U.S. patent application number 11/203253 was filed with the patent office on 2006-02-23 for integrin antagonists with enhanced antibody dependent cell-mediated cytoxicity activity.
This patent application is currently assigned to MEDIMMUNE, INC.. Invention is credited to Changshou Gao, Herren Wu.
Application Number | 20060040325 11/203253 |
Document ID | / |
Family ID | 35968077 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040325 |
Kind Code |
A1 |
Wu; Herren ; et al. |
February 23, 2006 |
Integrin antagonists with enhanced antibody dependent cell-mediated
cytoxicity activity
Abstract
The present invention relates to novel Fc variants of antibodies
that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3. The Fc variants comprise a variable
region that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 and a Fc region that further comprises at
least one novel amino acid residue which may provide for enhanced
effector function. More specifically, this invention provides Fc
variants that have modified binding affinity to one or more
Fc.gamma.R and/or C1q. Additionally, the Fc variants have altered
antibody dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) activity. The invention
further provides methods and protocols for the application of said
Fc variants of an antibody that immunospecifically binds to
Integrin .alpha..sub.v.beta..sub.3, particularly for therapeutic
purposes.
Inventors: |
Wu; Herren; (Boyds, MD)
; Gao; Changshou; (Potomac, MD) |
Correspondence
Address: |
JOHNATHAN KLEIN-EVANS
ONE MEDIMMUNE WAY
GAITHERSBURG
MD
20878
US
|
Assignee: |
MEDIMMUNE, INC.
Gaithersburg
MD
|
Family ID: |
35968077 |
Appl. No.: |
11/203253 |
Filed: |
August 15, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60601634 |
Aug 16, 2004 |
|
|
|
60608852 |
Sep 13, 2004 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/320.1; 435/334; 435/69.1; 530/388.22 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/00 20130101; A61P 35/02 20180101; A61P 35/04 20180101; G01N
33/574 20130101; A61P 43/00 20180101; C07K 2317/732 20130101; C07K
2317/72 20130101; C07K 16/2866 20130101; G01N 2500/00 20130101;
C07K 2319/30 20130101; C07K 16/2848 20130101; C07K 2317/52
20130101; A61P 9/00 20180101; A61P 11/06 20180101; C07K 2317/92
20130101; C07K 2317/622 20130101; A61P 11/00 20180101; A61P 29/00
20180101; A61P 1/04 20180101; A61P 17/06 20180101; G01N 33/566
20130101 |
Class at
Publication: |
435/007.1 ;
530/388.22; 435/334; 435/320.1; 435/069.1 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/53 20060101 G01N033/53; C12P 21/06 20060101
C12P021/06; C12N 5/06 20060101 C12N005/06 |
Claims
1. An antibody that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 comprising an IgG.sub.1 Fc region,
wherein the Fc region comprises at least the high effector function
amino acid residue 332E, as numbered by the EU index as set forth
in Kabat, wherein the antibody comprising at least the high
effector function amino acid residue 332E has an altered binding
affinity for one or more Fc.gamma.Rs as compared to the same
antibody not comprising at least the high effector function amino
acid residue 332E.
2. The antibody of claim 1, wherein the Fc region further comprises
at least the high effector function amino acid residues 239D and
330L, as numbered by the EU index as set forth in Kabat.
3. The antibody of claim 1, wherein the high effector function
amino acid residue is selected from the group consisting of: 234E,
235R, 235A, 235W, 235P, 235V, 235Y, 236E, 239D, 265L, 269S, 269G,
2981, 298T, 298F, 327N, 327G, 327W, 328S, 328V, 329H, 329Q, 330K,
330V, 330G, 330Y, 330T, 330L, 330I, 330R, 330C, 332E, 332H, 332S,
332W, 332F, 332D, and 332Y, wherein the numbering system is that of
the EU index as set forth in Kabat.
4. The antibody of claim 1, wherein said altered binding affinity
for said one or more Fc.gamma.Rs is increased as compared to the
same antibody not comprising at least the high effector function
amino acid residue 332E.
5. The antibody of claim 4, wherein said Fc.gamma.R is
Fc.gamma.RIIIA.
6. The antibody of claim 4, wherein said Fc.gamma.R is
Fc.gamma.RIIB.
7. The antibody of claim 1, wherein said altered binding affinity
for said one or more Fc.gamma.Rs is decreased as compared to the
same antibody not comprising at least the high effector function
amino acid residue 332E.
8. The antibody of claim 5, wherein the equilibrium dissociation
constant (K.sub.D) of binding for Fc.gamma.RIIIA is decreased at
least 2 fold as compared to the same antibody not comprising at
least the high effector function amino acid residue 332E.
