient in need
thereof. In an additional embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering a
therapeutically effective amount of CDP7657 comprising at least one
MRD to a patient in need thereof.
[0670] In another embodiment, the invention provides a method of
treating a disorder of the immune system comprising administering a
therapeutically effective amount of a TNFSF12 (TWEAK)-binding
MRD-containing antibody to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating a disorder
of the immune system comprising administering a therapeutically
effective amount of the Biogen TNFSF12 (TWEAK) antibody (that has
entered phase 1 clinical trials) comprising at least one MRD to a
patient in need thereof. In one embodiment, the invention provides
a method of treating an autoimmune disease by administering a
therapeutically effective amount of the Biogen TNFSF12 (TWEAK)
antibody comprising at least one MRD to a patient in need thereof.
In one embodiment, the invention provides a method of treating
rheumatoid arthritis, by administering a therapeutically effective
amount of the Biogen TNFSF12 (TWEAK) antibody comprising at least
one MRD to a patient in need thereof. In a further embodiment, the
invention provides a method of treating systemic lupus erythematous
comprising administering a therapeutically effective amount of the
Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a
patient in need thereof. In another embodiment, the invention
provides a method of treating multiple sclerosis comprising
administering a therapeutically effective amount of the Biogen
TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient
in need thereof. In another embodiment, the invention provides a
method of treating an inflammatory disorder, by administering a
therapeutically effective amount of the Biogen TNFSF12 (TWEAK)
antibody comprising at least one MRD to a patient in need thereof.
In an additional embodiment, the invention provides a method of
treating Crohn's disease, by administering a therapeutically
effective amount of the Biogen TNFSF12 (TWEAK) antibody comprising
at least one MRD to a patient in need thereof. In another
embodiment, the invention provides a method of treating ulcerative
colitis, by administering a therapeutically effective amount of the
Biogen TNFSF12 (TWEAK) antibody comprising at least one MRD to a
patient in need thereof. In a further embodiment, the invention
provides a method of treating psoriatic arthritis, ankylosing
spondylitis, psoriasis, or juvenile idiopathic arthritis by
administering a therapeutically effective amount of the Biogen
TNFSF12 (TWEAK) antibody comprising at least one MRD to a patient
in need thereof.
[0671] In an additional embodiment, the invention provides a method
of treating a disorder of the immune system comprising
administering a therapeutically effective amount of a CD25-binding
MRD-containing antibody to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating a disorder
of the immune system comprising administering a therapeutically
effective amount of daclizumab comprising at least one MRD to a
patient in need thereof. In one embodiment, the invention provides
a method of treating an autoimmune disease by administering a
therapeutically effective amount of daclizumab comprising at least
one MRD to a patient in need thereof. In another embodiment, the
invention provides a method of treating rheumatoid arthritis, by
administering a therapeutically effective amount of daclizumab
comprising at least one MRD to a patient in need thereof. In a
further embodiment, the invention provides a method of treating
systemic lupus erythematous comprising administering a
therapeutically effective amount of a daclizumab-MRD-containing
antibody to a patient in need thereof. In another embodiment, the
invention provides a method of treating multiple sclerosis
comprising administering a therapeutically effective amount of a
daclizumab-MRD-containing antibody to a patient in need thereof. In
one embodiment, the invention provides a method of treating an
inflammatory disorder, by administering a therapeutically effective
amount of daclizumab comprising at least one MRD to a patient in
need thereof in another embodiment, the invention provides a method
of treating Crohn's disease, by administering a therapeutically
effective amount of daclizumab comprising at least one MRD to a
patient in need thereof. In a further embodiment, the invention
provides a method of treating ulcerative colitis, by administering
a therapeutically effective amount of daclizumab comprising at
least one MRD to a patient in need thereof. In an additional
embodiment, the invention provides a method of treating psoriatic
arthritis, ankylosing spondylitis, psoriasis, or juvenile
idiopathic arthritis by administering a therapeutically effective
amount of daclizumab comprising at least one MRD to a patient in
need thereof.
[0672] Antibody-MRD fusion proteins having antibodies and/or MRDs
that bind cancer antigens or other targets associated with cancer
establishment, progression, and/or metastasis are described herein
or otherwise known in the art and may be used according to the
methods of the invention to treat cancer. In specific embodiments
the antibody-MRD fusion proteins comprise an antibody and/or MRD
that hind to a target identified herein.
[0673] In another embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of an EGFR-binding MRD-containing antibody to a
patient in need thereof. In a specific embodiment, the invention
provides a method of treating cancer comprising administering a
therapeutically effective amount of cetuximab comprising at least
one MRD to a patient in need thereof. In one embodiment, the
invention provides a method of treating cancer by administering a
therapeutically effective amount of cetuximab comprising at least
one MRD to a patient having colorectal cancer. In another
embodiment, therapeutic effective amounts of cetuximab comprising
at least one MRD are administered to a patient to treat metastatic
colorectal cancer, metastatic breast cancer, metastatic pancreatic
cancer, or metastatic non-small cell lung carcinoma. In one
embodiment, the invention provides a method of treating cancer by
administering a therapeutically effective amount of cetuximab
comprising at least one MRD to a patient having squamous cell
carcinoma of the head and neck.
[0674] In another embodiment, a therapeutically effective amount of
an EGFR-binding MRD-containing antibody is administered in
combination with irinotecan, FOLFIRI, platinum-based chemotherapy,
or radiation therapy. In a specific embodiment, a therapeutically
effective amount of cetuximab comprising at least one MRD is
administered in combination with irinotecan, FOLFIRI,
platinum-based chemotherapy, or radiation therapy.
[0675] In certain embodiments, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of an MRD-antibody described herein to a patient
in need thereof.
[0676] In one embodiment, the invention provides a method of
treating a solid cancer by administering a therapeutically
effective amount of a solid cancer binding MRD-antibody described
herein (e.g., an MRD-antibody that binds a validated solid tumor
associated target as described herein to a patient in need
thereof.
[0677] In some embodiments, the invention provides a method of
treating a solid cancer by administering a therapeutically
effective amount of an MRD-antibody that binds to a member
selected, from the group consisting of: IGFR1, ALK1, p-cadherin,
CRYPTO, and alpha5 b1 integrin. In other embodiments, the antibody
component of the administered MRD-antibody is a member selected
from: figitumumab, CP-870893, PF-3,732,010, PF-3,446,962,
volociximab, BIIB022, and the Biogen CRYPTO antibody.
[0678] In some embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) described herein are
useful for treating cancer. Thus, in some embodiments, the
invention provides methods of treating cancer comprise
administering a therapeutically effective amount of a
MRD-containing antibody to a patient (e.g., a patient (subject) in
need of treatment). In certain embodiments, the cancer is a cancer
selected from the group consisting of colorectal cancer, pancreatic
cancer, lung cancer, ovarian cancer, liver cancer, breast cancer,
brain cancer, kidney cancer, prostate cancer, gastrointestinal
cancer, melanoma, cervical cancer, bladder cancer, glioblastoma,
and head and neck cancer. In certain embodiments, the cancer is
breast cancer. In certain embodiments, the patient, is a human.
[0679] Other examples of cancers or malignancies that may be
treated with MRD containing antibodies and MRDs include, but are
not limited to: Acute Childhood Lymphoblastic Leukemia, Acute
Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid
Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular
Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult
Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft
Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of
the Renal Pelvis and Ureter, Central Nervous System (Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma,
Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood
Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia,
Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma,
Childhood Cerebral Astrocytoma, Childhood Extra cranial Germ Cell
Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood
Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood
Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial
Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,
Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancel,
Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma,
Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic
Cancer, Extra cranial Germ Cell Tumor, Extra gonadal Germ Cell
Tumor, Extra hepatic Bile Duct Cancer, Eye Cancer, Female Breast
Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ
Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia,
Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease,
Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,
Islet Cell Pancreatic Cancer, Kaposit's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung
Cancer, Lymphoproliferative Disorders, Macrogiobulinemia Male
Breast Cancer, Malignant Mesothelioma, Malignant Thymoma,
Medulloblastoma Melanoma, Mesothelioma, Metastatic Occult Primary
Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous
Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer,
Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma
Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant
Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian
Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant
Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary
Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,
Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck
Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal
and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic
Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer,
Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, and Wilms' Tumor.
[0680] In some embodiments, multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are useful for
inhibiting tumor growth. In certain embodiments, the method of
inhibiting the tumor growth comprises contacting the cell with a
MRD-containing antibody in vitro. For example, an immortalized cell
line or a cancer cell line that expresses an MRD target and/or an
antibody target is cultured in medium to which is added the
MRD-containing antibody to inhibit tumor growth. In some
embodiments, tumor cells are isolated from a patient sample for
example, a tissue biopsy, pleural effusion, or blood sample and
cultured in, medium to which is added a MRD-containing antibody to
inhibit tumor growth.
[0681] In some embodiments, the method of inhibiting tumor growth
comprises contacting the tumor or tumor cells with a
therapeutically effective amount of the MRD-containing antibody in
vivo. In certain embodiments, contacting a tumor or tumor cell is
undertaken in an animal model. For example, multivalent and
multispecific compositions (e.g., MRD containing antibodies) can be
administered to xenografts in immunocompromised mice (e.g.,
NOD/SCID mice) to inhibit tumor growth. In some embodiments, cancer
stem cells are isolated from a patient sample for example, a tissue
biopsy, pleural effusion, or blood sample and injected into
immunocompromised mice that are then administered a MRD-containing
antibody to inhibit tumor cell growth. In some embodiments, the
MRD-containing antibody is administered at the same time or shortly
after introduction of tumorigenic cells into the animal to prevent
tumor growth. In some embodiments, the MRD-containing antibody is
administered as a therapeutic after the tumorigenic cells have
grown to a specified size.
[0682] In certain embodiments, the method of inhibiting tumor
growth comprises administering to a patient (subject) a
therapeutically effective amount of a MRD-containing antibody. In
certain embodiments, the patient is a human. In certain
embodiments, the patient has a tumor or has had a tumor removed. In
certain embodiments, the tumor expresses an antibody target. In
certain embodiments, the tumor overexpresses the MRD target and/or
the antibody target.
[0683] In certain embodiments, the inhibited tumor growth is
selected from the group consisting of brain tumor, colorectal
tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor,
breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor,
melanoma, cervical tumor, bladder tumor, glioblastoma, and head and
neck tumor. In certain embodiments, the tumor is a breast
tumor.
[0684] In additional embodiments, multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are useful for
reducing tumorigenicity. Thus, in some embodiments, the method of
reducing the tumorigenicity of a tumor in a patient, comprises
administering a therapeutically effective amount of a multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) to the patient. In certain embodiments, the tumor
comprises cancer stem cells. In certain embodiments, the frequency
of cancer stem cells in the tumor is reduced by administration of
the multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody).
[0685] In other embodiments, multivalent and multispecific
compositions (e.g., MRD containing antibodies) are useful for
diagnosing, treating or preventing a disorder of the immune system.
In one embodiment, the disorder of the immune system is
inflammation or an inflammatory disorder. In a more specific
embodiment, the inflammatory disorder is selected from the group
consisting of asthma, allergic disorders, and rheumatoid arthritis.
In further embodiment, the disorder of the immune system is an
autoimmune disease. Autoimmune disorders, diseases, or conditions
that may be diagnosed, treated or prevented using multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
include, but are not limited to, autoimmune hemolytic anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic
anemia, antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g.,
IgA nephropathy), multiple sclerosis, neuritis, uveitis ophthalmia,
polyendocrinopathies, purpura (e.g., Henloch-scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary
inflammation, myocarditis, IgA glomerulonephritis, dense deposit
disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin
dependent diabetes mellitus, and autoimmune inflammatory eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis, systemic lupus erythematous, discoid lupus,
Goodpasture's syndrome, Pemphigus, Receptor autoimmunities for
example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, rheumatoid arthritis, scleroderma with
anti-collagen antibodies, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's disease, infertility, glomerulonephritis such as, primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes mellitus, and adrenergic drug
resistance (including adrenergic drug resistance with asthma or
cystic fibrosis), chronic active hepatitis, primary biliary
cirrhosis, other endocrine gland failure, vitiligo, vasculitis,
post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulomatous,
degenerative, and atrophic disorders.
[0686] In another embodiment the disorder of the immune system
diagnosed, treated or prevented using multivalent and multispecific
compositions (e.g., MRD-containing antibodies) is selected from the
group consisting of Crohn's disease, Systemic lupus erythematous
(SLE), inflammatory bowel disease, psoriasis, diabetes, ulcerative
colitis, multiple sclerosis, and rheumatoid arthritis. In, a
preferred embodiment, the autoimmune disease is rheumatoid
arthritis
[0687] In other embodiments, a therapeutically effective amount of
a multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) is administered, to a patient to treat a
metabolic disease or disorder.
[0688] In other embodiments, a therapeutically effective amount of
a multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) is administered to a patient to treat a
cardiovascular disease or disorder. In one embodiment, the
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) is administered to a patient to treat thrombosis,
atherosclerosis, heart attack, or stroke.
[0689] In another embodiments, a therapeutically effective amount
of a multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibodies) is administered to a patient to treat a
musculoskeletal disease or disorder.
[0690] In further embodiments, a therapeutically effective amount
of a multivalent and monovalent multispecific composition (e.g.,
MRD-containing antibody) is administered to a patient to treat a
skeletal disease or disorder. In one embodiment, the multivalent
and monovalent multispecific composition (e.g., MRD-containing
antibody) is administered to a patient to treat osteoporosis.
[0691] In additional embodiments, the multivalent and monovalent
multispecific composition binds (1) a target on a cell or tissue of
interest (e.g., a tumor antigen on a tumor cell) and (2) a target
on a leukocyte, such as, a T-cell receptor molecule. According to
one embodiment, the binding of one or more targets by the
multivalent and monovalent multispecific composition is used to
direct an immune response to an infectious agent, cell, tissue, or
other location of interest in, a patient. For example, in some
embodiments an MRD of the multivalent and monovalent multispecific
composition binds a target on the surface of an effector cell.
Thus, in some embodiments, an MRD of the multivalent and monovalent
multispecific composition binds a target on the surface of a T
cell. In specific embodiments an MRD of the multivalent and
monovalent multispecific composition binds CD3. In other
embodiments, an MRD of the multivalent and monovalent multispecific
composition binds CD2. In further embodiments, an MRD of the
multivalent and monovalent multispecific composition binds the
T-cell receptor (TCR). According to additional embodiments, an MRD
of the multivalent and monovalent multispecific composition binds a
target on the surface of a Natural Killer Cell. Thus, in some
embodiments, an MRD of the multivalent and monovalent multispecific
composition binds a NKG2D (Natural Killer Group 2D) receptor. In
additional embodiments an MRD of the multivalent and monovalent
multispecific composition binds CD16 (i.e., Fc gamma RIII) CD64
(i.e., Fc gamma RI), or CD32 (i.e., Fc gamma RII). In additional
embodiments, the multispecific composition contains more than one
monospecific binding site for different targets.
[0692] Thus, in some embodiments, a multivalent and monovalent
multispecific composition (e.g., an MRD-containing antibody) binds
a target on a leukocyte and a tumor antigen on a tumor cell. In
some embodiments, the MRD-containing antibody binds NKG2D. In
further embodiments, an MRD-containing antibody binds NKG2D and a
target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and
EPCAM. In some embodiments, the MRD-containing antibody binds CD3.
In particular embodiments, the MRD-containing antibody binds CD3
epsilon. In further embodiments, an MRD-containing antibody binds
CD3 and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80
and EPCAM. In some embodiments, the MRD-containing antibody binds
CD4. In further embodiments, an MRD-containing antibody binds CD4
and a target selected from ErbB2, EGFR, IGF1R, CD19, CD20, CD80 and
EPCAM.
[0693] In further embodiments, the multivalent and monovalent
multispecific composition has a single binding site (i.e., is
monospecific) for a target. In some embodiments, the multivalent
and monovalent multispecific composition has a single binding site
(i.e., is monospecific) for a target on a leukocyte, such as, a
T-cell (e.g., CD3) and binds a target on a cell or tissue of
interest (e.g., a tumor antigen on a tumor cell, such as, a target
disclosed herein).
[0694] In further embodiments, the invention is directed to
treating a disease or disorder by administering a therapeutically
effective amount of a multivalent and monovalent multispecific
composition that has a single binding site (i.e., is monospecific)
for a target. In some embodiments, the administered multivalent and
monovalent multispecific composition has a single binding site
(i.e., is monospecific) for a target on a leukocyte, such as, a
T-cell (e.g., CD3) and binds a target on a cell or tissue of
interest (e.g., a tumor associated antigen on a tumor cell). In
some embodiments, the tumor cell is from a cancer selected from
breast cancer, colorectal cancer, endometrial cancer, kidney (renal
cell) cancer, lung cancer, melanoma, Non-Hodgkin Lymphoma,
leukemia, prostate cancer, bladder cancer, pancreatic cancer, and
thyroid cancer.
[0695] Additional embodiments are directed to administering a
therapeutically effective amount of a multivalent and monovalent
multispecific composition to treat a neurological disease or
disorder selected from brain cancer, a neurodegenerative disease,
schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease,
Huntington's disease, ALS, multiple sclerosis, Neuromyelitis optica
and Neuro-AIDS (e.g., HIV-associated dementia). In another
embodiment, the multivalent and monovalent multispecific
composition is administered to a patient to treat a brain cancer,
metastatic cancer of the brain, or primary cancer of the brain. In
a further embodiment, the multivalent and monovalent multispecific
composition is administered to a patient to treat brain injury,
stroke, spinal cord injury, or pain management. In further
embodiments, the multivalent and monovalent multispecific
composition is administered to a patient to treat brain injury,
stroke, or spinal cord injury, or for pain management.
[0696] In one embodiment, a therapeutically effect amount of the
multivalent and monovalent multispecific composition is
administered to a patient to treat an infection or a symptom
associated with an infection caused by an infectious agent. In some
embodiments, the infection is caused by a member selected from
apovavirus (e.g., JC polyomavirus), trypanosomes, West Nile virus,
HIV, Streptococcus pneumoniae and Haemophilus influenzae, bovine
spongiform encephalopathy, meningitis, Progressive multifocal
leukoencephalopathy (PML), Late-stage neurological trypanosomiasis,
Encephalitis, and rabies.
[0697] According to some embodiments, the multivalent and
monovalent multispecific composition (e.g., MRD-containing
antibody) is able to cross the blood brain barrier (BBB) and bind a
target located on the brain side of the BBB. In additional
embodiments, the multivalent and monovalent multispecific
composition has a single binding site that binds a target (e.g.,
ligand, receptor, or accessory protein) associated with an
endogenous BBB receptor mediated transport system. In some
embodiments, a single binding site of the composition is an MRD. In
other embodiments, a single binding site of the composition is an
antibody antigen binding domain. In some embodiments, the
multivalent and monovalent multispecific composition contains 1, 2,
3, 4, 5, or more single binding sites (i.e., monovalently binds)
for a target associated with an endogenous BBB receptor mediated
transport system and the composition is able to cross to the
cerebrospinal fluid side of the BBB. In additional embodiments, the
multivalent and monovalent multispecific composition contains 1, 2,
3, 4, 5, or more, multiple binding sites (i.e., multivalently
binds) for a target associated with an endogenous BBB receptor
mediated transport system and the composition is able to cross to
the cerebrospinal fluid side of the BBB. In additional embodiments,
a therapeutically effective amount of an MRD-containing antibody is
administered to a patient to treat a neurological disease or
disorder selected from brain cancer, a neurodegenerative disease,
schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease,
Huntington's disease, ALS, multiple sclerosis, neuromyelitis optica
and beuro-AIDS (e.g., HIV-associated dementia). In some
embodiments, the multivalent and monovalent multispecific
composition has a single binding site (i.e., is monovalent for
binding a particular target (antigen)) or two Or more binding sites
(i.e., is monovalent for binding a particular target) for a target
selected from alpha-synuclein, RGM A, NOGO A, NgR, OMGp MAG, CSPG,
neurite inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin
4) an ephrin, A-beta, AGE (S100 A, amphoterin), NGF, soluble A-B,
aggrecan, midkine, neurocan, versican, phosphacan, Te38 and PGE2.
In some embodiments, the multivalent and monovalent multispecific
composition additionally has a single binding site or multiple
binding sites for a target selected from IL1, IL1R, IL6, IL6R,
IL12, IL18, IL23, TNFSF12 (TWEAK), TNFRSF5 (CD40), TNFSF5 (CD40
LIGAND), CD45RB, CD52, CD200, VEGF, VLA4, TNF alpha, Interferon
gamma, GMCSF, FGF, C5, CXCL13, CCR2, CB2, MIP 1a, and MCP-1.
[0698] In additional embodiments, the multivalent and monovalent
multispecific composition is capable of transferring to the
cerebrospinal fluid side of the BBB and is administered to a
patient to treat a neurological disease or disorder selected from:
brain cancer, a neurodegenerative disease, schizophrenia, epilepsy,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
ALS, multiple sclerosis, neuromyelitis optica and neuro-AIDS (e.g.,
HIV-associated dementia). In further embodiments, the invention is
directed to treating a disease or disorder by administering an
MRD-containing antibody that has a single binding site (i.e., is
monospecific) for a target to a patient in need thereof. In some
embodiments, the administered MRD-containing antibody has a single
binding site (i.e., is monospecific) for a target on a leukocyte,
such as, a T-cell (e.g., CD3) and binds a target on a cell or
tissue of interest (e.g., a tumor associated antigen on a tumor
cell).
[0699] In some embodiments, the multivalent and monovalent
multispecific composition is administered to a patient to treat a
neurological disease or disorder selected from brain cancer, a
neurodegenerative disease, schizophrenia, epilepsy, Alzheimer's
disease, Parkinson's disease, Huntington's disease, ALS, multiple
sclerosis, Neuromyelitis optica and Neuro-AIDS (e.g. HIV-associated
dementia). In additional embodiments, the multivalent and
monovalent multispecific composition is administered to a patient
to treat a brain cancer, metastatic cancer of the brain, or primary
cancer of the brain. In additional embodiments, the multivalent and
monovalent multispecific composition is administered to a patient
to treat brain injury, stroke, spinal cord injury, or pain. Thus,
according to some embodiments, the disease, disorder, or injury
treated or prevented with an MRD-containing antibody or MRD of the
invention is neurological. In one embodiment, the neurological
disease, disorder or injury is associated with pain such as, acute
pain or chronic pain.
[0700] In some embodiments the multivalent and monovalent
multispecific composition binds at least 1, 2, 3, 4, or 5 targets
associated with a neurological disease or disorder. In one
embodiment, the multivalent and monovalent multispecific
composition (e.g., MRD-containing antibody) binds 1, 2, or all 3 of
the targets RGM A; NgR, and NogoA. In another embodiment, the
multivalent and monovalent multispecific composition hinds 1, 2, 3,
or all 4 of RGM A, RGM B, and Semaphorin 3A or Semaphorin 4. In a
further embodiment, the multivalent and monovalent multispecific
composition binds at least 1, 2, 3, 4 or 5 targets selected from
aggrecan, midkine, neurocan, versican, phosphacan, Te38, TNF alpha,
NogoA, RGM A, MAG, and OMGp. In another embodiment, the multivalent
and monovalent multispecific composition binds at least 1, 2, 3, 4
or 5 targets selected from aggrecan, midkine, neurocan, versican,
phosphacan, Te38 and TNF alpha. In an alternative embodiment, the
multivalent and monovalent multispecific composition binds at least
1, 2, 3, 4 or 5 targets selected from NgR-.alpha.75, NgR-Troy,
NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG and Omgp.
In another embodiment, the multivalent and monovalent multispecific
composition binds at least 1, 2, 3, 4 or 5 targets selected from
NGF, prostaglandin E2 (PGE2), TNF-alpha, IL1 beta, and IL6R.
[0701] In an additional embodiment, the multivalent and monovalent
multispecific composition binds at least 1, 2, 3, 4 or 5 targets
selected from alpha-synuclein, RGM A and one or more
pro-inflammatory mediators (e.g., TNF alpha, IL1, and MCP-1). Such
compositions have applications in, for example, treating
neurodegenerative diseases such as, Parkinson's.
[0702] In another embodiment, the multivalent and monovalent
multispecific composition binds and antagonizes (i.e., is an
antagonist of e.g., inhibits the activity of) 1, 2, 3, 4 or 5
targets selected from RGM A, NOGO A, neurite inhibiting semaphorins
(e.g., Semaphorin 3A and Semaphorin 4), ephrins and
pro-inflammatory targets (e.g., IL12, TNFSF12 (TWEAK), IL23,
CXCL13, TNFRSF5 (CD40), TNFSF5 (CD40 LIGAND), IL18, VEGF, VLA4, TNF
alpha, CD45RB, CD200, interferon gamma, GMCSF, FGF, C5, CD52, and
CCR2). In an additional embodiment, the multivalent and monovalent
multispecific composition binds and antagonizes 1, 2, 3, 4 or 5
targets selected from CD3, IL2, IL2R, IL6, IL6R, IL10, IL12p40,
IL23, TGF beta, TNFRSF21 (DR6), fn14, CD20, LINGO, CXCL13 and CCL2.
The compositions have applications in treating for example,
inflammation, neuroregeneration and neurodegenerative disorders,
such as MS). Multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) that bind at least 1, 2, 3, 4, 5 or more
of these targets are also encompassed by the invention. In specific
embodiments, the antibody component of the MRD-containing antibody
binds CD3, CD20, CD52, VLA4, TNF, TNFRSF21 (DR6), LINGO, CD3,
interferon gamma or IL6.
[0703] In another embodiment, the multivalent and monovalent
multispecific composition binds and antagonizes (i.e., is an
antagonist of) 1, 2, 3, 4 or 5 targets selected from AGE (S100 A,
amphoterin), pro-inflammatory cytokines (e.g., IL1, IL6, and TNF),
chemokines (e.g., MCP 1), and molecules, that inhibit neural
regeneration (e.g., Nogo and RGM A). These compositions have
applications in treating, for example, chronic neurodegenerative
diseases such as, Alzheimer's. In an additional embodiment, the
composition of the invention binds at least 1, 2, 3, 4 or 5 targets
that influence neural generation and survival including, for
example, NGF agonists, IL1 or IL1R antagonists, and A-beta. These
compositions have applications in treating, for example, chronic
neurodegenerative diseases such as, Alzheimer's.
[0704] In an additional embodiment, the composition of the
invention binds to and antagonizes 1, 2, 3, 4, or 5 targets that
targets that interfere with neural regeneration or recovery,
including NogoA, OMgp MAG, RGM A, CSPG, one or more astrocyte
inhibiting semaphorins (e.g., Semaphorin 3A and Semaphorin 4),
ephrins, and pro-inflammatory cytokines (e.g., IL1, IL6, and TNF).
These compositions have applications in treating neurodegenerative
diseases and neural injury or trauma.
[0705] In additional embodiment, the multivalent and monovalent
multispecific composition binds and antagonize (i.e., is an
antagonist of) 1, 2, 3, 4, or 5 targets associated with pain,
including, but not limited to, NGF and SCN9A/NAV1.7. Such
compositions have applications in for example, treating or
alleviating pain and pain associated conditions.
[0706] In additional embodiments, the targets bound by the
compositions of the invention binds and antagonizes 1, 2, 3, 4, 5
or more mediators and or soluble or cell surface targets implicated
in the inhibition of neurite growth or recovery. In specific
embodiments, compositions of the invention bind to and antagonizes
1, 2, 3, 4, 5 or more targets selected from Nogo, Ompg, MAG, RGM A,
semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines
(e.g., IL1 and TNF alpha), chemokines (e.g., MIP 1a).
[0707] In some embodiments, the invention provides a method of
treating or ameliorating pain by administering a therapeutically
effective amount of a pain target binding MRD-antibody, to a
patient in need thereof. In additional embodiments, the invention
provides a method of treating or ameliorating pain by administering
a therapeutically effective amount of an NGF binding MRD-antibody,
to a patient in need thereof. In further embodiments, the invention
provides a method of treating or ameliorating pain by administering
a therapeutically effective amount of tanezumumab (e.g., Pfizer)
comprising an MRD, to a patient in need thereof.
[0708] In additional embodiments, the invention provides a method
of treating or ameliorating Alzheimer's by administering a
therapeutically effective amount of an Alzheimer's target binding
MRD-antibody, to a patient in need thereof in additional
embodiments, the invention provides a method of treating or
ameliorating Alzheimer's by administering a therapeutically
effective amount of a beta amyloid binding MRD-antibody, to a
patient in need thereof. In additional embodiments, the invention
provides a method of treating or ameliorating Alzheimer's by
administering a therapeutically effective amount of RN1219
(PF-4,360,365; Pfizer) comprising an MRD, to a patient in need
thereof.
[0709] In additional embodiments, the invention provides a method
of treating or ameliorating multiple sclerosis by administering a
therapeutically effective amount of a multiple sclerosis target
binding MRD-antibody, to a patient in need thereof. In additional
embodiments, the invention provides a method of treating or
ameliorating multiple sclerosis by administering a therapeutically
effective amount of a LINGO binding MRD-antibody, to a patient in
need thereof. In another embodiment, the invention provides a
method of treating or ameliorating multiple sclerosis by
administering a therapeutically effective amount of an MRD-antibody
that binds LINGO and TNFRSF21 (DR6) to a patient in need thereof.
In additional embodiments, the invention provides a method of
treating or ameliorating multiple sclerosis by administering a
therapeutically effective amount of the Biogen LINGO antibody
comprising an MRD, to a patient in need thereof. In further
embodiments, the invention provides a method of treating or
ameliorating multiple sclerosis by administering a therapeutically
effective amount of the natalizumab (e.g., TYSABRI.RTM.; Biogen)
comprising an MRD, to a patient in need thereof. In an additional
embodiment, the invention provides a method of treating or
ameliorating multiple sclerosis by administering a therapeutically
effective amount of the Biogen LINGO antibody comprising an MRD, to
a patient in need thereof
[0710] In an additional embodiment, the invention provides a method
of treating or ameliorating multiple sclerosis by administering a
therapeutically effective amount of a CD20 binding MRD-antibody, to
a patient in need thereof. In one embodiment, the invention
provides a method of treating or ameliorating multiple sclerosis by
administering a therapeutically effective amount of the ocrelizumab
(Biogen Idec) comprising an MRD, to a patient in need thereof.
[0711] In other embodiments, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are useful for
treating or preventing an infectious disease. Infectious diseases
that may be treated or prevented with multivalent and multispecific
compositions (e.g., MRD-containing antibodies) include, but are not
limited to, diseases associated with yeast, fungal, viral and
bacterial infections. Viruses causing viral infections which can be
treated or prevented with multivalent and multispecific
compositions (e.g., MRD-containing antibodies) include, but are not
limited to, retroviruses (e.g., human T-cell lymphotrophic virus
(HTLV) types I and II and human immunodeficiency virus (HIV)),
herpes viruses (e.g., herpes simplex virus (HSV) types I and II,
Epstein-Barr virus, HHV6-HHV8, and cytomegalovirus), adenoviruses
(e.g., lassa fever virus), paramyxoviruses (e.g., morbilbiviras
virus, human respiratory syncytial virus, mumps, and pneumovirus),
adrenoviruses, bunyaviruses (e.g., hantavirus), cornaviruses,
filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C
virus (HCV), yellow fever virus, and Japanese encephalitis virus),
hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses
(e.g., influenza viruses A, B and C (including avian influenza,
e.g., H5N1 subtype)), papovaviruses (e.g., papillomaviruses),
picornaviruses (e.g., rhinoviruses, enteroviruses and hepatitis A
viruses), poxviruses, reoviruses (e.g., rotaviruses), togaviruses
(e.g., rubella virus), rhabdoviruses (e.g., rabies virus).
Microbial pathogens causing bacterial infections include, but are
not limited to, Streptococcus pyogenes, Streptococcus pneumoniae,
Neisseria gonorrhoea, Neissetia meningitidis, Corynebacterium
diphtheriae, Clostridium botulinum, Clostridium pefringens,
Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae,
Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus
aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa,
Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas
hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia
enterocolitica Yersinia pestis, Yersinia pseudotuberculosis,
Shigella dysenteriae, Shigella flexneri, Shigella sonnei,
Salmonella typhimurium, Treponema pallidum, Treponema pertenue,
Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,
Leptospira icterohernorrhagiae, Mycobacterium tuberculosis,
Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis,
Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma
spp., Rickettsia prowazeki, Rickettsia Lsutsugamushi, Chlamydia
spp., and Helicobacter pylori.
[0712] In a preferred embodiment, the multivalent and multispecific
compositions (e.g., MRD-containing antibodies) are administered to
a patient to treat or prevent human immunodeficiency virus (HIV)
infection or AIDS, botulism, anthrax, or clostridium difficile.
VIII MRD LINKED COMPOUNDS THAT ARE NOT ANTIBODIES
[0713] In a distinct group of embodiments, one or more MRDs of the
invention are operably linked to the amino and/or carboxy terminus
of an immunoglobulin fragment, such as Fab, Fab',
F(ab.sup.').sub.2, pFc', or Fc. In some embodiments, the MRDs, are
operably linked to a Fab or Fc polypeptide containing an additional
Ig domain. In some embodiments, the MRDs are operably linked to the
amino and/or carboxy terminus of an immunoglobulin fragment that is
also operably linked to an scFv. In other embodiments, the MRDs of
the invention are operably linked to an Fc-fusion protein.
[0714] According to this group of embodiments, one two, three,
four, five, six, seven to ten, or more than ten MRDs are operably
linked to the amino terminus and/or carboxy terminus of the
immunoglobulin fragment. These MRDs are optionally linked to one
another or to the immunoglobulin fragment via a linker. In one
embodiment, one, two, three, four, five, six, seven to ten, or more
than ten, of the MRDs operably linked to the amino terminus and/or
carboxy terminus of the immunoglobulin fragment are the same. In
another embodiment, one, two, three, four, five, six, seven to ten,
or more than ten, of the MRDs operably linked to the amino terminus
and/or carboxy terminus of the immunoglobulin fragment are
different.
[0715] The MRDs operably linked to the immunoglobulin fragment can
be monomeric (i.e., containing one MRD at the terminus of a peptide
chain optionally connected by a linker) or multimeric (i.e.,
containing more than one MRD in tandem optionally connected by a
linker). The MRDs can be homo-multimeric (i.e., containing more
than one of the same MRD in tandem optionally connected by
linker(s) (e.g., homodimers, homotrimers, homotetramers etc.)) or
hetero-multimeric (i.e., containing two or more MRDs in which there
are at least two different MRDs optionally connected by linker(s)
where all or some of the MRDs linked to a particular terminus are
different (e.g., heterodimer)). In one embodiment, two different
monomeric MRDs are located at different termini of the
immunoglobulin fragment. In another embodiment, three, four, five,
six, or more different monomeric MRDs are located at different
termini of the immunoglobulin fragment.
[0716] In an alternative embodiment, the MRD-containing antibody
contains at least one dimeric and one monomeric MRD located at
different immunoglobulin termini. In another alternative
embodiment, the MRD-containing antibody contains at least one
homodimeric and one monomeric MRD located at different
immunoglobulin termini. In another alternative embodiment, the
MRD-containing antibody contains at least one heterodimeric and one
monomeric MRD located at different immunoglobulin termini.
[0717] In an alternative embodiment, the MRD-containing antibody
contains at least one multimeric and one monomeric MRD located at
different immunoglobulin termini. In another alternative
embodiment, the MRD-containing antibody contains at least one
homomultimeric and one monomeric MRD located at different
immunoglobulin termini. In another alternative embodiment, the
MRD-containing antibody contains at least one heteromultimeric and
one monomeric MRD located at different immunoglobulin termini.
[0718] Multiple MRDs that are operably linked to the immunoglobulin
fragment can target the same target binding site, or two or more
different target binding sites. Where the MRDs bind to different
target binding sites, the binding sites may be on the same or
different targets. Similarly, one or more of the MRDs may bind to
the same target as the immunoglobulin fragment.
[0719] In some embodiments, at least one of the MRDs and if
applicable, the immunoglobulin fragment (e.g., where the
immunoglobulin fragment is an Fab), bind to their targets
simultaneously. In additional embodiments, two, three, four, five,
six, seven, eight, nine, ten, or more than ten MRDs, and if
applicable the immunoglobulin fragment, bind to their targets
simultaneously.
[0720] The synthesis of MRDs operably linked to an immunoglobulin
fragment and the assay of these MRDs and immunoglobulin fragment
for their ability to bind, or compete for binding with one or more
targets simultaneously can be routinely accomplished using methods
disclosed herein or otherwise known in the art.
[0721] In a specific embodiment, one or more of the operably linked
MRDs or the immunoglobulin fragment, binds to VEGF. In another
specific embodiment, one or more of the operably linked MRDs or the
immunoglobulin fragment, binds, to the same epitope as ranibizumab
(LUCENTIS.RTM., Genentech). In another specific embodiment, one or
more of the operably linked MRDs or the immunoglobulin fragment,
competitively inhibits ranibizumab binding to VEGF. In an
additional embodiment, the immunoglobulin fragment is a Fab. In a
further specific embodiment, the immunoglobulin fragment is
ranibizumab.
[0722] In another embodiment, the invention provides a method of
treating macular degeneration comprising administering a
therapeutically effective amount of a VEGFA or EGFR binding
MRD-immunoglobulin fragment fusion to a patient in need thereof. In
a specific embodiment, the invention provides a method of treating
macular degeneration comprising administering a therapeutically
effective amount of a VEGFA or VEGFR binding MRD-Fab fusion to a
patient in need thereof. In a specific embodiment, the invention
provides a method of treating macular degeneration comprising
administering a therapeutically effective amount of MRD-ranibizumab
to a patient, in need thereof.
[0723] In other embodiments the one or more MRDs of the invention
are operably linked to the amino and/or carboxyl terminus of an Fc
fusion protein. The Fc fusion protein can contain fusions to any
protein or polypeptide sequence of therapeutic value, for example,
any of the targets or receptors of the targets described herein.
For example, the fusions can contain the extracellular domain of
receptors or ligands that typically function or display improved
cognate-partner binding in multimeric form, including for example,
receptors corresponding to the TNF-R superfamily (e, g., TNFR2,
TACI, BCMA, HVEM, etc.), IL receptor superfamily (e.g.,
IL1-R-IL6R), EGFR superfamily (e.g., VEGFR1-VEGR3), FGRFR
superfamily (e.g., FGFR1-FGFR4), and B7 superfamily (e.g.,
CTLA)).
[0724] In a specific embodiment, one, two, three, four, five, six,
or more MRDs are operably linked to a VEGR1/VEGFR2-Fc fusion
protein. In another specific embodiment, one or more of the
operably linked MRDs bind to the same epitope as aflibercept
(Regeneron). In another specific embodiment, one or more of the
operably linked MRDs competitively inhibit aflibercept binding to
VEGFA or PLGF. In a further specific embodiment, the MRDs are
operably linked to aflibercept.
[0725] In another embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of an MRD-VEGFR1-VEGFR2-Fc fusion protein to a
patient in need thereof in a specific embodiment, the invention
provides a method of treating colorectal cancer, prostate cancer,
or non-small cell lung cancer comprising administering a
therapeutically effective amount of a VEGFA or PLGF binding MRD-Fc
fusion protein to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating macular
degeneration comprising administering a therapeutically effective
amount of a VEGFA or PLGF binding MRD-Fc fusion protein and
irinotecan, 5FU, oxaliplatin, doxetaxel, or FOLFOX6, to a patient
in need thereof.
[0726] In another embodiment, the invention provides a method of
treating cancer comprising administering a therapeutically
effective amount of MRD-aflibercept to a patient in need thereof.
In a specific embodiment, the invention provides a method of
treating colorectal cancer, prostate cancer, or non-small cell lung
cancer comprising administering a therapeutically effective amount
of MRD-aflibercept to a patient in need thereof. In a specific
embodiment, the invention provides a method of treating macular
degeneration comprising administering a therapeutically effective
amount of MRD-aflibercept and irinotecan, 5FU, oxaliplatin,
doxetaxel, or FOLFOX6, to a patient in need thereof.
[0727] In a specific embodiment, one, two, three, four, five, six,
or more MRDs are operably linked to a CTLA4-Fc fusion protein. In
another specific embodiment, one or more of the operably linked
MRDs bind to the same epitope as abatacept (ORENCIA.RTM.). In
another specific embodiment, one or more of the operably linked
MRDs competitively inhibits abatacept binding to CD80 (B7-1) or
CD86 (B7-2). In a further specific embodiment, the MRDs are
operably linked to abatacept. In another specific embodiment, one
or more of the operably linked MRDs bind to the same epitope as
belatacept (Bristol Myers Squibb). In another specific embodiment,
one or more of the operably linked MRDs competitively inhibits
belatacept binding to CD80 (B7-1) or CD86 (B7-2). In an additional
embodiment, the immunoglobulin fragment is a Fab. In a further
specific embodiment, the MRDs are operably linked to
belatacept.
[0728] In another embodiment, the invention provides a method of
suppressing an immune response comprising administering a
therapeutically effective amount of an MRD-CTLA4-Fc fusion protein
to a patient in need thereof. In a specific embodiment, the
invention provides a method suppressing an immune response
comprising administering a therapeutically effective amount of
MRD-abatacept to a patient in need thereof. In another specific
embodiment, the invention provides a method of treating rheumatoid
arthritis comprising administering a therapeutically effective
amount of MRD-abatacept to a patient in need thereof. In another
specific embodiment, the invention provides a method of suppressing
an immune response to a graft rejection comprising administering a
therapeutically effective amount of MRD-abatacept to a patient in
need thereof.
[0729] In a specific embodiment, the invention provides a method of
suppressing an immune response comprising administering a
therapeutically effective amount of MRD-belatacept to a patient in
need thereof. In another specific embodiment, the invention
provides a method of suppressing an immune response to a graft
rejection comprising administering a therapeutically effective
amount of MRD-belatacept to a patient in need thereof.
[0730] In another specific embodiment, one two, three, four, five,
six, or more MRDs are operably linked to a TNFR2-Fc fusion protein.
In another specific embodiment, one or more of the operably linked
MRDs bind to the same epitope as etanercept (ENBREL.RTM.). In
another specific embodiment, one or more of the operably linked
MRDs competitively inhibits etanercept binding to TNF alpha. In
another embodiment, one or more of the operably linked MRDs binds
ANG2. In a further specific embodiment, the MRDs are operably
linked to etanercept.
[0731] In another embodiment, the invention provides a method of
suppressing an immune response comprising administering a
therapeutically effective amount of a MRD-TNFR2-Fc fusion protein
to a patient in need thereof. In one embodiment, the invention
provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of a MRD-TNFR2-Fc
fusion protein to a patient in need thereof. In one embodiment, the
invention provides a method of treating rheumatoid arthritis, by
administering a therapeutically effective amount of an MRD-TNFR2-Fc
fusion protein to a patient in need thereof. In one embodiment, the
invention provides a method of treating an inflammatory disorder,
by administering a therapeutically effective amount of an
MRD-TNFR2-Fc fusion protein to a patient in need thereof. In
another embodiment, the invention provides a method of treating
Crohn's disease, by administering a therapeutically effective
amount of an MRD-TNFR2-Fc fusion protein to a patient in need
thereof. In another embodiment, the invention provides a method of
treating ulcerative colitis, by administering a therapeutically
effective amount of an MRD-TNFR2-Fc fusion protein to a patient in
need thereof. In another embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering a
therapeutically effective amount of an MRD-TNFR2-Fc fusion protein
to a patient in need thereof.
[0732] In another embodiment, the invention provides a method of
suppressing an immune response comprising administering a
therapeutically effective amount of a MRD-etanercept-Fc fusion
protein to a patient in need thereof. In one embodiment the
invention provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of MRD-etanercept
to a patient in need thereof. In one embodiment, the invention
provides a method of treating rheumatoid arthritis, by
administering a therapeutically effective amount of MRD-etanercept
to a patient in need thereof. In one embodiment, the invention
provides a method of treating an inflammatory disorder, by
administering a therapeutically effective amount of MRD-etanercept
to a patient in need thereof. In another embodiment, the invention
provides a method of treating Crohn's disease, by administering a
therapeutically effective amount of MRD-etanercept to a patient in
need thereof. In another embodiment, the invention provides a
method of treating ulcerative colitis, by administering a
therapeutically effective amount of MRD-etanercept to a patient in
need thereof. In another embodiment, the invention provides a
method of treating psoriatic arthritis, ankylosing spondylitis,
psoriasis, or juvenile idiopathic arthritis by administering, a
therapeutically effective amount of MRD-etanercept to a patient in
need thereof.
[0733] In another specific embodiment, one, two, three, four, five,
six, or more MRDs are operably linked to a TACI-Fc fusion protein.
In another specific embodiment, one or more of the operably linked
MRDs bind to the same epitope as atacicept (Merck/Serono). In
another specific embodiment, one or more of the operably linked
MRDs competitively inhibits atacicept binding to BLyS or APRIL. In
a further specific embodiment, the MRDs are operably linked to
atacicept.
[0734] In another embodiment, the invention provides a method of
suppressing an immune response comprising administering a
therapeutically effective amount of a MRD-TACI-Fc fusion protein to
a patient in need thereof. In one embodiment, the invention
provides a method of treating an autoimmune disease by
administering a therapeutically effective amount of a MRD-TACI-Fc
fusion protein to a patient in need thereof. In one embodiment, the
invention provides a method of treating rheumatoid arthritis, by
administering a therapeutically effective amount of a MRD-TACI-Fc
fusion protein to a patient in need thereof. In one embodiment, the
invention provides a method of treating systemic lupus erythematous
by administering a therapeutically effective amount of a
MRD-TACI-Fc fusion protein to a patient in need thereof. In another
embodiment, the invention provides a method of suppressing an
immune response comprising administering a therapeutically
effective amount of an MRD-atacicept fusion protein to a patient in
need thereof. In one embodiment, the invention provides a method of
treating an autoimmune disease by administering a therapeutically
effective amount of an MRD-atacicept fusion protein to a patient in
need thereof. In one embodiment, the invention provides a method of
treating rheumatoid arthritis, by administering a therapeutically
effective amount of an MRD-atacicept protein fusion protein to a
patient in need thereof. In one embodiment, the invention provides
a method of treating systemic lupus erythematous, by administering
a therapeutically effective amount of an MRD-atacicept fusion
protein to a patient in need thereof.
[0735] In another specific embodiment, one two, three, four, five,
six, or more MRDs are operably linked to an IL1R-Fc fusion protein.
In another specific embodiment, one or more of the operably linked
MRDs bind to the same epitope as rilonacept (Regeneron). In another
specific embodiment, one or more of the operably linked MRD
competitively inhibits rilonacept binding to IL1R. In a further
specific embodiment, the MRDs are operably linked to
rilonacept.
[0736] In another embodiment, the invention provides a method of
preventing gout comprising administering a therapeutically
effective amount of a MRD-IL1R-Fc fusion protein to a patient in
need thereof. In, a specific embodiment, the invention provides a
method of preventing gout comprising administering a
therapeutically effective amount of an MRD-rilonacept-Fc fusion
protein to a patient in need thereof.
[0737] In some embodiments, the invention encompasses a complex
comprising an antibody and at least one modular recognition domain
(MRD), wherein the MRD comprises at least two cysteines, wherein a
first cysteine is located within the first ten amino acids of the
MRD, a second cysteine is located within the last ten amino acids
of the MRD, and wherein the MRD comprises at least five amino acids
between said first cysteine and said second cysteine. In additional
embodiments, the MRD comprises at least 10, 15, 20, or 25 amino
acids between the first cysteine and the second cysteine. In some
embodiments, the MRD comprises at least one proline between the
first cysteine and the second cysteine. In other embodiments, the
MRD comprises at least two proline between the first cysteine and
the second cysteine. In some embodiments, the first cysteine is no
more than 5, 3, 3, 2, or 1 amino acids away from the N-terminus of
the complex. In some embodiments, the second cysteine is no more
than 5, 3, 3, 2, or 1 amino acids away from the C-terminus of the
complex. In some embodiments, the in vivo half-life of an MRD in a
complex of the invention is increased compared to the half-life of
an MRD in a corresponding complex wherein at least one of the
cysteines is mutated or deleted. In some embodiments, the binding
affinity of the MRD is at least equal to the binding affinity of an
MRD in a corresponding complex wherein at least one of the
cysteines is mutated or deleted.
[0738] In some embodiments, the invention encompasses a complex
comprising an antibody and at least one modular recognition domain
(MRD), wherein the antibody and the MRD bind to different targets
or epitopes on the same cell or molecule, wherein the MRD binding
agonizes or antagonizes the MRD target under physiological
conditions, and wherein said MRD does not bind to and agonize or
antagonize said MRD target under physiological conditions in the
absence of said antibody. In some embodiments, an MRD in the
complex of the invention binds the MRD target in the absence of the
antibody with an EC50 of greater than 0.01 nM, 0.1 nM, 0.5 nM, or
0.7 nM under physiological conditions.
[0739] In some embodiments, the invention encompasses a complex
comprising an antibody and at least one MRD, wherein the antibody
and the MRD bind to different targets or epitopes on a heteromeric
or homomeric protein, wherein the MRD binding agonizes or
antagonizes the MDR target under physiological conditions, and
wherein said MRD does not bind to and agonize or antagonize said
MRD target under physiological conditions in the absence of the
antibody. In some embodiments, an MRD in the complex of the
invention binds the MRD target with an EC50 of greater than 0.01
nM, 0.1 nM, 0.5 nM, or 0.7 nM under physiological conditions.
[0740] In some embodiments, the invention encompasses a method for
inhibiting the growth of a cell comprising contacting the cell with
a multispecific and multivalent complex comprising an antibody and
at least one modular recognition domain (MRD), and a protein kinase
inhibitor. In some embodiments, the antibody binds to a target
selected from: VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and
FGFR2. In some embodiments, the protein kinase inhibitor inhibits a
target of the MRD containing antibody. In some embodiments, the
protein kinase inhibitor inhibits a different target than the MRD
containing antibody. In some embodiments, the protein kinase
inhibitor inhibits more than one protein kinase. In some
embodiments, the protein kinase inhibitor is a member selected
from: imatinib, gefitinib, vandetanib, erlotinib, sunitinib,
lapatinib, and sorafenib.
[0741] In some embodiments, the invention encompasses a method for
inhibiting angiogenesis in a patient, comprising administering to
said patient a therapeutically effective amount of a multispecific
and multivalent complex comprising an antibody and at least one
modular recognition domain (MRD), and a protein kinase inhibitor.
In some embodiments, the antibody binds to a target selected from:
VEGF, VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and FGFR2. In some
embodiments, the protein kinase inhibitor inhibits a target of the
MRD containing antibody. In some embodiments, the protein kinase
inhibitor inhibits a different target than the MRD containing
antibody. In some embodiments, the protein kinase inhibitor
inhibits more than one protein kinase. In some, embodiments, the
protein kinase inhibitor is a member selected from imatinib,
gefitinib, vandetanib, erlotinib, sunitinib, lapatinib, and
sorafenib.
[0742] In some embodiments, the invention encompasses a method for
treating a patient having cancer comprising administering to said
patient a therapeutically effective amount of a multispecific and
multivalent complex comprising an antibody and at least one modular
recognition domain (MRD), and a protein kinase inhibitor. In some
embodiments, the antibody binds to a target selected from VEGF,
VEGFR1, EGFR, ErbB2, IGF-IR, cMET, FGFR1, and FGFR2. In some
embodiments, the protein kinase inhibitor inhibits a target of the
MRD containing antibody. In some embodiments, the protein kinase
inhibitor inhibits a different target than the MRD containing
antibody. In some embodiments, the protein kinase inhibitor
inhibits more than one protein kinase. In some embodiments, the
protein kinase inhibitor is a member selected from imatinib,
gefitinib, vandetanib, erlotinib, sunitinib, lapatinib, and
sorafenib.
[0743] In some embodiments, the invention encompasses a method for
treating a patient having a disease or disorder of the immune
system comprising administering to said patient a therapeutically
effective amount of a multispecific and multivalent complex
comprising an antibody and at least one modular recognition domain
(MRD), and a protein kinase inhibitor. In some embodiments, the
disease or disorder of the immune system is inflammation or an
autoimmune disease. In further embodiments, the autoimmune disease
is rheumatoid arthritis, Crohn's disease, systemic lupus
erythematous, inflammatory bowel disease, psoriasis, diabetes,
ulcerative colitis, or multiple sclerosis. In additional
embodiments, the antibody binds TNF. In some embodiments, the
protein kinase inhibitor inhibits a target that is not a target of
the MRD-containing antibody. In some embodiments, the protein
kinase inhibitor inhibits more than one protein kinase. In further
embodiments, the protein kinase inhibitor is a member selected from
lestaurtinib, tofacitinib, ruxolitinib, SB1518, CYT387, LY3009104,
TG101348, fostamatinib, BAY 61-3606, and sunitinib.
[0744] In some embodiments, the invention encompasses a multivalent
and multispecific complex comprising an antibody and at least one
modular recognition domain (MRD), wherein the complex has a single
binding site for a cell surface target. In some embodiments, the
multivalent and multispecific complex comprises 2 single binding
sites for different epitopes on the same target. In some
embodiments, the multivalent and multispecific complex has 2, 3, 4,
5 or more single binding sites for different targets. In some
embodiments, the multivalent and multispecific complex has a single
binding site for a target on a leukocyte. In some embodiments, the
multivalent and multispecific complex has a single binding site for
a target on a T-cell. In some embodiments, the multivalent and
multispecific complex has a single binding site for CD3. In further
embodiments, complex has a single binding site for CD3 epsilon. In
additional embodiments, the complex has a single binding site for a
target on a natural killer cell. In additional embodiments, the
complex has multiple binding sites for a target on a diseased cell.
In some embodiments, the complex has multiple binding sites for 2,
3, 4, 5 or more targets on a diseased cell. In additional
embodiments, the complex has multiple binding sites for a target on
a tumor cell. In further embodiments, the complex has multiple
binding sites for 2, 3, 4, 5 or more targets on a tumor cell. In
some embodiments, the complex has multiple binding sites for a
target on an immune cell. In further embodiments, the complex has
multiple binding sites for 2, 3, 4, 5 or more targets on an immune
cell. In some embodiments, the complex has a single binding site
for a target on a natural killer cell. In some embodiments, the
complex binds a target on a leukocyte and a target on a tumor cell.
In some embodiments, the complex binds CD3 and CD19. In further
embodiments, the complex has multiple binding sites for a target on
an infectious agent or a cell infected with an infectious agent. In
further embodiments, the complex has multiple binding sites for 2,
3, 4, 5 or more targets on an infectious agent or a cell infected
with an infectious agent. In some embodiments, the complex has a
single binding site for a target associated with an endogenous
blood brain barrier (BBB) receptor mediated transport system. In
further embodiments, the complex has multiple binding sites for a
target associated with an endogenous BBB receptor mediated
transport system. In some embodiments, the complex has multiple
binding sites for 2, 3, 4, 5 or more targets associated with an
endogenous BBB receptor mediated transport system. In some
embodiments, the single binding site is an MRD. In some
embodiments, the single binding site is an antigen binding
domain.
[0745] In some embodiments, the complexes of the invention comprise
a cytotoxic agent.
[0746] Polynucleotide encoding a heavy chain or light chain of the
MRD containing antibody of the invention, vectors comprising these
polynucleotides and host cells containing these vectors and/or
polynucleotides are also encompassed by the invention
[0747] The following examples are intended to illustrate but not
limit the invention.
EXAMPLES
Example 1
Integrin Targeting Antibody-MRD Molecules
[0748] Novel antibody-MRD fasion molecules were prepared by fusion
of an integrin .alpha.v.beta.3 targeting peptides to catalytic
antibody 38C2. Fusions at the N-termini and C-termini of the light
chain and the C-termini of the heavy chain were most effective.
Using flow cytometry, the antibody conjugates were shown to bind
efficiently to integrin .alpha.v.beta.3-expressing human breast
cancer cells. The antibody conjugates also retained the retro-aldol
activity of their parental catalytic antibody 38C2, as measured by
methodol and doxorubicin prodrug activation. This demonstrates that
cell targeting and catalytic antibody capability can be efficiently
combined for selective chemotherapy.
Example 2
Angiogenic Cytokine Targeting Antibody-MRD Molecules
[0749] Angiogenic cytokine targeting antibody-MRD fusion molecules
were constructed. The antibody used was 38C2, which was fused with
a MRD containing the 2xCon4 peptide
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID
NO:10)). The MRD-containing peptide was fused to either the N- or
C-terminus of the light chain and the C-terminus of the heavy
chain. Similar results were found with the other Ang2 MRD peptides.
Additional Ang2 MRD peptides include: MGAQTNFMPMDNDELLL
YEQFILQQGLEGGSGSTASSGSGSSLGAQTNFMPMDNDELLLY (SEQ ID NO:20)
(LM-2x-32); and AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEH
MLE (SEQ ID NO:10) (2xCon4).
[0750] One of skill in the art, given the teachings herein, will
appreciate that other such combinations will create functional Ang2
binding MRDs as described herein.
Example 3
Antibody-MRD Fusions with Non-Catalytic Antibodies
[0751] A humanized mouse monoclonal antibody, LM609, directed
towards human integrin .alpha.v.beta.3 has been previously
described (Rader, C. et. al., PNAS 95:8910-5 (1998)).
[0752] A human non-catalytic monoclonal Ab, JC7U was fused to an
anti-Ang2 MRD containing 2xCon4
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEH MLE (SEQ ID
NO:10)) at either the N- or C-terminus of the light chain. 2xCon4
(AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID
NO:10)) was studied as an N-terminal fusion to the Kappa chain of
the antibody (2xCon4-JC7U) and as a C-terminal fusion
(JC7U-2xCon4). Both fusions maintained integrin and Ang2 binding.
As shown in the left panel of FIG. 3, both antibody constructs
(2xCon4-JC7U and JC7U-2xCon4) specifically bound to recombinant
Ang2 as demonstrated by ELISA studies. Binding to Ang2, however, is
significantly higher with JC7U-2xCon4, which has the 2xCon4 (SEQ ID
NO:10) fusion at the C-terminus of the light chain of the antibody.
The right panel of FIG. 3 depicts the binding of Ang2-JC7U and
JC7U-Ang2 to integrin .alpha.v.beta.3. The results show that fusion
of 2xCon4 (SEQ ID NO:10) to either the N--Of the C-light chain
terminus does not affect mAb JC7U binding to integrin
.alpha.v.beta.3. FIG. 4 depicts another ELISA study using the same
antibody-MRD fusion constructs.
Example 4
HERCEPTIN.RTM.--MRD Fusion Molecules
[0753] Another example of MRD fusions to a non-catalytic antibody
are HERCEPTIN.RTM.-MRD fusion constructs. The HERCEPTIN.RTM.-MRD
fusions are multifunctional, both small molecule .alpha.v integrin
antagonists and the chemically programmed integrin-targeting
antibody show remarkable efficacy in preventing the breast cancer
metastasis by interfering with .alpha.v-mediated cell adhesion and
proliferation. MRD fusions containing HERCEPTIN.RTM.-2xCon4 (which
targets ErbB2 and Ang2) and HERCEPTIN.RTM.-V114 (which targets
ErbB2 and VEGF targeting) and HERCEPTIN.RTM.-RGD-4C-2xCon4 (which
targets ErbB2, ang2, and integrin targeting) are effective.
Example 5
VEGF Targeting Antibody-MRD Molecules
[0754] An antibody containing an MRD that targets VEGF was
constructed. A MRD which targets vl 14 (SEQ ID NO:13) was fused at
the N-terminus of the kappa chain of 38C2 and HERCEPTIN.RTM. using
a linker. Expression and testing of the resulting antibody-MRD
fusion constructs demonstrated strong VEGF binding.
Example 6
IGF1R Targeting Antibody MRD Molecules
[0755] Fusion of an MRD which targets IGF1R(SFYSCLESLVNGPAEKSRG
QWDGCRKK (SEQ ID NO:14)) to the N-terminus of the kappa chain of
38C2 and HERCEPTIN.RTM. using the long linker sequence as a
connector was studied. Expression, and testing of the resulting
antibody-MRD fusion constructs demonstrated strong IGF1R binding.
Additional clones showing high binding to IGR1R were identified
after several rounds of mutagenesis and screening of the regions
described in Table 4. The preferred sequences listed in Table 5
bind IGF1R and show no significant or no binding affinity to the
insulin receptor, thereby suggesting specificity for IGF1R.
TABLE-US-00004 TABLE 4 Template for further mutagenesis. Name DNA
AA Rm2-2-218 GTGGAGTGCAGGGCGCCG VECRAP (SEQ ID NO: 50) (SEQ ID NO:
51) Rm2-2-316 GCTGAGTGCAGGGCTGGG AECRAG (SEQ ID NO: 52) (SEQ ID NO:
53) Rm2-2-319 CAGGAGTGCAGGACGGGG QECRTG (SEQ ID NO: 54) (SEQ ID NO:
55)
TABLE-US-00005 TABLE 5 SEQ ID Mutant Amino acid sequence Template
NO Rm4-31 NFYQCIEMLASHPAEKSRGQWQECRTGG Rm2-2-319 35 Rm4-33
NFYQCIEQLALRPAEKSRGQWQECRTGG Rm2-2-319 36 Rm4-39
NFYQCIDLLMAYPAEKSRGQWQECRTGG Rm2-2-319 37 Rm4-310
NFYQCIERLVTGPAEKSRGQWQECRTGG Rm2-2-319 38 Rm4-314
NFYQCIEYLAMKPAEKSRGQWQECRTGG Rm2-2-319 39 Rm4-316
NFYQCIEALQSRPAEKSRGQWQECRTGG Rm2-2-319 40 Rm4-319
NFYQCIEALSRSPAEKSRGQWQECRTGG Rm2-2-319 41 Rm4-44
NFYQCIEHLSGSPAEKSRGQWQECRTG Rm2-2-319 42 Rm4-45
NFYQCIESLAGGRAEKSRGQWQECRTG Rm2-2-319 43 Rm4-46
NFYQCIEALVGVPAEKSRGQWQECRTG Rm2-2-319 44 Rm4-49
NFYQCIEMLSLPPAEKSRGQWQECRTG Rm2-2-319 45 Rm4-410
NFYQCIEVFWGRPAEKSRGQWQECRTG Rm2-2-319 46 Rm4-411
NFYQCIEQLSSGPAEKSRGQ WQFCRTG Rm2-2-319 47 Rm4-415
NFYQCIELLSARPAEKSRGQWAECRAG Rm2-2-316 48 Rm4-417
NEYQIEALARTPAEKSRGQWVECRAP Rm2-2-218 49
Example 7
ErbB2 Binding, Ang2-Targeting Antibody-MRD Molecules
[0756] An antibody was constructed which contains an MRD that
targets Ang2 (L17) (SEQ ID NO:7) fused to the light chain of an
antibody which binds to ErbB2. Either the short linker sequence,
the long linker sequence, or the 4th loop in the light chain
constant region was used as a linker. FIG. 5 depicts the results of
an ELISA using constructs containing an N-terminal fusion of an
Ang2 targeting MRD with the ErbB2 antibody with the short linker
peptide (GGGS (SEQ ID NO:1)) (L17-sL-Her), a C-terminal fusion of
Ang2 targeting MRD with the ErbB2 antibody with the short linker
peptide (Her-sL-L17), a C-terminal fusion of Ang2 targeting MRD
with the ErbB2 antibody with the 4th loop in the light chain
constant region (Her-lo-L17), or an N-terminal fusion of Ang2
targeting MRD with the ErbB2 antibody with the long linker peptide
(SSGGGGSGGGGGGSSRSS (SEQ ID NO:19)) (L17-1L-Her). ErbB2 was bound
with varying degrees by all of the constructs. However, Ang2 was
bound only by Her-sL-L17 and L17-1L-Her.
Example 8
Hepatocyte Growth Factor Receptor Binding, Ang2-Targeting
Antibody-MRD Molecules
[0757] Fusion of an MRD which targets Ang2 (L17) (SEQ ID NO:7) was
made to either the N-terminus or C-terminus of the light chain of
the Met antibody, which binds to hepatocyte growth factor receptor.
Either the short linker sequence or the long linker sequence were
used as a connector. FIG. 6 depicts the results of an ELISA using
constructs containing N-terminal fusion of Ang2 targeting MRD with
the Met antibody with the short linker peptide (GGGS (SEQ ID NO:1))
(L17-sL-Met), N-terminal fusion of Ang2 targeting MRD with the Met
antibody with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID
NO:19)) (L17-1L-Met), and C-terminal fusion of Ang2 targeting MRD
with the Met antibody with the long linker peptide (Met-ILL17).
Expression and testing of the resulting antibody-MRD fusion
constructs demonstrated strong Ang2 binding when the long linker
peptide was used Fusion of the Ang2 targeting MRD to the C-light
chain terminus of the antibody resulted in slightly higher binding
to Ang2 then fusion of the Ang2 targeting to the N-light chain
terminus of the antibody.
Example 9
ErbB2 Binding Integrin-Targeting Antibody-MRD Molecules
[0758] An antibody was constructed which contains an MRD that
targets integrin .alpha.v.beta.3 (RGD4C) with the sequence
CDCRGDCFC (SEQ ID NO:106) fused to the light chain of an antibody
HERCEPTIN.RTM. which binds to ErbB2 (Her). Either the short linker
sequence, the long linker sequence, or the 4th loop in the light
chain constant region was used as a linker. FIG. 7 depicts the
results of an ELISA using constructs containing an N-terminal
fusion of integrin .alpha.v.beta.3 targeting MRD with the ErbB2
antibody with the short linker peptide (GGGS (SEQ ID NO:1))
(RGD4C-sL-Her), a C-terminal fusion of integrin .alpha.v.beta.3
targeting MRD with the ErbB2 antibody with the short linker peptide
(Her-sL-RGD4C), a C-terminal fusion of integrin .alpha.v.beta.3
targeting MRD with the ErbB2 antibody with the 4th loop in the
light chain constant region (Her-lo-RGD4C), or an N-terminal fusion
of integrin .alpha.v.beta.3 targeting MRD with the ErbB2 antibody
with the long linker peptide (SSGGGGSGGGGGGSSRSS (SEQ ID NO:19))
(RGD4C-1L-Her). ErbB2 was bound with varying degrees by all of the
constructs. However, integrin .alpha.v.beta.3 was bound only by
RGD4C-1L-Her.
Example 10
Hepatocyte Growth Factor Receptor Binding, Integrin-Targeting
Antibody-MRD Molecules
[0759] An antibody was constructed which contains an MRD that
targets integrin .alpha.v.beta.3 (RGD4C) (SEQ ID NO:106) fused to
the light chain of an antibody which binds to the hepatocyte growth
factor receptor (Met). Antibody-MRD constructs containing the long
linker sequence were used FIG. 8 depicts the results of an ELISA
using constructs containing an N-terminal fusion of integrin
.alpha.v.beta.3 targeting MRD with the hepatocyte growth factor
receptor antibody (RGD4C-1L-Met), or a C-terminal fusion of
integrin .alpha.v.beta.3 targeting MRD with the hepatocyte growth
factor receptor antibody (Met-1L-RGD4C). The RGD4C-1L-Met
demonstrated strong integrin .alpha.v.beta.3 binding.
Example 11
ErbB2 Binding, Insulin-line Growth Factor-I Receptor--Targeting
Antibody-MRD Molecules
[0760] Antibodies were constructed which contains an MRD that
targets insulin-like growth factor-I receptor (RP) (SEQ ID NO:14)
fused to the light chain of an antibody which binds to ErbB2 (Her).
Either the short linker peptide, the long linker peptide, or the
4th loop in the light chain constant region was used as a linker
(Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992);
U.S. Pat. No. 5,677,171; and ATCC Deposit 10463, each of which is
herein incorporated by reference). FIG. 9 depicts the results of an
ELISA using constructs containing an N-terminal fusion of
insulin-like growth factor-I receptor targeting MRD with the ErbB2
antibody with the short linker peptide (RP-sL-Her), a C-terminal
fusion of insulin-like growth factor-I receptor targeting MRD with
the ErbB2 antibody and the short linker peptide (Her-sL-RP), a
C-terminal fusion of insulin-like growth factor-I receptor
targeting MRD with the ErbB2 antibody with the 4th loop in the
light chain constant region (Her-lo-RP), an N-terminal fusion of
insulin-like growth factor-I receptor targeting MRD with the ErbB2
antibody with the long linker peptide (RP-1L-Her), or a C-terminal
fusion of insulin-like growth factor-I receptor targeting MRD with
the ErbB2 antibody with the long linker peptide (Her-1L-RP). ErbB2
was bound with varying degrees by all of the constructs.
Insulin-like growth factor-I receptor was bound by RP-1L-Her.
Example 12
ErbB2 Binding, VEGF-Targeting Antibody-MRD Molecules
[0761] Fusion of an MRD which targets VEGF (Vl 14) (SEQ ID NO:13)
(Fairbrother W. J., et al., biochemistry 37:177754-177764 (1998))
was made to the N-terminus of the light chain of a ErbB2-binding
antibody (Her). A medium linker peptide (SSGGGGSGGGGGGSS (SEQ ID
NO:2)) was used as a connector. FIG. 10 depicts the results of an
ELISA using a construct containing an N-terminal fusion of VEGF
targeting MRD with the Erb132-binding antibody with the medium
linker peptide (Vl 14-mL-Her). Expression and testing of the
resulting antibody-MRD fusion construct demonstrated strong VEGF
and ErbB2 binding.
Example 13
Integrin Targeting Antibody-MRD Molecules
[0762] Fusion of an MRD which targets integrin .alpha.v.beta.3
(RGD) (SEQ ID NO:106) to the N-terminus of the light chain of 38C2
using the medium linker peptide as a connector was studied. FIG. 11
demonstrates that expression and testing of the resulting
antibody-MRD fusion construct had strong integrin .alpha.v.beta.3
binding.
Example 14
Ang2 Targeting Antibody-MRD Molecules
[0763] Fusion of an MRD which targets Ang2 (L 17) (SEQ ID NO:7) to
the C-terminus of the light chain of 38C2 using the short linker
sequence as a connector was studied. FIG. 12 demonstrates that
expression and testing of the resulting antibody-MRD fusion
construct had strong Ang2 binding.
Example 15
ErbB2 Binding, Integrin and Ang2 Targeting Antibody-MRD
Molecules
[0764] An MRD which targets integrin .alpha.v.beta.3 (RGD4C) was
connected to the N-terminus of the light chain of an ErbB2
targeting antibody (Her) with a medium linker, and an Ang2 (L17)
targeting MRD was connected by a short linker to the C-terminus of
the same ErbB2 targeting antibody (RGD4C-mL-Her-sL-L17). FIG. 13
demonstrates that the resulting antibody-MRD fusion construct bound
to integrin, Ang2, and ErbB2.
[0765] Similarly, ErbB2 targeting antibodies (e.g., Her) with an
IGF-1R MRD fused to the C-terminus of the heavy chain or the
N-terminus of the light chain bound to immobilized IGF-1R at
comparable rates. In addition, ErbB2 targeting antibodies
containing an IGF-1R MRD fused to the N-terminus of the light chain
and an Ang2 MRD fused to the C-terminus of the heavy chain bound to
immobilized IGF-1R at comparable rates. Each of these three
multivalent and multispecific compositions (e.g., MRD-containing
antibodies) also inhibited the binding of IGF-1 to immobilized
IGF-1R. The trispecific molecule (HERCEPTIN with IGF-1R and Ang2
MRDs) bound to both cell surface ErbB2 and soluble Ang2.
Example 16
ErbB2 Binding, Integrin-Targeting Antibody-MRD Molecules
[0766] An antibody was constructed which contains an MRD that
targets integrin .alpha.v.beta.3 (RGD4C) fused to the N-terminus of
the heavy chain of an antibody which binds to ErbB2 (Her) using the
medium linker as a connector (RGD4C-mL-her-heavy). FIG. 14 depicts
the results of an ELISA using the construct. Both integrin and
ErbB2 were bound by the construct.
Example 17
ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin,
Ang2 Or Insulin-Like Growth Factor-I Receptor-Targeting
Antibody-MRD Molecules with the Short Linker Peptide
[0767] Antibody-MRD molecules were constructed which contain ErbB2
or hepatocyte growth factor receptor binding antibodies, and
integrin .alpha.v.beta.3, Ang2 or insulin-like growth factor-I
receptor-targeting MRD regions were linked with the short linker
peptide to the light chain of the antibody. FIG. 15 depicts the
results of an ELISA using constructs containing an N-terminal
fusion of Ang2 targeting MRD fused to the ErbB2 antibody
(L17-sL-Her), an N-terminal fusion of integrin-targeting MRD with
the ErbB2 antibody (kGD4C-sL-Her), an N-terminal fusion of
insulin-like growth factor-I receptor targeting MRD with the
ErbB2-binding antibody (RP-sL-Her), a C-terminal fusion of Ang2
targeting MRD with the hepatocyte growth factor receptor binding
antibody (L17-sL-Met), a C-terminal fusion of Ang2 targeting MRD
with the ErbB2-binding antibody (Her-sL-L17), a C-terminal fusion
of integrin targeting MRD with the ErbB2-binding antibody
(Her-sL-RGD4C), or a C-terminal fusion of insulin-like growth
factor-I receptor targeting MRD with the ErbB2-binding antibody
(Her-sL-RP). ErbB2 was bound with varying degrees by the
antibody-MRD constructs, with the exception of the construct
containing the hepatocyte growth factor receptor-binding antibody.
Antigen was bound only by the Her-sL-L17 construct.
Example 18
ErbB2 or Hepatocyte Growth Factor Receptor Binding, and Integrin,
Ang2 or Insulin-Like Growth Factor-I Receptor-Targeting
Antibody-MRD Molecules with the Long Linker Peptide
[0768] Antibody-MRD molecules were constructed which contain ErbB2
or hepatocyte growth factor receptor binding antibodies, and
integrin .alpha.v.beta.3, Ang2 or insulin-like growth factor-I
receptor-targeting MRD regions linked with the long linker peptide
to the light chain of the antibody. FIG. 16 depicts the results of
an ELISA using constructs containing an N-terminal fusion of Ang2
targeting MRD fused to the ErbB2 antibody (L17-1L-Her), an
N-terminal fusion of integrin-targeting MRD with the ErbB2 antibody
(RGD4C-1L-Her), an N-terminal fusion of insulin-like growth
factor-I receptor-targeting MRD with the ErbB2-binding antibody
(RP-1L-Her), a C-terminal fusion of Ang2 targeting MRD with the
hepatocyte growth factor receptor binding antibody (L17-1L-Met), a
C-terminal fusion of integrin targeting MRD with the hepatocyte
growth factor receptor binding antibody (RGD4C-1L-Met), a
C-terminal fusion of Ang2 targeting MRD with the insulin-like
growth factor-I receptor binding antibody (Her-1L-RP), a C-terminal
fusion of Ang2 targeting MRD with the hepatocyte growth factor
receptor binding antibody (Met-1L-L17), or a C-terminal fusion of
integrin targeting MRD with the hepatocyte growth factor receptor
binding antibody (Met-1L-RGD4C). As shown in FIG. 16, antibody-MRD
fusions are effective to bind antigen and ErbB2. Lu et al., J.
Biol. Chem. 20:280(20):19665-19672 (2005); Lu et al., J. Biol.
Chem. 2004 Jan. 23:279(4):2856-65.
Example 19
Cloning and Expression of Ang2 MRDs Fused to Maltose Binding
Protein
A. Cloning of MBP Fusions
[0769] Monomer and dimer peptides were expressed as protein fusions
to maltose binding protein (MBP) using a modified form of the
pMAL-p2 vector and expression system from New England Biolabs (NEB;
Beverly, Mass.) The PCR-generated MRD sequence was inserted into a
pMAL vector down-stream from the malE gene, which encodes MBP. This
results in a vector that encodes an MRD-MBP-fusion protein. The
pMAL vector contains a strong Ptac promoter and is inducible by
IPTG. The pMAL-p2 series contains the normal malE signal sequence,
which directs the fusion protein through the cytoplasmic membrane.
pMAL-p2 fusion proteins capable of being exported can be purified
from the periplasm through osmotic shock. Further purification can
be performed, for example by binding to amylose resin.
B. Expression of MBP Fusion Proteins and Osmotic Shock
Fractionation
[0770] For expression of fusion proteins, bacterial cultures grown
overnight were back-diluted into fresh media to an OD A600 of
approximately 0.1. Cultures were grown to an OD of approximately
0.8 and induced with IPTG at a concentration of 0.3 mM. Cultures
were incubated with shaking for approximately 4 hours, after which
bacteria were centrifuged for 15 minutes at 4700 g. Pelleted
bacteria were resuspended in 30 mM Tris-HCL pH 7.4, 20% sucrose, 1
mM EDTA. Cells were incubated for 20 minutes at room temperature
(RT) prior to centrifugation for 15 minutes at 4700 g. Pelleted
bacteria were then resuspended in ice cold MgSO.sub.4, and
incubated for 20 minutes on ice, with periodic mixing. Cell
suspensions were sonicated (Misonix XL2020) for 90 seconds. Cells
were centrifuged at 4.degree. C. for 20 minutes at 4700 g. The
supernatant ("osmotic shock fraction") was adjusted to 1.times.PBS
using 10.times.PBS (Quality Biologics, cat #119-069-131) and
filtered through 0.2 micron filter. These osmotic shock fractions
were assayed directly for binding to Ang2.
C. Direct Binding of MBP Fusion Proteins
[0771] For detection of, direct binding of MRD-MBP fusions to Ang2,
the following ELISA was performed. Ninety-six-well plates were
coated overnight with rhAng2 (R&D cat#623-AN) at 320 ng/ml (100
.mu.l/well). Wells were blocked for 3.25 hours with 250 .mu.l
Blocking buffer (Thermo Cat# N.sub.5O.sub.2), followed by 4 washes
with 300 .mu.l wash buffer (PBS, 0.1% tween). MBP fusion proteins
were serially diluted in Blocking buffer and added to wells for 2
hours at RT. After washing (8.times.300 .mu.l wash buffer), samples
were treated with HRP-mouse anti MBP mAb (NEB, cat #E80385),
diluted 1:4000 in Blocking buffer. After incubation for 1 hour at
RT, wells were washed (8.times.300 .mu.l wash buffer) prior to
receiving 100 .mu.l of TMB substrate (KPL Laboratories). Color
development was stopped with 100 .mu.l of H.sub.2SO.sub.4, and
absorbance was read at 450 nm.
D. Results
[0772] MRD-MBP fusions were assayed for direct binding to Ang2.
Osmotic shock fractions of induced bacterial cultures were serially
diluted and added to Ang2 coated wells. Bound fusion proteins were
detected with anti-MBP mAb. The dose response curves are presented
in FIG. 17A. Assayed proteins represent mutational variants of the
sequence MGAQTNFMPMDDDE LLLYEQFILQQGLE (L17D) (SEQ ID NO:107). In
this series, the motif MDD within L17D was mutated at the first D
to all other possible amino acids (except cysteine). Other MRDs
tested were "Lm32 KtoS" and a dimer of Lm32 (2xLm32). As presented
in FIG. 17B, several MXD mutants exhibit binding in the 0.1 to 100
nm range. The Lm32 dimer (2XLm32) exhibits greater than 10 fold
higher affinity for Ang2 than either L17D or "Lm32 KtoS".
Example 21
Expression and Purification of Antibodies Containing MRDs
[0773] Molecular recognition domains were constructed and expressed
in a pcDNA 3.3 vector as fusion proteins with either the heavy or
light chains of antibodies. For protein production, plasmid DNAs
encoding the heavy and light chains of the antibodies containing
MRDs were first transformed into chemically competent bacteria in
order to produce large amounts of DNA for transient transfection.
Single transformants were propagated in LB media and purified using
Qiagen's Endotoxin Free Plasmid Kits. Briefly, cells from an
overnight culture were lysed; lysates were clarified and applied to
an anion-exchange column, and then subjected to a wash step and
eluted with high salt. Plasmids were precipitated, washed, and
resuspended in sterile water.
[0774] HEK293T cells were expanded to the desired final batch size
(about 5 L) prior to transfection. The purified plasmid (1 mg per
liter of production) was complexed with the polyethylenimine (PEI)
transfection reagent, added to the shake flask culture, and
incubated at 37.degree. C. The culture was monitored daily for cell
count, cell diameter, and viability. The conditioned medium was
harvested and stored at -80.degree. C. until purification.
[0775] Antibodies containing MRDs were purified from the
conditioned medium using affinity chromatography. Culture
supernatant was filter clarified and applied directly to a
chromatography column containing recombinant Protein A Sepharose
(GE Healthcare). The column was washed, and bound antibodies
containing MRDs were eluted by lowering buffer pH. Following
elution, eluate fractions were immediately adjusted to physiologic
pH. Following Protein A affinity purification, an additional
optional polishing chromatographic step can be performed as
needed.
[0776] Purified proteins were dialyzed into PBS, concentrated to
.about.1-4 mg/ml, sterile filtered, aliquoted aseptically, and
stored frozen at -80.degree. C. All steps of the purification were
monitored by SDS-PAGE-Coomassie, and precautions were taken during
the purification to keep endotoxin levels as minimal as
possible.
[0777] The final product was analyzed for endotoxin levels
(EndoSafe), purity (SDS-PAGE-coomassie, analytical SEC-HPLC),
protein identity (Western blot), and yield (Bradford assay). An
additional size exclusion HPLC analysis was performed to assess the
level of aggregates.
[0778] The data presented in Table 6 indicate that the antibodies
containing MRDs can be expressed and purified using conventional
techniques.
TABLE-US-00006 TABLE 6 Endotoxin Zybody Yield (mg) Purity
Aggregates (%) (EU/ml) HER2xCon4(H) 36 >90% 4.6 <1
HER-lm32(H) 57 >90% 1 2.02 HER-lm32(L) 98 >90% 2 3.26
AVA-lm32(H) 12 >90% 0 <1
Example 22
Simultaneous Binding of HER Lm32(H) and HER Lm32 (L) to Her2 and
Ang2
A. Methods
[0779] Ninety-six-well plates were coated overnight with rHER2-Fc
(R&D cat#1129-ER-050) at 20 ng/ml (100 .mu.l/well). Wells are
blocked for 3.25 hours with 250 .mu.l Blocking buffer (Thermo Cat#
N502), followed by 4 washes with 300 .mu.l wash buffer (PBS, 0.1%
tween). Antibodies containing MRDs (HER-lm32(H), HER-lm32(L), and
AVA-lm32(H)) and antibodies (HERCEPTIN.RTM.) were serially diluted
in Blocking buffer, containing 1.94 .mu.g/ml biotinylated Ang2
(R&D cat#BT633) and added to wells for 2 hours at RT. After
washing (8.times.300 .mu.l wash buffer), parallel samples received
either HRP-conjugated anti-human kappa chain mAb-(Abcam, cat
#ab79115-1) diluted 1:1000 in Blocking buffer or HRP-conjugated
streptavidin (Thermo Scientific cat#N100) diluted 1:4000 diluted in
Blocking buffer. After incubation for 1 hour at RT, wells were
washed (8.times.300 .mu.l wash buffer) prior to receiving 100 .mu.l
of TMB substrate (KPL Laboratories). Color development was stopped
with 100 .mu.l of H.sub.2SO.sub.4, and absorbance was read at 450
nm.
B. Results
[0780] As detected with anti-human kappa chain mAb, both a
HERCEPTIN.RTM.-based antibody or HERCEPTIN.RTM.-based antibodies
containing MRDs bind to Her2 Fc in the presence of Ang2 in a dose
dependent manner (FIG. 18A). Only the HERCEPTIN.RTM.-based
antibodies containing MRDs (HER-lm32(H) and HER-lm32(L)) exhibit
simultaneous binding to Her2 Fc and Ang2, as detected by
HRP-conjugated streptavidin (FIG. 18B).
Example 23
Simultaneous Binding of AVA-Lm32(H) to VEGF and Ang2
A. Methods:
[0781] Ninety-six-well plates were coated overnight with human VEGF
(PeproTech, Inc. cat#100-20) at 30 ng/ml (100 .mu.l/well). Wells
were blocked for 3.25 hours with 250 .mu.l Blocking buffer (Thermo
Cat# N502), followed by 4 washes with 300 .mu.l wash buffer (PBS,
0.1% tween). Antibodies containing MRDs (HER-lm32(H) and
AVA-lm32(H)) and antibodies (AVASTIN.RTM.) were serially diluted in
Blocking buffer, containing 3.876 .mu.g/ml biotinylated Ang2
(R&D cat#BT633) and added to wells for 2 hours at RT. After
washing (8.times.300 .mu.l wash buffer), parallel samples received
either HRP-conjugated anti-human kappa chain mAb (Abeam, cat
#ab79115) diluted 1:1000 in Blocking buffer or HRP-conjugated
streptavidin (Thermo Scientific cat#N100) diluted 1:4000 diluted in
Blocking buffer. After incubation for 1 hour at RT, wells were
washed (8.times.300 .mu.l wash buffer) prior to receiving 100 .mu.l
of TMB substrate (KPL Laboratories). Color development was stopped
with 100 .mu.l of H.sub.2SO.sub.4, and absorbance was read at 450
nm.
B. Results
[0782] As detected with anti-human kappa chain mAb, both
ANASTIN.RTM. and AVASTIN.RTM.-based antibodies containing MRDs bind
to VEGF in the presence of Ang2 in a dose dependent manner (FIG.
19A). Only the AVASTIN.RTM.-based antibodies containing MRDs
(AVA-lm32(H)) exhibited simultaneous binding to VEGF and Ang2, as
detected by HRP-conjugated streptavidin (FIG. 19B).
Example 24
Simultaneous Binding of HER-Lm32 (H) and HER-Lm32 (L) to HER2 and
Angiopoietin-2
[0783] The ability of HER-lm32 (H) and HER-lm32 (L) simultaneously
bind to Her2 expressed on the surface of breast carcinoma cells
BT-474, and to Ang2 in solution, was determined by flow cytometry.
Mouse anti-human Ig-FITC was used for detection of the heavy chain
of the antibodies containing MRDs, and Ang2-biotin/streptavidin-PE
was used for detection of the lm32 MRD. Cells that bind Her2 and
Ang2 simultaneously are expected to be detected as double positive
for FITC and PE fluorescence.
[0784] One million HER2 positive breast carcinoma cells BT-474 were
incubated with 1 .mu.g HER-lm32(F1 or HER-lm32(L) for 25 minutes at
RT. After washing, cells were incubated with 200 ng/mL Ang2 biotin
(R&D systems) for 25 minutes at RT and then with 20 .mu.L of
mouse anti-human Ig FITC and Streptavidin-PE for 15 minutes. After
washing with 2 mL buffer, cells were analyzed by flow cytometry
(FACS Canto II, BD).
[0785] In order to confirm the specificity of binding of
HER-lm32(H) and HER-lm32(L) to HER2 on BT-474 cells, binding was
determined in the presence of 10-fold excess of HERCEPTIN.RTM.. In
these experiments, antibodies containing MRDs (1 .mu.g) were
incubated with one million BT-474 cells in the absence or presence
of 10 .mu.g HERCEPTIN.RTM. for 25 minutes at RT. Binding of
antibodies containing MRDs to HER2 was determined by incubating
with 200 ng/mL Ang2 biotin followed by detection with
streptavidin-PE.
[0786] The data presented in FIG. 20A demonstrate that both
HER-lm32(H) and HER-lm32(L), bind simultaneously to HER2 and Ang2.
In both cases, the cells exhibited bright dual fluorescence in the
FITC and PE fluorescence channels. The fact that HER-lm32(H) and
HER-lm32(L) binding to HER2 is completely inhibited by
HERCEPTIN.RTM. (FIG. 20B) indicates that the binding is
specific.
[0787] In addition, tri-specific binding was demonstrated using an
antibody containing two distinct MRDs. An EGFR-binding affibody and
an Ang2-binding peptide (LM32) were fused to the C-terminus of the
light and heavy chains of HERCEPTIN, respectively. The
MRD-containing antibody was incubated with the EGFR+, Her2-A431
human epithelial cell line. Cell-bound MRD-containing antibody was
detected with biotinylated Ang2/strepavidin-PE, alexafluor labeled
ErbB2-Fc, or the combination of Ang2/strepavidin-PE and alexafluor
labeled ErbB2-Fc. The results demonstrated that the MRD-containing
antibody simultaneously bound EGFR cell surface receptor and two
soluble ligands (ErbB2 and Ang2).
[0788] Additional experiments demonstrated that a fusion of lm32 to
the C-terminus of the heavy chain of HERCEPTIN retained the binding
specificity and Fc function of HERCEPTIN. HERCEPTIN and the
HERCEPTIN-lm32 fusion bound to FcRn with similar affinities (EC50s
for HERCEPTIN and HERCEPTIN lm-32 were 2.17 and 2.84 .mu.g/ml,
respectively). The HERCEPTIN and the HERCEPTIN-lm32 fusion
displayed comparable ADCC activity on SK-BR-3 cells. The
HERCEPTIN-lm32 fusion and HERCEPTIN bound to Fc.gamma.-RI and
Fc.gamma.-RIII with similar affinities. The HERCEPTIN-lm32 fusion
and HERCEPTIN bound to complement receptor C1q with similar
affinities. In addition, the HERCEPTIN-lm32 fusion bound Ang2 with
subnanomolar affinity and antagonized Ang2 binding to the Tie-2
receptor. The HERCEPTIN-lm32 fusion bound the extracellular domain
of ErbB2 and also inhibited Ang2-induced proliferation of primary
bovine lymphoendothelial cells. The HERCEPTIN-lm32 fusion and
HERCEPTIN bound to the extracellular domain of Her2 with similar
kinetic parameters. Additional experiments demonstrated that the
HERCEPTIN-lm32 fusion was as effective as HERCEPTIN in inhibiting
the proliferation of several cultured breast cancer cell lines
(BT-474, MDA-MB-361 and SK-BR-3). The anti-proliferative effect of
the HERCEPTIN-lm32 fusion on SK-BR-3 cells was not affected by the
presence of Ang2 (2 .mu.g/ml) in the culture.
[0789] Simultaneous target binding has been observed for other
multivalent and multispecific compositions (e.g., multivalent and
multispecific compositions (e.g., MRD-containing antibodies)). For
example, a fusion of lm32 to the C-terminus of HUMIRA heavy chain
(HUM-lm32(H)) was able to simultaneously bind to Ang2 and
TNF.alpha.. The same fusion was able to bind 293 cells transiently
transfected with full length human TNF.alpha. with similar affinity
to the HUMIRA antibody. HUM-lm32(H) was also able to inhibit the
interaction of TNF with its receptors. HUM-lm32(H) also bound to
cell surface expressed and plate bound FcRn as well as to
Fc.gamma.-RI and Fc.gamma.-RIII.
Example 25
Antibody-MRDs Containing Heavy Chain Fusions Bind to Targets
[0790] To assess the ability of lm32-containing antibodies to block
the interaction of Ang2 with its receptor TIE2, their effect on the
binding of soluble TIE2 to plate-bound Ang2 was determined by
ELISA.
[0791] Ang2 (R&D Systems, catalog#623-AN) was coated on a
96-well plate (Thermo Electron, cat#3855) at 200 ng/mL in PBS
overnight at 4.degree. C. The plate was then incubated with 100
.mu.L of blocking solution (Thermo Scientific, cat#N502) for 1 hour
at RT. After washing the plate 4 times with 0.1% Tween-20 in PBS,
the plate bound Ang2 was incubated with 0.5 .mu.g/mL soluble TIE2
(R&D Systems, cat#313-TI,) in the absence or presence of
various concentrations of serially diluted antibodies containing
MRDs for 1 hour at RT. After washing 4 times, 100 .mu.L of 0.5
.mu.g/mL anti TIE2 antibody (cat#BAM3313, R&D Systems) was
added and incubated at RT for 1 hour. TIE2 binding to Ang2 was
detected by incubation with 1:1000 diluted goat anti-mouse-HRP (BD
Pharmingen, cat#554002) for 1 hour at RT. The plate was washed 4
times and incubated with 100 .mu.L TMB reagent for 10 minutes at
RT. After stopping the reaction with 100 .mu.L of
0.36NH.sub.2SO.sub.4, the plate was read at 450 nm using a
spectrophotometer.
[0792] As presented in FIG. 21A, HER-lm32(H), HER-lm32(L), and
AVA-lm32(H) inhibited TIE2 binding to plate-bound Ang2 in a
dose-dependent fashion. All tested lm32-containing antibodies
demonstrated comparable inhibitory effects with IC-50 values of 4
nM for HER-lm32 (H), 8 nM for HER-lm32(L) and 3.3 nM for
AVA-lm32(H).
Example 26
Antibody-MRDs Containing Heavy Chain Fusions Bind to Target
[0793] To determine the specificity and relative affinity of
AVA-lm32 (H) binding to VEGF, a competitive binding assay was
performed using biotin labeled AVASTIN.RTM..
[0794] AVASTIN.RTM. was labeled with biotin using EZ-Link
NHS-LC-Biotin (Pierce, cat#21336). VEGF (Peprotech, cat#100-20) was
coated on a 96-well plate (Thermo Electron, cat#3855) at 100 ng/mL
in PBS overnight at 4.degree. C. The plate was then incubated with
100 .mu.L of blocking, solution (Thermo Scientific, cat#N502) for 1
hour at RT. After washing the plate 4 times with 0.1% Tween 20 in
PBS, 50 .mu.L of AVASTIN.RTM.-biotin at 150 ng/mL and 50 .mu.L of
various concentrations of AVA-lm32(H) or unlabeled AVASTIN.RTM.
were added and incubated at RT for 1 hour. The plate was washed 4
times and incubated with Streptavidin-HRP (Thermo, cat#N100) at
1:1000 dilution for 1 hour at RT. The plate was washed 4 times and
100 .mu.L of TMB reagent was added. After 10 minutes incubation at
RT, 100 .mu.L of 0.36N H.sub.2SO.sub.4 was added to stop the
reaction and the plate was read at 450 nm.
[0795] The data presented, in FIG. 22 demonstrate that AVA-lm32(H)
specifically binds to VEGF-2. It inhibits binding of biotinylated
AVASTIN.RTM. to VEGF in a dose dependent manner. The dose response
curves generated by AVA-lm32(H) and unlabeled AVASTIN.RTM. are
superimposable and indicate similar binding affinities.
Example 27
Binding HER-lm32(H) and HER-lm32(L) to HER2 Expressed on Breast
Cancer Cells
[0796] To determine the relative binding affinity of
HERCEPTIN.RTM.-based antibodies containing MRDs to cell surface
HER2 compared to HERCEPTIN.RTM., a competitive binding assay was
performed with Eu-labeled HERCEPTIN.RTM..
[0797] HERCEPTIN.RTM. as labeled with Eu3+ using a
dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA)
Europium-labeling kit (Perkin Elmer Life Sciences, cat#1244-302)
following the manufacturer's instructions. The labeling agent is
the Eu-chelate of N1-(p-isothiocynateobenzyl) diethylenetriamine
N1, N2, N3, N3-tetraacetic acid (DTTA). The DTTA group forms a
stable complex with Eu3+, and the isothiocynate group reacts with
amino groups on the protein at alkaline pH to form a stable,
covalent thio-urea bond. HERCEPTIN.RTM. (0.2 mg in 200 mL sodium
bicarbonate buffer pH 9.3) was labeled with 0.2 mg of labeling
agent at 4.degree. C. overnight. Eu-labeled HERCEPTIN.RTM. was
purified by spin column using 50 mmol/L tris-HCl pH 7.5 and 0.9%
NaCl elution buffer.
[0798] The Eu-HERCEPTIN.RTM. binding assay was performed by
incubating 0.5-1 million BT-474 or SK-BR3 breast cancer cells per
well in a 96-well plate with 2-5 nM Eu-HERCEPTIN.RTM. in the
presence of various concentrations of unlabeled
HERCEPTIN.RTM.-based antibodies containing MRDs or HERCEPTIN.RTM.
for 1 hour at RT. Unbound Eu-HERCEPTIN.RTM. as removed by washing
using 200 .mu.L complete medium, Cells were then resuspended in 100
.mu.L complete medium and 80 .mu.L of cell suspension transferred
to a 96-well isoplate. Cells were incubated with 100 .mu.L Delfia
enhancer solution at RT for 10 minutes and cell bound
Eu-HERCEPTIN.RTM. was detected by Envison (Perkin Elmer).
[0799] The inhibition of binding curves obtained using BT-474 cells
are presented in FIG. 23. Eu-HERCEPTIN.RTM. binding to BT-474 was
inhibited by HERCEPTIN.RTM. and HERCEPTIN.RTM.-based antibodies
containing MRDs in a dose-dependent fashion. Comparable IC-50
values were observed: 4.7 nM for HER-lm32(H), 5.7 nM for
HER-lm32(L), and 3.7 nM for unlabeled HERCEPTIN.RTM..
Example 28
Inhibition of Breast Cancer Cells Proliferation by
HERCEPTIN.RTM.-Based Antibodies Containing MRDs
[0800] HERCEPTIN sensitive breast cancer cells SK-BR-3 expressing
HER2neo receptor were also tested in a bioassay. SK-BR-3 cells
(2000 cell/well) were plated in 96 well plates (Costar) in complete
McCoy's growth medium containing 2 mM glutamine, pen/strep
(Invitrogen) and 10% FBS (HyClone). The cells were cultured for 24
hours at 37.degree. C., 5% CO.sub.2, 85% humidity. On the following
day, the growth medium was replaced with starvation medium (McCoy's
medium containing 2 mM glutamine, pen/strep, 0.5% FBS). Nine serial
dilutions (concentration range 5000-7.8 ng/ml) of HERCEPTIN.RTM.
and HERCEPTIN.RTM.-based antibodies containing MRDs were prepared
in complete growth medium. After 24 hours of incubation, the
starvation medium was removed, and the serial dilutions of
HERCEPTIN.RTM. and HERCEPTIN.RTM.-based antibodies containing MRDs
were transferred to the plates in triplicates. The cells were
cultured for 6 days. The proliferation was quantified using the
CellTiter Glo luminescence method.
[0801] The IC50 values determined using a four-parameter logistic
model were as follows: 0.49+/-0.17 nm for HER-lm32(H), 0.81+/-0.19
nm for HER-lm32(L), and 0.67+/-0.15 nm for HER-con4(H). All tested
HERCEPTIN.RTM.-based antibodies containing MRDs were able to
inhibit the proliferation of the SK-BR-3 breast carcinoma cells
with subnanomolar IC-50 values. The representative fitted dose
response, curves shown in FIGS. 24A-C demonstrate that
HERCEPTIN.RTM.-based antibodies containing MRDs inhibit cell
proliferation with similar potency to HERCEPTIN.RTM..
Example 29
Antibody Dependent Cytotoxicity HERCEPTIN.RTM.-Based Antibodies
Containing MRDs
[0802] To assess the ability of antibodies containing MRDs to
mediate ADCC in vitro, a cytotoxicity assay based on the "DELFIA
EUTDA Cytotoxicity reagents AD0116" kit (PerkinElmer) was used. In
this assay, the target cells were labeled with a hydrophobic
fluorescence enhancing ligand (BADTA, his (acetoxymethyl)
2,2':6',2''-terpyridine-6,6''-dicarboxylate). Upon entering the
cells, BADTA is converted to a hydrophilic compound (TDA,
2,2':6',2''-terpyridine-6,6''-dicarboxylic acid) by cytoplasmic
esterases mediated cleavage and no longer can cross the membrane.
After cell lysis, TDA is released into a medium containing Eu3+
solution to form a fluorescent chelate (EuTDA). The fluorescence
intensity is directly proportional to the number of lysed
cells.
[0803] HERCEPTIN.RTM. and HERCEPTIN.RTM.-based antibodies
containing MRDs can mediate ADCC on Her2 positive breast cancer
cells by binding to the HER2 receptor on the surface of the target
cells and activating the effector cells present in human PBMCs by
interacting with their Fc.gamma.RIII receptors. A HER2 positive
human breast cancer cell line SK-BR-3 was used as a target cell
line in the ADCC assay to demonstrate this.
[0804] SK-BR-3 cells were detached with 0.05% trypsin-versene and
resuspended at 1.times.10.sup.6 cells/mL in RPMI1640 medium
containing 2 mM glutamine, pen/strep and 10% FBS (complete growth
medium). 2.times.10.sup.6 cells in 2 mL of media were transferred
into 15 mL tube and 10 .mu.l of BADTA reagent was added. The cell
suspension was mixed gently and placed in the incubator at
37.degree. C., 5% CO.sub.2 and 85% humidity for 15 minutes. Seven
10.times. serial dilutions starting with 5 .mu.g/mL of
HERCEPTIN.RTM. or HERCEPTIN.RTM.-based antibodies containing MRDs
were prepared during cell labeling.
[0805] After incubation with BADTA, cells were washed 4 times in
complete growth medium containing 2.5 mM Probenecid. Between
washes, cells were spun down by centrifugation at 1000 rpm for 3
minutes. After the last wash, labeled SK-BR-3 cells were
resuspended in 10 mL complete growth medium and 50 .mu.l of cells
were added to each well of 96 well plate, except background wells.
50 .mu.l of serial dilutions of HERCEPTIN.RTM. or
HERCEPTIN.RTM.-based antibodies containing MRDs were added to the
designated wells. The plates were transferred to the incubator at
37.degree. C., 5% CO.sub.2 and 85% humidity for 30 minutes.
[0806] PBMCs that were purified from human peripheral blood one day
prior the ADCC assay, were washed once in RPMI1640 with 2 mM
glutamine, pen/strep, 10% FBS. 10 mL of the PBMCs suspension with
2.5.times.10.sup.6 cells/mL was prepared. 100 .mu.l of PBMC
suspension was transferred into wells containing target cells and
HERCEPTIN.RTM. or HERCEPTIN.RTM.-based antibodies containing MRDs
in triplicate. The following controls were placed in designated
wells: Spontaneous release (target cells without effector cells),
Maximum release (lysed target cells) and Background (media without
cells). The plates were incubated for 2.5 hours an incubator with
37.degree. C., 5% CO.sub.2 and 85% humidity.
[0807] After incubation 20 .mu.l of the supernatant was transferred
to another plate and 200 .mu.l of Europium solution was added. The
plates were incubated on a plate shaker at KT for 15 minutes. The
time resolved fluorescence was measured using PerkinElmer EnVision
2104 Multilabel Reader.
[0808] The following formula was used to calculate percentage of
Specific release:
Experimental release (counts)-Spontaneous release
(counts).times.100 Maximum release (counts)-Spontaneous release
(counts)
[0809] The IC50 values calculated by a four-parameter logistic
model were as follows: 0.213+/-0.077 nM for HER-lm32(H),
0.204+/-0.036 nM for HER-lm32(L), and 0.067+/-0.015 nM for
HER-con4(H). All tested antibodies containing MRDs demonstrated
robust ADCC activity with subnanomolar IC-50 values. The
representative fitted dose response curves shown in FIGS. 25A and
25B demonstrate that antibodies containing MRDs are able to mediate
cell dependent cytotoxicity with comparable potency to
HERCEPTIN.RTM..
[0810] A similar experiment was conducted in the presence of Ang2.
Human PBMCs were activated with 20 ng/ml of IL2 overnight and added
to freshly plated (10,000 cells/well) BADTA labeled SK-BR-3 cells.
The effector/target ratio was 25/1. After a 4-hour incubation with
serial dilutions of HER-lm32(H) or HUMIRA in the presence of 2
.mu.g/ml Ang2, Eu was added to the medium and TRFI measured on
Envision reader (Perkin-Elmer). HER-lm32 was more potent in
mediating ADCC in the presence of Ang2.
Example 30
Inhibition of Endothelial Cell Proliferation by AVA-lm32(H)
[0811] The biological activities of the AVASTIN.RTM.-based
antibodies containing MRDs AVA-lm32(H) were tested to determine if
they could inhibit VEGF-induced proliferation of Human Umbilical
Vein Endothelial Cells (HUVEC) assay.
[0812] HUVEC were obtained from GlycoTech (Gaithersburg, Md.) and
Lonza on passage 1 and passage 3 respectively. Cells were grown on
Endothelial cell basal medium (EBM-2) with addition of 2% fetal
bovine serum (FBS) and single quotes (Lonza) at 37.degree. C., 5%
CO.sub.2, 85% humidity. For inhibition of proliferation
experiments, cells were plated in 96-well plates (Costar) at 2000
cells per well in EBM-2 medium with 2% FBS and cultivated for 24
hours. Nine serial dilutions of AVASTIN.RTM. or AVA-lm32(H) were
prepared starting with 5 .mu.g/mL on EBM-2 medium with 2% FBS. VEGF
(R & D Systems) was added at a final concentration of 10 ng/mL,
to all serial dilutions. After incubation for 15 minutes at
37.degree. C., 5% CO.sub.2, 85% humidity, serial dilutions were
added to the cells. After 96 hours, CellTiter Glo was added to the
cells. After incubation at RT for 15 minutes, the cell suspension
was transferred into 96 well white opaque plates, and luminescence
was measured using PerkinElmer EnVision 2104 Multilabel Reader.
[0813] As shown in FIGS. 26A and 26B, AVA-lm32(H) exhibited dose
dependent anti-proliferative activity on HUVECs from both sources.
IC50 values calculated from 4 PL fitted curves indicate similar
potency for AVA-lm32(H) and AVASTIN.RTM. (IC50 values 0.36+/-0.42
nM and 0.33+/-0.38 nM, respectively).
Example 31
MRD-Containing Antibodies Inhibit Tumor Proliferation In Vivo
[0814] In order to determine the effectiveness of multivalent and
multispecific compositions (e.g., MRD-containing antibodies) in
vivo, their efficacy in a mouse Colo5 tumor model was assessed. In
these experiments, tumors were implanted into the right flank of
six-week old female athymic nude mice by injecting 5.times.10.sup.6
Colo205 cells suspended in 100 .mu.L PBS. Three groups of eight
animals each received intraperitoneal injections of 5 mg/kg of
antibody (HERCEPTIN, Rituxan) or an MRD-containing antibody
(HER-2xCon4; "H2xCon4") in 100 .mu.L PBS every third day starting
at day 6 after tumor implantation. The results, shown in FIG. 27,
demonstrate that the MRD-containing antibody was more efficient at
inhibiting tumor growth than either Rituximab.RTM. or
HERCEPTIN.RTM..
[0815] HERCEPTIN with lm32 fused to the C-terminus of the heavy
chain also inhibited tumor growth in both Her2 dependent and
angiogenesis dependent xenograft tumor models. The HERCEPTIN-lm32
fusion had a similar PK to HERCEPTIN in both mice and monkeys after
single dose injections. Furthermore, the HERCEPTIN-lm32 fusion was
stable in whole blood at 37.degree. C. for up to 72 hours.
Example 32
Assays to Evaluate MRD-Containing Antibodies
[0816] Novel multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are generated by altering the sequence
of the MRD and/or the antibody, by altering the location at which
the antibody is linked to the MRD, and/or by altering the e linker
through which the MRD is connected to the antibody. The binding
potential, structure, and functional properties of the multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
are evaluated using known techniques to measure protein binding and
function. The multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) are compared to the MRD alone, the
antibody alone, and to other multivalent and multispecific
compositions (e.g., MRD-containing antibodies).
[0817] An MRD-containing antibody is tested using a solid phase
assay in which a target of the MRD and/or antibody is immobilized
on a solid surface and then exposed to increasing concentrations of
a fluorescently labeled MRD-containing antibody. The solid surface
is washed to remove unbound MRD-containing antibody and the amount
of target-bound MRD-containing antibody is determined directly by
quantitating fluorescence. In another experiment, the immobilized
target is exposed to increasing concentrations of an unlabeled
MRD-containing antibody and the amount of target-bound
MRD-containing antibody is determined indirectly by use of a
labeled reagent that binds to the MRD-containing antibody
[0818] An MRD-containing antibody is tested using a liquid phase
assay in which a target of the MRD and/or antibody is added to
various concentrations of an MRD-containing antibody is a solution.
The interaction of the target with the MRD-containing antibody is
detected by the appearance of a molecular complex comprised of a
target and MRD-containing antibody that differs in molecular mass
(and mobility) from unbound target and unbound MRD-containing
antibody.
[0819] An MRD-containing antibody is also assayed in a cell based
assay in which target-expressing cells are incubated in the
presence at increasing concentrations of MRD-containing antibody.
The binding of the MRD-containing antibody is detected by
fluorescence activated cell sorting. In addition, cellular
proliferation, cellular differentiation, protein phosphorylation,
protein expression, mRNA expression, membrane composition,
signaling pathway activity, and cellular viability are
assessed.
[0820] Useful multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) bind to both the MRD target and to the
antibody target. In addition, useful multivalent and multispecific
compositions (e.g., MRD-containing antibodies) affect at least one
cellular process.
Example 33
Identification of MRDs with Improved Characteristics
[0821] Two potential T cell epitopes were identified in LM32. In
order to identify LM32 variants that did not contain T cell
epitopes, and therefore, were less likely to produce immunogenic
responses, mutational and deletional variants of the LM32 peptide
were created. The LM32 variants listed in Table 7 MRDs were
expressed as MBP fusion proteins and tested for the ability to bind
Ang2.
TABLE-US-00007 TABLE 7 SEQ EC50 ID MRD expressed as a MBP fusion
protein (nM) NO SGGGSMGAQTNFMPMDNDELLLYEQFI 1.080 32
SGGGSMGAQTNFMPMDNEELLLYEQFI 20.700 33
SGGGSMGAQTNFMPMDNDEGLLYEQFILQQGLE 1.040 88
KSLSLSPGSGGGSMGAQTNFMPMDNDELGLYEQFILQQGLE na 89
KSLSLSPGSGGGSMGAQTNFMPMDNDEALLYEQFILQQGLE 0.182 90
KSLSLSPGSGGGSMGAQTNFMPMDNDELTLYEQFILQQGLE 1.420 91
KSLSLSPGSGGGSMGAQTNFMPMDNDELLLYEQFIYQQGLE na 92
KSLSLSPGSGGGSMGAQTNFMPMDNDEGLLYEQFIYQQGLE 0.902 93
KSLSLSPGSGGGSMGAQTNFMPMDNDEALLYEQFIYQQGLE 0.392 94
KSLSLSPGSGGGSMGAQTNFMPMDNEELTLYEQFIFQQG na 95
KSLSLSPGSGGGSMGAQTNFMPMDNDEGLLYEEFILQQGLE 0.922 96 KSLSLS
GSGGGSMGAQTNFMPMDNDEALLYEEFILQQGLE 0.426 97
KSLSLSPGSGGGSMGAQTNFMPMDNEELTLYEEFILQQGLE na 98
KSLSLSPGSGGGSMGAQTNFMPMDQDELLLYEQFILQQGLE 0.383 99
KSLSLSPGSGGGSMGAQTNFMPMDDDELLLYEQFILQQGLE 0.240 100
[0822] The LM32 variants are then tested for their ability to
induce proliferation and/or cytokine release. LM32 variants that
are functionally active and have reduced immunogenic potential are
identified. An MRD-containing antibody comprising the LM32 variant
fused to the light chain of HERCEPTIN.RTM., an MRD-containing
antibody comprising the LM32 variant fused to the heavy chain of
HERCEPTIN.RTM., an MRD-containing antibody comprising the LM32
variant fused to the light chain of HUMIRA.RTM., an MRD-containing
antibody comprising the LM32 variant fused to the heavy chain of
HUMIRA.RTM., MRD-containing antibody comprising the LM32 variant
fused to the light chain of AVASTIN.RTM., and an MRD-containing
antibody comprising the LM32 variant fused to the heavy chain of
AVASTIN.RTM. are created. The LM32-variant containing antibodies
are administered to animal models and the plasma protein
representation and plasma and tissue residence are measured and
compared to those of HERCEPTIN.RTM., HUMIRA.RTM., and AVASTIN.RTM..
In addition, the effects of the LM32-variant containing antibodies
on cellular proliferation, angiogenesis, tumorigenicity, arthritic
indicators are compared to the effects of HERCEPTIN.RTM.,
HUMIRA.RTM., and AVASTIN.RTM..
Example 34
In Vivo Assays to Evaluate MRD-Containing Antibodies
[0823] In order to determine the efficacy of multivalent and
multispecific compositions (e.g., MRD-containing antibodies) in
vivo, animal models are treated with an antibody and an
MRD-containing antibody and the results are compared.
[0824] MRD-containing anti-HER2 antibodies are tested in the
following in vivo model. NIH 3T3 cells transfected with a HER2
expression plasmid are injected into nu/nu athymic mice
subcutaneously at a dose of 10.sup.6 cells in 0.1 ml of
phosphate-buffered saline as described in U.S. Pat. No. 6,399,063,
which is herein incorporated by reference in its entirety. On days,
0, 1, 5, and every 4 days thereafter 100 .mu.g, of a HER2 antibody,
an ang2-containing HER2 antibody, an igf1r-containing HER2 antibody
and an ang2-igf1r-containing HER2 antibody are injected
intraperitoneally. Tumor occurrence and size are monitored for one
month. Increases in efficacy of multivalent and multispecific
compositions (e.g., MRD-containing antibodies) compared to
antibodies are observed.
[0825] MRD-containing anti-VEGF antibodies are tested in the
following in vivo model, RIP-T.beta.Ag mice are provided with
high-sugar chow and 5% sugar water as described in U.S. Published
Application No. 2008/0248033, which is herein incorporated by
reference in its entirety. At 9-9, 5 or 11-12 weeks of age, the
mice are treated twice-weekly with intra-peritoneal injections of 5
mg/kg of an anti-VEGF antibody, ang2-containing VEGF antibody,
ifg1r-containing VEGF antibody or ang2- and igf1r-containing
antibody. The 9-9.5 week mice are treating for 14 days and then
examined. The 11-12 week mice are examined after 7, 14, and 21 days
of treatment. The pancreas and spleen of the mice are removed and
analyzed. Tumor number is determined by dissecting out each
spherical tumor and counting. Tumor burden is determined by
calculating the sum of the volume of all tumors within the pancreas
of a mouse. The effect on angiogenesis is determined by calculating
the mean number of angiogenic islets observed. Increases in
efficacy of multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) compared to antibodies are observed.
[0826] MRD-containing anti-TNF antibodies are tested in the
following in vivo model. Transgenic mice (Tg197) are treated with
three intra-peritoneal injections of anti-TNF antibody or
ang2-containing TNF antibody at 1.5 .mu.g/g, 15 .mu.g/g, or 30
.mu.g/g as in U.S. Pat. No. 6,258,562, which is incorporated herein
by reference in its entirety. Injections continue for about 10
weeks and macroscopic changes in joint morphology are recorded each
week. At 10 weeks, mice are sacrificed and microscopic examination
of tissue is performed. Joint size is established as an average
measurement on the hind right ankle using a micrometer device and
arthritic scores are recorded as follows: 1=no arthritis; +/-=mild
(joint distortion); ++=moderate arthritis (swelling, joint
deformation); and +++=heavy arthritis (ankylosis detected on
flexion and severely impaired movement). Histopathological scoring
based on haematoxylinleosin staining of joint sections is based as
follows; 0=No detectable disease; 1=proliferation of the synovial
membrane; 2=heavy synovial thickening 3=cartilage destruction and
bone erosion. Increases in efficacy of multivalent and
multispecific compositions (e.g., MRD-containing antibodies)
compared to antibodies are observed.
Example 35
MRD-Containing Antibodies are Superior to Combinations of
Antibodies and MRDs
[0827] In order to compare the efficacy of multivalent and
multispecific compositions (e.g., MRD-containing antibodies) to
combinations of antibodies and MRDs, their effect on SK-BR-3 cells
treated with EGF was studied.
[0828] SK-BR-3 cells were treated with HERCEPTIN, HERCEPTIN
containing an EGFR-MRD, HUMIRA containing an EGFR-MRD ("MRD
alone"), or HERCEPTIN in combination with HUMIRA containing an
EGFR-MRD ("antibody plus MRD alone") for 10 minutes or 3 hours and
stimulated with EGF for 5 minutes. Cell lysates were collected and
western blots were used to determine phosphotyrosine or phospho-Akt
levels. The results are shown in FIG. 28. The HERCEPTIN containing
an EGFR-MRD completely inhibited EGF-induced receptor
phosphorylation and Akt activation, whereas HERCEPTIN, the MRD
alone, and the HERCEPTIN antibody plus MRD alone had little or no
effect.
[0829] In addition, the effect of multivalent and multispecific
compositions (e.g., MRD-containing antibodies) on cellular
proliferation was compared to the effect of the combination of
antibodies and MRDs. In these experiments, MCF-7 derived breast
carcinoma cells were treated with a HERCEPTIN antibody containing
an igf1r-targeting MRD fused to the C-terminus of the light chain,
HERCEPTIN alone, or a HUMIRA antibody containing the same
igf1r-targeting MRD ("MRD alone"). As shown in FIG. 29A, the
MRD-containing antibody inhibited cell proliferation better than
HERCEPTIN alone or the MRD alone.
[0830] MCF-7 cells were also treated with a penta-specific
MRD-containing antibody. First, these penta-specific multivalent
and multispecific compositions (e.g., MRD-containing antibodies)
were shown to bind to five targets. An Ang2-targeting MRD (lm32),
and an EGFR-targeting MRD were fused to the N- and C-termini,
respectively of a HERCEPTIN antibody. In addition, a
.alpha.v.beta.3-targeting MRD (eeti) and an igf1r-targeting MRD
were fused to the N- and C-termini, respectively of the same
HERCEPTIN antibody. Ang2, ErbB2, EGFR, IGF1R and .alpha.v.beta.3
were coated on separate wells of a 96-well plate and incubated with
serial dilutions of the MRD-containing antibody. Bound
MRD-containing antibody vas detected using anti-human IgG-kappa
detector. The results demonstrated that the MRD-containing antibody
bound to Ang2, ErbB2, EGFR, IGF1R and .alpha.v.beta.3 with low
nanomolar or sub-nanomolar affinities that are comparable to the
binding-affinities of the MRDs or antibodies individually.
[0831] Then, the ability of these penta-specific multivalent and
multispecific compositions (e.g., MRD-containing antibodies) to
inhibit proliferation of MCF-7 cells was tested as described above.
The results, shown in FIG. 29B demonstrate that the HERCEPTIN
penta-specific multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) decrease proliferation more efficiently
than the HERCEPTIN antibody.
Example 36
MRD-Containing Ant-TNF Antibodies
[0832] An MRD-containing antibody that targets TNF was created by
fusing an Ang2-binding MRD (lm32) to the C-terminus of HUMIRA heavy
chain and was expressed in both transient and stable expression
systems. The MRD-containing antibody bound simultaneously to
TNF.alpha. and Ang2. The MRD-containing antibody bound soluble and
cell surface TNF.alpha. and retained the binding specificity and Fc
functions of HUMIRA (e.g., FcRn, FcgammaR1 and FcgammaR3 binding)
and also inhibited Ang2 mediated signaling through Tie-2 receptor
in a dose dependent manner with sub-nanomolar affinities.
[0833] The HUMIRA-lm32 fusion also inhibited TNF.alpha. mediated
cytotoxicity in L929 cells. The cells were cultured in 96-well
plates overnight and treated with 1 ng/mL TNF plus 1 mg/ml of
Actinomycin D in the presence of HUMIRA or HUMIRA-lm32 for 24 hours
at 37.degree. C. After incubation, 100 .mu.L of cell Titer-Glow
reagent was added, and luminescence was measured using InVision
(Perkin-Elmer) after 15 minutes at room temperature. The results
demonstrated that HUMIRA and HUMIRA-lm32 displayed equal potency in
inhibiting TNF.alpha.-mediated cytotoxicity in L929 cells (HUMIRA
IC50=19.0 ng/ml; HUMIRA-lm32 IC50-18.9 ng/ml).
[0834] Furthermore, the HUMIRA-lm32 fusion displayed a
dose-dependent protection of hTNF-transgenic mice from clinical
signs of arthritis in a well-established mouse model. See, e.g.,
Keffer et al., EMBO J. 10:4025-4031 (1991). A single-dose PK study
in mice demonstrated that the HUMIRA-lm32 fusion and HUMIRA have
similar PK and immunogenicity profiles. However, the HUMIRA-lm32
fusion showed increased efficacy in this model compared to that of
HUMIRA alone both when measured by clinical symptoms or by
histology. See FIG. 30.
Example 37
Zybodies Inhibit EGF-Induced Signaling
[0835] SK-BR3 cells were plated at 0.5.times.10.sup.6 cells/well in
6-well plates and incubated (37.degree. C., 5% CO.sub.2) for 24
hours at which time cells were treated with 10 .mu.g/mL bi- and
tri-specific zybodies or Herceptin in 1 mL complete DMEM medium
with 10% FBS for 24 hr. at 37.degree. C. Cells were then stimulated
with 100 ng/mL EGF for 5 minutes. After stimulation, cells were was
disrupted in 200 .mu.L cell lysis buffer (10 mM Tris-HCl (pH 7.5),
1% Triton X-100, 150 mM NaCl, 10% Glycerol, 1 mM sodium vanadate, 5
mM EDTA and protease inhibitors). The cell lysates were centrifuged
at 14000 RPM for 10 minutes at 4.degree. C. to remove cell debris.
Equal volume of 2.times. sample buffer and cell lysates were mixed
and boiled at 100.degree. C. for 5 minutes, proteins were resolved
on a 10% SDS-PAGE and transferred to PVDF (catalog#LC2005,
Invitrogen) membranes. Membranes were block with 3% BSA, 0.1% Tween
20 overnight and incubated with antibodies to phospho-AKT
(catalog#AF887, R&D systems), phospho-ERK (Catalog#AF1018,
R&D systems), and total ERK (MAB1576, R&D system).
Horseradish or AP conjugated anti-rabbit and anti-mouse secondary
antibodies (Invitrogen) were used to visualize immune-reactive
proteins using chemiluminescence or AP detection reagents
respectively.
[0836] One bispecific antibody used in this example comprised
Herceptin and an EGFR-binding MRD (Her-egfr). Another bispecific
antibody comprised Herceptin with a Pertuzumab-scfv (which targets
a different HER2 epitope than the Herceptin antibody) on the
C-terminus of the heavy chain (Her-Pertuzumab(H)). One trispecific
antibody comprised Herceptin with an EGFR-binding MRD on the
C-terminus of the heavy chain and a Pertuzumab-scfv on the
C-terminus of the light chain. Another trispecific antibody
comprised Herceptin with an EGFR-binding MRD on the C-terminus of
the light chain and a Pertuzumab-scfv on the C-terminus of the
heavy chain (Her-zEGFR(L)-Pert(H)). Another trispecific antibody
comprised an EGFR-binding MRD, and a Pertuzumab-scfv which targets
a different HER2 epitope than the Herceptin antibody
(Her-Pert(L)zEGFR(H) and Her-zEGFR(L)-Pert(H).
[0837] SK-BR3 cells express very high levels of HER2 and are
sensitive to anti-proliferative effects of Herceptin. Inhibition of
constitutively activated AKT is one of the mechanisms for the
anti-proliferative effects of Herceptin in HER2 over-expressing
cells. Such inhibition can be overcome by the addition of growth
factors such as EGF, IGF-1 and Heregulin (HRG) through the
induction of intracellular pro-mitogenic signaling. As shown in
FIG. 31, EGF-induced activation of AKT and ERK pathways in SK-BR3
cells as shown by increase in phoshorylated AKT and ERK levels.
Herceptin had no effect on EGF-induced activation of signaling
pathways in SK-BR3 cells whereas Her-egfr and the two tri-specific
zybodies with an EGFR targeting peptide inhibited EGF effects.
Compared to the bi-specific antibody which inhibited only
EGF-induced signaling, tri-specific antibodies also inhibited
constitutively active levels of Akt and ERK in SK-BR3 cells.
Example 38
Zybodies Inhibit Heregulin-Induced Signaling
[0838] SK-BR3 cells were plated at 0.5.times.10.sup.6 cells/well in
6-well plates and cultured overnight. The next day, cells were
treated with 10 .mu.g/mL bi- and tri-specific zybodies or Herceptin
in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37.degree.
C. Cells were then stimulated with 200 ng/mL Heregulin for 10
minutes. Western blot analysis was performed to detect
phosphorylated and total Akt and ERK levels as described in Example
37.
[0839] Heregulin binds to HERS and induces activation of signaling
pathways via HER2-HER3 heterodimer formation. As shown in FIG. 32,
Heregulin induced activation of Akt and ERK in SK-BR3 cells.
Herceptin and HER-egfr had no effect on Heregulin-induced Akt
activation. Pertuzumab, but not Herceptin, blocks Heregulin
mediated HER2-HER3 heterodimer formation and signaling. FIG. 32
shows that bi-and tri-specific zybodies comprising a
Pertuzumab-scfv completely inhibited Heregulin-induced Akt
activation in SK-BR3 cells.
Example 39
Zybodies Inhibit EGF- and Heregulin-Induced Signaling
[0840] SK-BR3 cells were plated at 0.5.times.10.sup.6 cells/well in
6-well plates and cultured overnight. The next day, cells were
treated with 10 .mu.g/mL bi and tri-specific zybodies or Herceptin
in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37.degree.
C. Cells were then stimulated with 100 ng/mL EGF and 200 ng/mL
Heregulin for 10 minutes. Western blot analysis was performed to
detect phosphorylated and total Akt and ERK levels as described in
Example 37.
[0841] Combined stimulation of SK-BR3 cells with EGF and Heregulin
resulted in both AKT and ERK activation (FIG. 33). Herceptin,
HER-efgr and HER-pertuzuScfv were ineffective in blocking the
combined effects of EGF and Heregulin. Tri-specific zybodies that
contained both an EGFR targeting peptide and a Pertuzumab-scfv
completely inhibited EGF and Heregulin induced Akt and ERK
activation in SK-BR3 cells.
Example 40
Zybodies Down-Regulate Cell-Surface EGFR
[0842] SK-BR3 cells were plated at 0.5.times.10.sup.6 cells/well in
6-well plates and cultured overnight. The next day, cells were
treated with 10 .mu.g/mL bi- and tri-specific zybodies, Herceptin,
Erbitux, EGF, and combinations thereof in 1 mL complete DMEM medium
with 10% FBS for 24 hr. at 37.degree. C. as indicated. Cells were
detached and the levels of EGFR on the cells were determined by
Flow cytometry using PE conjugated anti-EGFR antibody.
[0843] The zybodies used in this experiment were a Herceptin
antibody with an EGFR-binding MRD on the C-terminal of the heavy
chain (HER-egfr(H)); a Humira antibody with an EGFR-binding MRD on
the C-terminal of the heavy chain (HUM-egfr(H)); Herceptin with a
Pertuzumab-scfv on the C-terminal of the heavy chain
(HER-perscfv(H)); Humira with a Pertuzumab-scfv on the C-terminal
of the heavy chain (HUM-perscfv(H)); Herceptin with a
Pertuzumab-scfv on the C-terminal of the light chain and an
EGFR-binding MRD on the C-terminal of the heavy chain
(HER-perscfv(L)-egfr(H)); Herceptin with a Pertuzumab-scfv on the
C-terminal of the heavy chain and an EGFR-binding MRD on the
C-terminal of the light, chain (HER-perscfv(H)-egfr(L)); and a
Humira antibody with an EGFR-binding MRD (HUM-egfr).
[0844] Erbitux, EGF, and bi- and tri-specific zybodies containing
egfr-targeting peptides down regulated EGFR receptor levels on the
cell surface. FIGS. 34A and B. Treatment with HER-egfr for 24 hr.
was more effective (61%) than HUM-egfr alone (27%) or in
combination of HUM-egfr plus Herceptin (41%) and Erbitux plus
Herceptin (51%) (FIGS. 34A and B). This indicates that simultaneous
targeting of EGFR and HER2 in zybody format was more effective in
down regulating EGFR than a combination of two individual
antibodies. Also, treatment with tri-specific antibodies that
target EGFR and two different epitopes of HER2 completely down
regulated (99%) EGFR levels on SK-13R3 cells.
Example 41
Zybodies Down-Regulate EGFR Expression
[0845] SK-BR3 cells were plated at 0.5.times.10.sup.6 cells/well in
6-well plates and cultured overnight. The next day, cells were
treated with 10 .mu.g/mL bi and tri-specific zybodies or Herceptin
in 1 mL complete DMEM medium with 10% FBS for 24 hr. at 37.degree.
C. Cell lysates were prepared and Western blot analysis was
performed to detect EGFR using EGFR antibody (catalog# MAB 10951,
R&D system) and total ERK levels as described in Example
37.
[0846] Treatment with Herceptin and Her-Pertuzumab scfv had no
effect on EGFR levels in SK-BR3 cells. HER-egfr partially down
regulated EGFR protein and almost complete degradation of EGFR was
observed in cells treated with tri-specific zybodies.
Example 42
Zybodies Increase MRD Potency
[0847] The following experiments were performed to examine the
effect that antibody fusion has on MRD potency. Individual wells of
a 96-well plate were coated with IGF1R and ErbB2-Fc (HER2) (1
.mu.g/ml) at varying ratios. Wells were washed, and then serial
dilutions of zybodies were added in the presence of biotin-labeled
IGF-1. The zybodies used were a Herceptin antibody containing an
IGFR-targeting MRD on, the C-terminus of the heavy chain
(HER-igfr(H)) and a Humira antibody containing an IGFR-targeting
MRD on the C-terminus of the heavy chain (HUM-igfr(H)). Bound IGF-1
was quantitated by addition of streptavidin-HRP. The results are
shown in the table below.
TABLE-US-00008 TABLE 8 Inhibition of IGF-1 Binding, IC.sub.50 (nM)
Ratio of IGF1R:HER2 Zybody 1:0 3:1 1:1 1:3 HER-igfr(H) 220 20.91
1.44 0.14 HUM-igfr(H) 240 189.9 186 111
[0848] The inhibition of IGF-1 binding was similar for both
zybodies tested when the plates did not contain ErbB2-Fc (HER2)
(IC50 HER-igfr(H)=220 nM; IC50 HUM-igfr(H)=240 nM). When the plates
contained both the antibody target and the MRD target, binding to
MRD target drastically decreased. For example, using the
HER-igfr(H) zybody, inhibition of IGF-1 binding dropped from
IC.sub.50=220 nm when the antibody target was not on the plates
(ratio of IGFR1R:HER2=1:0) to IC.sub.50=1.44 when the antibody
target and MRD target were present on the plates in equivalent
amounts (ratio of IGFR1R:HER2=1:1). The data indicate that
engagement of both antibody and MRD targets by HER-igf1r(H)
enhances the potency of the low affinity IGF-1R MRD>1000-fold.
In contrast, this drastic effect was not observed when the same
experiment was performed using a zybody containing the Humira
antibody, which does not bind to HER2 even though the zybody
contained the same IGF1R-targeting MRD. Thus, bi-specific zybodies
display enhanced MRD potency through heterotypic avidity-driven
binding.
Example 43
Conformational Constraints Can Increase MRD Binding and
Stability
[0849] In order to determine the effect of conformational
constraints on MRDs in the context of an MRD-containing antibody,
several modified MRD constructs containing cysteines at various
locations were developed. The cysteines form intermolecular
disulfide bonds and therefore contain the three-dimensional
conformation of the proteins. The MRD that was altered in this
experiment was MPM. The sequence of MPM, which is shown in the
table below, is similar to the sequence of lm32 (described above),
but contains four amino acid changes: M5G, N16Q, L19A, and Q24E.
The G at position five in MPM minimizes potential oxidation of the
methionine residue, and L19A and Q24E remove potentially
immunogenic sequences. In these experiments, all MRDs were fused to
the C-terminus of the Herceptin heavy chain.
[0850] The resulting multivalent and multispecific compositions
(e.g., MRD-containing antibodies) were then tested for target
binding and stability. Stability was determined by administering
the MRD-containing antibody by IV injection to mice and comparing
the levels of MRD in the plasma 15 minutes post-injection to the
levels of MRD in the plasma 2, 3, or 4 days post-injection. The
results are shown below in Table 9.
TABLE-US-00009 TABLE 9 Binding and Stability of Constrained MRDs
EC50 MRD % Construct Sequence mm 48 hrs HER-2x-con4
AQQEECEWDPWTCEHMGSGSATGGS 0.018 70 GSTASSGSGS THQEECEWDPWTCEH MLE
(SEQ ID NO: 136) HER-lm32(H) KSLSLSPGKGGGSMGAQTNFMPMDND 0.03 23
ELLLYEQFILQQGLE (SEQ ID NO: 34) HER-mpm(H)
KSLSLSPGSGGGSGGAQTNFMPMDQD 19 EALLYEEFILQQGLE (SEQ ID NO: 56)
HER-lm32 (MPM KSLSLSPGSGGGSGGACTNFMPMDQD 4.06 Q8C G30C) (H)
EALLYEEFILQQCLE (SEQ ID NO: 57) HER-lm32 (MPM
KSLSLSPGSGGGSGGAQCNFMPMDQD 0.598 T9C G30C) (H) EALLYEEFILQQCLE (SEQ
ID NO: 58) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTCFMPMDQD 0.177 9 N10C
G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 67) HER-lm32 (MPM
KSLSLSPGSGGGSGGAQTNCMPMDQD 5.91 F11C G30C) (H) EALLYEEFILQQCLE (SEQ
ID NO: 68) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTCFMPMDQD 0.403 33 N10C
L28C) (H) EALLYEEFICQQGLE (SEQ ID NO: 69) HER-lm32 (MPM
KSLSLSPGSGGGSCGAQTNFMPMDQD 0.0641 60 M5C G30C) (H) EALLYEEFILQQCLE
(SEQ ID NO: 70) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTNFMPMDQD 0.109 14
M5C L28C) (H) EALLYEEF CQQGLE (SEQ ID NO: 101) HER-lm32 MPM
KSLSLSPGSGGGSGGCQTNFMPMDQD 0.174 82 A7C G30C) (H) EALLYEEFILQQCLE
(SEQ ID NO: 108) HER-lm32 (MPM KSLSLSPGSGGGSGGAQTNFMCMDQD P13C
G30C) (H) EALLYEEFILQQCLE (SEQ ID NO: 109) HER-lm32 (MPM
KSLSLSPGSGGGSCGAQTCFMPMDQD 0.2 13 M5C N10C) (H) EALLYEEFILQQGLE
(SEQ ID NO: 110) HER-lm32 (MPM KSLSLSPGSGGGSCGAQTNCMPMDQD M5C F11C)
(H) EALLYEEFILQQGLE (SEQ ID NO: 111) HER-lm32 (MPM
KSLSLSPGSGGGSCFMPMDQDEALLYE 0.751 3 D5 N10C G30C) EFILQQCLE (SEQ ID
NO: 112) (H) HER-lm32 (MPM KSLSLSPGSGGGSCFMPMDQDEALLYE 0.697 36 D5
N10C L28C) EFICQQGLE (SEQ ID NO: 113) (H)
[0851] The results demonstrated that adding two cysteine residues
outside the core target-binding domain (e.g., PMDQDEALLY in MPM) of
an MRD can increase the MRD half-life without substantially
decreasing the binding affinity.
[0852] Constructs containing one cysteine located near the terminus
of the molecule (e.g., about two amino acids away from the
terminus) and one cysteine located on the opposite end of the
target-binding domain (e.g., at least about 3 or about 4-7 amino
acids outside of the core binding domain) and near the protein
fusion (e.g., about 4-6 amino acids away from the linker or
antibody sequence) can show increased MRD half-life without
substantially decreasing the binding affinity. Furthermore. MRDs
that include cysteines within the target-binding site, in
particular on either end of the target-binding site can be MRDs
that have both high stability and efficient target binding.
[0853] Novel multivalent and multispecific compositions (e.g.,
MRD-containing antibodies) in which the MRDs have a long half-life
in vivo and efficient target-binding can be identified by altering
the sequence of the MRD to include at least two cysteines. The
cysteines can be at least about 6, about 7, about 8, about 9, about
10, about 11, about 12, about 13, about 14, about 15, about 16,
about 17, about 18, about 19, or about 20 amino acids away from
each other. The binding potential and half-life of the MRD in the
MRD-containing antibody is evaluated using known techniques and the
methods described herein. MRD-containing antibodies are
administered to mice intravenously. Plasma is collected from mice
shortly after administration (e.g., 15 minutes after
administration) and at a later time point (e.g., 2, 3, or 4 days
after administration). Useful multivalent and multispecific
compositions (e.g., MRD-containing antibodies) that both bind
efficiently to the MRD target and are stable in vivo (e.g., at
least about 50% of the MRD is present 48 hours after
administration) are identified.
Example 44
MRD-Containing Antibodies for Redirected T-Cell Killing
[0854] Antibody MRD-fusion molecules were prepared by fusion of a
CD3-targeting peptide to an anti-CD19 antibody and by fusion of a
CD19-targeting peptide to an anti-CD3 antibody. The resulting
MRD-containing antibodies are analyzed by flow cytometric analysis
on CD3-positive Jurkat cells, human PBMCs and a number of different
CD19-positive B cell lymphoma cell lines (e.g., SKW6.4, Blin I,
BJAB, Daudi and Raji) to determine their specific binding
affinities to each target. Since BL60 and the plasmacytoma cell
lines NCI and L363 are negative for both CD3 and CD19, they are
used as negative control cells to determine the specificity
MRD-containing antibodies. CD3-negative Jurkat cells can also be
used as a negative control cell population. Cell lines are cultured
in complete RPMI 1640 (Invitrogen) with 10% FCS (GIBCO).
[0855] Cells are washed with PBS and blocked by resuspension in PBS
with 10% human IgG (Innovative Research) and 0.1% NaN.sub.3
(blocking buffer) for 30 min at 4.degree. C. Cells are then
pelleted by centrifugation (100.times.g for 5 min) followed by
incubation with the MRD-containing antibodies in blocking buffer
for 30 min at 4.degree. C. The cells are washed three times with
PBS, and cell-surface bound MRD-containing antibodies are detected.
Flow cykometry can be performed with a BD FACScan.
Example 45
In Vitro Cytotoxicity of MRD-Containing Antibodies for Redirected
T-Cell Killing
[0856] The bispecific CD19/CD3 MRD-containing antibodies are
assayed with respect to their abilities to induce redirected T-cell
killing of CD19-positive lymphoma cells. Human peripheral blood
mononuclear cells (PBMCs) are isolated as effector cells from fresh
buffy coats of random donors using Lymphoprep.TM.
(Nycomed/Axis-Shield PoC) gradient centrifugation with subsequent
centrifugation at 100.times.g to remove platelets. CD19-positive B
cells are depleted using Dynabeads.RTM. CD19 Pan B (Life
Technologies). The PBMC populations are analyzed by flow cytometry
before and after CD19-positive B cell depletion by labeling with
FITC-conjugated mouse antibody against human CD19 and
counter-labeled with a PE-conjugated anti-CD45 antibody. The PBMCs
are incubated overnight at 37.degree. C. under 5% CO.sub.2.
CD19-positive B cell lines (e.g., SKW6.4, Blin I, BJAB, Daudi and
Raji) were used as target cells.
[0857] Target cells are incubated in 96-well plates using RPMI 1640
complete medium (Invitrogen) with 10% FCS (GIBCO) at different
densities, such that addition of the same number of unstimulated
PBMCs resulted in different effector-to-target cell (ET) ratios.
Various concentrations of bispecific CD19/CD3 MRD-containing
antibodies are then added to each well followed by the addition of
unstimulated PEMCs. Plates are incubated at 37.degree. C. under 5%
CO.sub.2 for 3 hrs. Cytotoxicity can be measured using the
DELFIA.RTM. EuTDA cytotoxicity assay (PerkinElmer) in round-bottom
96-well-plates following manufacturer's instructions. Spontaneous
cell death is measured by incubating the target cells without
effector cells or MRD-containing antibodies, and maximal cell death
is determined by incubating the target cells with 10% Triton X-100.
The fraction of specific cell lysis is calculated as the ratio
between effector mediated cytotoxicity ([experimental cell
death]-[spontaneous cell death]) and the maximum expected
cytotoxicity ([maximal cell death]-[spontaneous cell death]).
Example 45
In Vivo Efficacy of MRD-Containing Antibodies for Redirected T-Cell
Killing
[0858] Raji B lymphoma cells are removed from routine cell culture,
washed in PBS, and prepared as 1.times.10.sup.7 NOD/SCID mice are
then inoculated subcutaneously with 1.times.10.sup.6 Raji cells
with or without 5.times.10.sup.6 PBMCs (as prepared above) in a 50%
Matrigel solution. Bispecific CD19/CD3 MRD-containing antibodies
are administered intravenously 1 hr after lymphoma cell
inoculation. As negative controls, bispecific MRD-containing
antibodies directed to HER2 and CD3 (i.e., a CD3-binding MRD used
to an anti-HER2 antibody and a HER2-binding MRD fused to an
anti-CD3 antibody) and HER2 and CD19 ((i.e., a CD19-binding MRD
fused to an anti-HER2 antibody and a HER2-binding MRD fused to an
anti-CD19 antibody) and PBS are also administered intravenously 1
hr after lymphoma cell inoculation. MRD-containing antibodies or
PBS are administered once per day for four days after the initial
dose. Subcutaneous tumors are measured by caliper to determine
growth, rate for each treatment group. Body weight of mice is also
determined twice per week as an indicator of treatment
tolerability.
[0859] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims
[0860] This application claims the benefit under 35 U.S.C.
.sctn.119(e) to Provisional Application Nos. 61/489,249, filed May
24, 2011; 61/597,714, filed Feb. 10, 2012; and 61/610,831 filed
Mar. 14, 2012; each of which is herein incorporated by reference in
its entirety. Additionally, the disclosure of U.S. Appl. Publ. No.
2012/0100166 is herein incorporated by reference in its
entirety.
[0861] All publications, patents, patent applications, internet
sites, and accession numbers/database sequences (including both
polynucleotide and polypeptide sequences) cited are herein
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent, patent
application, internet site, or accession number/database sequence
were specifically and individually indicated to be so incorporated
by reference.
Sequence CWU 1
1
13514PRTArtificial Sequencelinker peptide 1Gly Gly Gly Ser 1
215PRTArtificial Sequencelinker peptide 2Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Gly Gly Ser Ser 1 5 10 15 37PRTArtificial
Sequenceintegrin targeting MRD 3Tyr Cys Arg Gly Asp Cys Thr 1 5
47PRTArtificial Sequenceintegrin targeting MRD 4Pro Cys Arg Gly Asp
Cys Leu 1 5 57PRTArtificial Sequenceintegrin targeting MRD 5Thr Cys
Arg Gly Asp Cys Tyr 1 5 67PRTArtificial Sequenceintegrin targeting
MRD 6Leu Cys Arg Gly Asp Cys Phe 1 5 728PRTArtificial
Sequencecytokine targeting MRD 7Met Gly Ala Gln Thr Asn Phe Met Pro
Met Asp Asp Leu Glu Gln Arg 1 5 10 15 Leu Tyr Glu Gln Phe Ile Leu
Gln Gln Gly Leu Glu 20 25 828PRTArtificial Sequencelm 32MRD 8Met
Gly Ala Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Leu Leu 1 5 10
15 Leu Tyr Glu Gln Phe Ile Leu Gln Gln Gly Leu Glu 20 25
927PRTArtificial SequenceANGa 9Gly Ala Gln Thr Asn Phe Met Pro Met
Asp Asp Leu Glu Gln Arg Leu 1 5 10 15 Tyr Glu Gln Phe Ile Leu Gln
Gln Gly Leu Glu 20 25 1054PRTArtificial Sequence2xCon4 10Ala Gln
Gln Glu Glu Cys Glu Trp Asp Pro Trp Thr Cys Glu His Met 1 5 10 15
Gly Ser Gly Ser Ala Thr Gly Gly Ser Gly Ser Thr Ala Ser Ser Gly 20
25 30 Ser Gly Ser Ala Thr His Gln Glu Glu Cys Glu Trp Asp Pro Trp
Thr 35 40 45 Cys Glu His Met Leu Glu 50 1118PRTArtificial
SequenceANGb 11Leu Trp Asp Asp Cys Tyr Phe Phe Pro Asn Pro Pro His
Cys Tyr Asn 1 5 10 15 Ser Pro 1218PRTArtificial SequenceANGc 12Leu
Trp Asp Asp Cys Tyr Ser Tyr Pro Asn Pro Pro His Cys Tyr Asn 1 5 10
15 Ser Pro 1319PRTArtificial SequenceVEGF targeting MRD 13Val Glu
Pro Asn Cys Asp Ile His Val Met Trp Glu Trp Glu Cys Phe 1 5 10 15
Glu Arg Leu 1427PRTArtificial SequenceMRD which targets IGF1R 14Ser
Phe Tyr Ser Cys Leu Glu Ser Leu Val Asn Gly Pro Ala Glu Lys 1 5 10
15 Ser Arg Gly Gln Trp Asp Gly Cys Arg Lys Lys 20 25
1518PRTArtificial SequenceANGd 15Leu Trp Asp Asp Cys Tyr Ser Phe
Pro Asn Pro Pro His Cys Tyr Asn 1 5 10 15 Ser Pro 1618PRTArtificial
SequenceANGe 16Asp Cys Ala Val Tyr Pro Asn Pro Pro Trp Cys Tyr Lys
Met Glu Phe 1 5 10 15 Gly Lys 1718PRTArtificial SequenceANGf 17Pro
His Glu Glu Cys Tyr Phe Tyr Pro Asn Pro Pro His Cys Tyr Thr 1 5 10
15 Met Ser 1818PRTArtificial SequenceANGg 18Pro His Glu Glu Cys Tyr
Ser Tyr Pro Asn Pro Pro His Cys Tyr Thr 1 5 10 15 Met Ser
1918PRTArtificial Sequencelong linker peptide 19Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg 1 5 10 15 Ser Ser
2060PRTArtificial SequenceLM-2x-32 20Met Gly Ala Gln Thr Asn Phe
Met Pro Met Asp Asn Asp Glu Leu Leu 1 5 10 15 Leu Tyr Glu Gln Phe
Ile Leu Gln Gln Gly Leu Glu Gly Gly Ser Gly 20 25 30 Ser Thr Ala
Ser Ser Gly Ser Gly Ser Ser Leu Gly Ala Gln Thr Asn 35 40 45 Phe
Met Pro Met Asp Asn Asp Glu Leu Leu Leu Tyr 50 55 60
2126PRTArtificial Sequencefusion protein 21Gly Tyr Tyr Val Cys Tyr
Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn 1 5 10 15 Phe Tyr Leu Tyr
Leu Arg Ala Arg Val Cys 20 25 227PRTArtificial Sequencefusion
protein 22Tyr Leu Tyr Leu Arg Ala Arg 1 5 2323PRTArtificial
Sequencefusion protein 23Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys
Asp Lys Glu Ser Thr Val 1 5 10 15 Gln Val His Tyr Arg Met Cys 20
249PRTArtificial Sequencefusion protein 24Asp Lys Glu Ser Thr Val
Gln Val His 1 5 2578PRTArtificial Sequencehuman CD3 delta mature
ECD 25Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys
Asn 1 5 10 15 Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu
Leu Ser Asp 20 25 30 Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu
Asp Pro Arg Gly Ile 35 40 45 Tyr Arg Cys Asn Gly Thr Asp Ile Tyr
Lys Asp Lys Glu Ser Thr Val 50 55 60 Gln Val His Tyr Arg Met Cys
Gln Ser Cys Val Glu Leu Asp 65 70 75 2689PRTArtificial
Sequencehuman CD3 gamma mature ECD 26Gln Ser Ile Lys Gly Asn His
Leu Val Lys Val Tyr Asp Tyr Gln Glu 1 5 10 15 Asp Gly Ser Val Leu
Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr 20 25 30 Trp Phe Lys
Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys 35 40 45 Lys
Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln 50 55
60 Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg
65 70 75 80 Met Cys Gln Asn Cys Ile Glu Leu Asn 85
27105PRTArtificial Sequencehuman CD3 epsilon mature ECD 27Gly Asn
Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val Ser 1 5 10 15
Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly Ser 20
25 30 Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu
Asp 35 40 45 Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu
Lys Glu Phe 50 55 60 Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys
Tyr Pro Arg Gly Ser 65 70 75 80 Lys Pro Glu Asp Ala Asn Phe Tyr Leu
Tyr Leu Arg Ala Arg Val Cys 85 90 95 Glu Asn Cys Met Glu Met Asp
Val Met 100 105 289PRTArtificial Sequencehuman CD3 zeta mature ECD
28Gln Ser Phe Gly Leu Leu Asp Pro Lys 1 5 2927PRTArtificial
SequenceMRD-binding polypeptide 29Gln Asp Gly Asn Glu Glu Met Gly
Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr
Thr Val Ile Leu Thr 20 25 3010PRTArtificial SequenceMRD-binding
polypeptide 30Gln Asp Gly Asn Glu Glu Met Gly Gly Ile 1 5 10
319PRTArtificial SequenceMRD-binding polypeptide 31Gln Asp Gly Asn
Glu Glu Met Gly Gly 1 5 3235PRTArtificial SequenceMRD expressed as
a MBP fusion protein 32Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly
Gly Ser Met Gly Ala 1 5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn
Asp Glu Leu Leu Leu Tyr Glu 20 25 30 Gln Phe Ile 35
3335PRTArtificial SequenceMRD expressed as a MBP fusion protein
33Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Glu Glu Leu Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile 35 3441PRTArtificial SequenceHER-lm32(H)
34Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Leu Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu Glu 35 40
3528PRTArtificial SequenceRm4-31 35Asn Phe Tyr Gln Cys Ile Glu Met
Leu Ala Ser His Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly Gly 20 25 3628PRTArtificial SequenceRm4-33
36Asn Phe Tyr Gln Cys Ile Glu Gln Leu Ala Leu Arg Pro Ala Glu Lys 1
5 10 15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly Gly 20 25
3728PRTArtificial SequenceRm4-39 37Asn Phe Tyr Gln Cys Ile Asp Leu
Leu Met Ala Tyr Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly Gly 20 25 3828PRTArtificial SequenceRm4-310
38Asn Phe Tyr Gln Cys Ile Glu Arg Leu Val Thr Gly Pro Ala Glu Lys 1
5 10 15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly Gly 20 25
3928PRTArtificial SequenceRm4-314 39Asn Phe Tyr Gln Cys Ile Glu Tyr
Leu Ala Met Lys Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly Gly 20 25 4028PRTArtificial SequenceRm4-316
40Asn Phe Tyr Gln Cys Ile Glu Ala Leu Gln Ser Arg Pro Ala Glu Lys 1
5 10 15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly Gly 20 25
4128PRTArtificial SequenceRm4-319 41Asn Phe Tyr Gln Cys Ile Glu Ala
Leu Ser Arg Ser Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly Gly 20 25 4227PRTArtificial SequenceRm4-44
42Asn Phe Tyr Gln Cys Ile Glu His Leu Ser Gly Ser Pro Ala Glu Lys 1
5 10 15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly 20 25
4327PRTArtificial SequenceRm4-45 43Asn Phe Tyr Gln Cys Ile Glu Ser
Leu Ala Gly Gly Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly 20 25 4427PRTArtificial SequenceRm4-46 44Asn
Phe Tyr Gln Cys Ile Glu Ala Leu Val Gly Val Pro Ala Glu Lys 1 5 10
15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly 20 25
4527PRTArtificial SequenceRm4-49 45Asn Phe Tyr Gln Cys Ile Glu Met
Leu Ser Leu Pro Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly 20 25 4627PRTArtificial SequenceRm4-410 46Asn
Phe Tyr Gln Cys Ile Glu Val Phe Trp Gly Arg Pro Ala Glu Lys 1 5 10
15 Ser Arg Gly Gln Trp Gln Glu Cys Arg Thr Gly 20 25
4727PRTArtificial SequenceRm4-411 47Asn Phe Tyr Gln Cys Ile Glu Gln
Leu Ser Ser Gly Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Gln
Glu Cys Arg Thr Gly 20 25 4827PRTArtificial SequenceRm4-415 48Asn
Phe Tyr Gln Cys Ile Glu Leu Leu Ser Ala Arg Pro Ala Glu Lys 1 5 10
15 Ser Arg Gly Gln Trp Ala Glu Cys Arg Ala Gly 20 25
4927PRTArtificial SequenceRm4-417 49Asn Phe Tyr Gln Cys Ile Glu Ala
Leu Ala Arg Thr Pro Ala Glu Lys 1 5 10 15 Ser Arg Gly Gln Trp Val
Glu Cys Arg Ala Pro 20 25 5018DNAArtificial SequenceRm2-2-218
50gtggagtgca gggcgccg 18516PRTArtificial SequenceRm2-2-218 51Val
Glu Cys Arg Ala Pro 1 5 5218DNAArtificial SequenceRm2-2-316
52gctgagtgca gggctggg 18536PRTArtificial SequenceRm2-2-316 53Ala
Glu Cys Arg Ala Gly 1 5 5418DNAArtificial SequenceRm2-2-319
54caggagtgca ggacgggg 18556PRTArtificial SequenceRm2-2-319 55Gln
Glu Cys Arg Thr Gly 1 5 5641PRTArtificial SequenceHER-mpm(H) 56Lys
Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Gly Gly Ala 1 5 10
15 Gln Thr Asn Phe Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu
20 25 30 Glu Phe Ile Leu Gln Gln Gly Leu Glu 35 40
5741PRTArtificial SequenceHER-lm32 (MPM Q8C G30C) (H) 57Lys Ser Leu
Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Gly Gly Ala 1 5 10 15 Cys
Thr Asn Phe Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20 25
30 Glu Phe Ile Leu Gln Gln Cys Leu Glu 35 40 5841PRTArtificial
SequenceHER-lm32 (MPM T9C G30C) (H) 58Lys Ser Leu Ser Leu Ser Pro
Gly Ser Gly Gly Gly Ser Gly Gly Ala 1 5 10 15 Gln Cys Asn Phe Met
Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20 25 30 Glu Phe Ile
Leu Gln Gln Cys Leu Glu 35 40 5912PRTArtificial SequenceVL-CDR1
59Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 1 5 10
607PRTArtificial SequenceVL-CDR2 60Ser Ala Ser Phe Leu Tyr Ser 1 5
619PRTArtificial SequenceVL-CDR3 61Gln Gln His Tyr Thr Thr Pro Pro
Thr 1 5 6210PRTArtificial SequenceVH-CDR1 62Gly Arg Asn Ile Lys Asp
Thr Tyr Ile His 1 5 10 6317PRTArtificial SequenceVH-CDR2 63Arg Ile
Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15
Gly 6411PRTArtificial SequenceVH-CDR1 64Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr 1 5 10 65109PRTArtificial SequenceVL 65Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105 66120PRTArtificial SequenceVH 66Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20
25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 6741PRTArtificial SequenceHER-lm32 (MPM N10C
G30C) (H) 67Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Gly
Gly Ala 1 5 10 15 Gln Thr Cys Phe Met Pro Met Asp Gln Asp Glu Ala
Leu Leu Tyr Glu 20 25 30 Glu Phe Ile Leu Gln Gln Cys Leu Glu 35 40
6841PRTArtificial SequenceHER-lm32 (MPM F11C G30C) (H 68Lys Ser Leu
Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Gly Gly Ala 1 5 10 15 Gln
Thr Asn Cys Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20 25
30 Glu Phe Ile Leu Gln Gln Cys Leu Glu 35 40 6941PRTArtificial
SequenceHER-lm32 (MPM N10C L28C) (H) 69Lys Ser Leu Ser Leu Ser Pro
Gly Ser Gly Gly Gly Ser Gly Gly Ala 1 5 10 15 Gln Thr Cys Phe Met
Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20 25 30 Glu Phe Ile
Cys Gln Gln Gly Leu Glu 35 40 7041PRTArtificial SequenceHER-lm32
(MPM M5C G30C) (H)
70Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Cys Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr
Glu 20 25 30 Glu Phe Ile Leu Gln Gln Cys Leu Glu 35 40
717PRTArtificial SequenceMRD 71Ala Thr Trp Leu Pro Pro Pro 1 5
7211PRTArtificial SequenceVL-CDR1 72Ser Ala Ser Gln Asp Ile Ser Asn
Tyr Leu Asn 1 5 10 737PRTArtificial SequenceVL-CDR2 73Phe Thr Ser
Ser Leu His Ser 1 5 749PRTArtificial SequenceVL-CDR3 74Gln Gln Tyr
Ser Thr Val Pro Trp Thr 1 5 7510PRTArtificial SequenceVH-CDR1 75Gly
Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 7617PRTArtificial
SequenceVH-CDR2 76Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe Lys 1 5 10 15 Arg 7714PRTArtificial SequenceVH-CDR3
77Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 1 5 10
78108PRTArtificial SequenceVL 78Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr
Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro
Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 79123PRTArtificial SequenceVH 79Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp
Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 8011PRTArtificial SequenceVL-CDR1 80Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala 1 5 10 817PRTArtificial SequenceVL-CDR2
81Ala Ala Ser Thr Leu Gln Ser 1 5 829PRTArtificial SequenceVL-CDR3
82Gln Arg Tyr Asn Arg Ala Pro Tyr Thr 1 5 835PRTArtificial
SequenceVH-CDR1 83Asp Tyr Ala Met His 1 5 8417PRTArtificial
SequenceVH-CDR2 84Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala
Asp Ser Val Glu 1 5 10 15 Gly 8512PRTArtificial SequenceVH-CDR3
85Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr 1 5 10
86108PRTArtificial SequenceVL 86Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 87121PRTArtificial SequenceVH 87Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 8840PRTArtificial SequenceMRD expressed as a MBP fusion protein
88Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Gly Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu 35 40
8941PRTArtificial SequenceMRD expressed as a MBP fusion protein
89Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Leu Gly Leu Tyr
Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu Glu 35 40
9041PRTArtificial SequenceMRD expressed as a MBP fusion protein
90Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Ala Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu Glu 35 40
9141PRTArtificial SequenceMRD expressed as a MBP fusion protein
91Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Leu Thr Leu Tyr
Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu Glu 35 40
9241PRTArtificial SequenceMRD expressed as a MBP fusion protein
92Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Leu Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Tyr Gln Gln Gly Leu Glu 35 40
9341PRTArtificial SequenceMRD expressed as a MBP fusion protein
93Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Gly Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Tyr Gln Gln Gly Leu Glu 35 40
9441PRTArtificial SequenceMRD expressed as a MBP fusion protein
94Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Asp Glu Ala Leu Leu Tyr
Glu 20 25 30 Gln Phe Ile Tyr Gln Gln Gly Leu Glu 35 40
9539PRTArtificial SequenceMRD expressed as a MBP fusion protein
95Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1
5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asn Glu Glu Leu Thr Leu Tyr
Glu 20 25 30 Gln Phe Ile Phe Gln Gln Gly 35 9641PRTArtificial
SequenceMRD expressed as a MBP fusion protein 96Lys Ser Leu Ser Leu
Ser Pro Gly Ser Gly Gly Gly Ser Met Gly Ala 1 5 10 15 Gln Thr Asn
Phe Met Pro Met Asp Asn Asp Glu Gly Leu Leu Tyr Glu 20 25 30 Glu
Phe Ile Leu Gln Gln Gly Leu Glu 35 40 9741PRTArtificial SequenceMRD
expressed as a MBP fusion protein 97Lys Ser Leu Ser Leu Ser Pro Gly
Ser Gly Gly Gly Ser Met Gly Ala 1 5 10 15 Gln Thr Asn Phe Met Pro
Met Asp Asn Asp Glu Ala Leu Leu Tyr Glu 20 25 30 Glu Phe Ile Leu
Gln Gln Gly Leu Glu 35 40 9841PRTArtificial SequenceMRD expressed
as a MBP fusion protein 98Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly
Gly Gly Ser Met Gly Ala 1 5 10 15 Gln Thr Asn Phe Met Pro Met Asp
Asn Glu Glu Leu Thr Leu Tyr Glu 20 25 30 Glu Phe Ile Leu Gln Gln
Gly Leu Glu 35 40 9941PRTArtificial SequenceMRD expressed as a MBP
fusion protein 99Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly
Ser Met Gly Ala 1 5 10 15 Gln Thr Asn Phe Met Pro Met Asp Gln Asp
Glu Leu Leu Leu Tyr Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu
Glu 35 40 10041PRTArtificial SequenceMRD expressed as a MBP fusion
protein 100Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Met
Gly Ala 1 5 10 15 Gln Thr Asn Phe Met Pro Met Asp Asp Asp Glu Leu
Leu Leu Tyr Glu 20 25 30 Gln Phe Ile Leu Gln Gln Gly Leu Glu 35 40
10141PRTArtificial SequenceHER-lm32 (MPM M5C L28C) (H) 101Lys Ser
Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Cys Gly Ala 1 5 10 15
Gln Thr Asn Phe Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20
25 30 Glu Phe Ile Cys Gln Gln Gly Leu Glu 35 40 102105PRTArtificial
SequenceMRD containing antibody 102Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Lys Leu Gly Thr 35 40 45 Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 50 55 60
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 65
70 75 80 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Leu
Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
10310PRTArtificial SequenceMRD encoded sequence 103Ser Leu Phe Val
Pro Arg Pro Glu Arg Lys 1 5 10 10410PRTArtificial SequenceMRD
encoded sequence 104Glu Ser Asp Val Leu His Phe Thr Ser Thr 1 5 10
1059PRTArtificial SequenceMRD encoded sequence 105Leu Arg Lys Tyr
Ala Asp Gly Thr Leu 1 5 1069PRTArtificial SequenceMRD encoded
sequence 106Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5
10728PRTArtificial SequenceL17D 107Met Gly Ala Gln Thr Asn Phe Met
Pro Met Asp Asp Asp Glu Leu Leu 1 5 10 15 Leu Tyr Glu Gln Phe Ile
Leu Gln Gln Gly Leu Glu 20 25 10841PRTArtificial SequenceHER-lm32
MPM A7C G30C) (H) 108Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly
Gly Ser Gly Gly Cys 1 5 10 15 Gln Thr Asn Phe Met Pro Met Asp Gln
Asp Glu Ala Leu Leu Tyr Glu 20 25 30 Glu Phe Ile Leu Gln Gln Cys
Leu Glu 35 40 10941PRTArtificial SequenceHER-lm32 (MPM P13C G30C)
(H) 109Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Gly Gly
Ala 1 5 10 15 Gln Thr Asn Phe Met Cys Met Asp Gln Asp Glu Ala Leu
Leu Tyr Glu 20 25 30 Glu Phe Ile Leu Gln Gln Cys Leu Glu 35 40
11041PRTArtificial SequenceHER-lm32 (MPM M5C N10C) (H) 110Lys Ser
Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Cys Gly Ala 1 5 10 15
Gln Thr Cys Phe Met Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20
25 30 Glu Phe Ile Leu Gln Gln Gly Leu Glu 35 40 11141PRTArtificial
SequenceHER-lm32 (MPM M5C F11C) (H) 111Lys Ser Leu Ser Leu Ser Pro
Gly Ser Gly Gly Gly Ser Cys Gly Ala 1 5 10 15 Gln Thr Asn Cys Met
Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu 20 25 30 Glu Phe Ile
Leu Gln Gln Gly Leu Glu 35 40 11236PRTArtificial SequenceHER-lm32
(MPM D5 N10C G30C) (H) 112Lys Ser Leu Ser Leu Ser Pro Gly Ser Gly
Gly Gly Ser Cys Phe Met 1 5 10 15 Pro Met Asp Gln Asp Glu Ala Leu
Leu Tyr Glu Glu Phe Ile Leu Gln 20 25 30 Gln Cys Leu Glu 35
11336PRTArtificial SequenceHER-lm32 (MPM D5 N10C L28C) (H) 113Lys
Ser Leu Ser Leu Ser Pro Gly Ser Gly Gly Gly Ser Cys Phe Met 1 5 10
15 Pro Met Asp Gln Asp Glu Ala Leu Leu Tyr Glu Glu Phe Ile Cys Gln
20 25 30 Gln Gly Leu Glu 35 11435PRTArtificial SequenceL1-7D MDD
114Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro
1 5 10 15 Met Asp Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu
Gln Gln 20 25 30 Gly Leu Glu 35 11535PRTArtificial SequenceL1-7D
MQD 115Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met
Pro 1 5 10 15 Met Gln Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile
Leu Gln Gln 20 25 30 Gly Leu Glu 35 11635PRTArtificial
SequenceL1-7D MVD 116Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln
Thr Asn Phe Met Pro 1 5 10 15 Met Val Asp Asp Glu Gln Arg Leu Tyr
Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
11735PRTArtificial SequenceL1-7D MHD 117Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met His Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
11835PRTArtificial SequenceL1-7D MND 118Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Asn Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
11935PRTArtificial SequenceL1-7D MKD 119Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Lys Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12035PRTArtificial SequenceL1-7D MAD 120Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Ala Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12135PRTArtificial SequenceL1-7D MSD 121Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Ser Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12235PRTArtificial SequenceL1-7D MRD 122Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Arg Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12335PRTArtificial SequenceL1-7D MGD 123Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Gly Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12435PRTArtificial
SequenceL1-7D MTD 124Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln
Thr Asn Phe Met Pro 1 5 10 15 Met Thr Asp Asp Glu Gln Arg Leu Tyr
Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12535PRTArtificial SequenceL1-7D MYD 125Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Tyr Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12635PRTArtificial SequenceL1-7D MPD 126Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Pro Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12735PRTArtificial SequenceL1-7D MID 127Pro Gly Lys Gly Gly Gly Ser
Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Ile Asp Asp Glu
Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly Leu Glu 35
12835PRTArtificial SequenceL1-7D MFD 14F11 128Pro Gly Lys Gly Gly
Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Phe Asp
Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30 Gly
Leu Glu 35 12935PRTArtificial SequenceL1-7D MWD 129Pro Gly Lys Gly
Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met Trp
Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25 30
Gly Leu Glu 35 13035PRTArtificial SequenceL1-7D MLD 130Pro Gly Lys
Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15 Met
Leu Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20 25
30 Gly Leu Glu 35 13135PRTArtificial SequenceL1-7D MED 131Pro Gly
Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10 15
Met Glu Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln 20
25 30 Gly Leu Glu 35 13235PRTArtificial SequenceL1-7D MMD 132Pro
Gly Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro 1 5 10
15 Met Met Asp Asp Glu Gln Arg Leu Tyr Glu Gln Phe Ile Leu Gln Gln
20 25 30 Gly Leu Glu 35 13335PRTArtificial SequenceL1-7D MFD 14G4
133Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met Pro
1 5 10 15 Met Phe Asp Asp Glu Gln Arg Leu Tyr Asp Gln Phe Ile Leu
Gln Gln 20 25 30 Gly Leu Glu 35 13435PRTArtificial SequenceLm32
KtoS 134Pro Gly Ser Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met
Pro 1 5 10 15 Met Asp Asn Asp Glu Leu Leu Leu Tyr Glu Gln Phe Ile
Leu Gln Gln 20 25 30 Gly Leu Glu 35 13567PRTArtificial SequenceLM32
2X 135Pro Gly Lys Gly Gly Gly Ser Met Gly Ala Gln Thr Asn Phe Met
Pro 1 5 10 15 Met Asp Asn Asp Glu Leu Leu Leu Tyr Glu Gln Phe Ile
Leu Gln Gln 20 25 30 Gly Leu Glu Gly Gly Gly Ser Met Gly Ala Gln
Thr Asn Phe Met Pro 35 40 45 Met Asp Asn Asp Glu Leu Leu Leu Tyr
Glu Gln Phe Ile Leu Gln Gln 50 55 60 Gly Leu Glu 65
* * * * *