9. The antibody of claim 8, wherein the equilibrium dissociation
constant (K.sub.D) of binding for Fc.gamma.RIIIA is decreased at
least 70 fold as compared to the same antibody not comprising at
least the high effector function amino acid residue 332E.
10. The antibody of claim 5, wherein said increased affinity for
Fc.gamma.RIIIA results in an enhanced ADCC activity relative to a
comparable molecule not comprising at least the high effector
function amino acid residue 332E.
11. The antibody of claim 10, wherein said enhanced ADCC activity
is at least 2 fold greater relative to a comparable molecule not
comprising at least the high effector function amino acid residue
332E.
12. The antibody of claim 1, wherein said antibody is humanized,
fully human, CDR-grafted, or chimeric.
13. The antibody of claim 12, wherein said antibody is
Vitaxin.RTM..
14. The antibody of claim 12, wherein said antibody variable
sequences comprise SEQ ID Nos. 3 and 4.
15. The antibody of claim 12, wherein said antibody is conjugated
to a detectable agent, therapeutic agent or drug.
16. A method of generating the antibody of claim 1, comprising (a)
isolating antibody coding regions; and (b) making one or more
desired substitutions in said Fc region of said isolated antibody
coding region.
17. A method of generating the antibody of claim 1, comprising
subcloning variable regions into a vector encoding said Fc region
comprising at least one or more high effector function amino acid
residues.
18. A formulation comprising a therapeutically effective amount of
the antibody of claim 1 in a pharmaceutically-acceptable
excipient.
19. A method of ameliorating, treating or preventing cancer by
administering the formulation of claim 18 to a patient in need
thereof.
20. The method of claim 19, wherein said cancer is of the head,
neck, eye, mouth, throat, esophagus, chest, bone, lung, colon,
rectum, colorectal, stomach, spleen, renal, skeletal muscle,
subcutaneous tissue, metastatic melanoma, endometrial, prostate,
breast, ovaries, testicles, skin, thyroid, blood, lymph nodes,
kidney, liver, pancreas, brain or central nervous system.
21. The method of claim 19, wherein said administration is oral,
parenteral, intramuscular, intranasal, vaginal, rectal, lingual,
sublingual, buccal, intrabuccal, intravenous, cutaneous,
subcutaneous or transdermal.
22. The method of claim 19, further comprising administering said
formulation in combination with other therapies, such as
chemotherapy, hormonal therapy, biological therapy, immunotherapy
or radiation therapy.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of the following U.S. Provisional Application Nos.
60/601,634, filed, Aug. 16, 2004 and 60/608,852, filed, Sep. 13,
2004. The priority applications are hereby incorporated by
reference herein in their entirety for all purposes.
2. FIELD OF THE INVENTION
[0002] The present invention provides novel antibodies comprising
immunologically active fragments of immunoglobulin molecules and an
Fc region that further comprises at least one novel amino acid
residue of the invention. The present invention also relates to
novel antibodies comprising a variable region, or fragment thereof,
that immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3
and a Fc region that further comprises at least one high effector
function amino acid residue (e.g., 239D, 330L, 332E). The present
invention further relates to novel variants of antibodies that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
(e.g., VITAXIN.RTM. (Wu et al., 1998, PNAS USA 95:6037-6042)) which
contain one or more substitutions in their Fc regions.
Collectively, these two types of novel antibodies are referred to
herein as "Fc variants of the invention" or "Fc variants." In one
embodiment, the Fc variants of the invention have enhanced effector
function. In another embodiment the Fc variants of the invention
have altered binding affinity to one or more Fc ligands (e.g.,
Fc.gamma.Rs, complement protein C1q). In another embodiment, the Fc
variants of the invention have enhanced binding to Fc.gamma.RIIIA
and increased ability to mediate antibody dependent cell-mediated
cytotoxicity (ADCC). In another embodiment, the Fc variants have
reduced binding to Fc.gamma.RIIIA and decreased ability to mediate
ADCC. In still another embodiment, the Fc variants have enhanced
binding to the C1q and increased ability to mediate complement
dependent cytotoxicity (CDC). In yet another embodiment, the Fc
variants have reduced binding to C1q and decreased ability to
mediate CDC. In particular, the present invention relates to Fc
variants that can act as inhibitors and/or antagonists of Integrin
.alpha..sub.v.beta..sub.3. In addition the present invention
provides methods and protocols for the application or use of Fc
variants, particularly for therapeutic purposes. Specifically, the
methods and protocols involve the administration of a
prophylactically or therapeutically effective amount of one or more
Fc variants alone or in combination with the administration of one
or more other therapies useful for cancer therapy. The Fc variants
utilized for therapeutic purposes may or may not be conjugated or
fused to a moiety (e.g., a therapeutic agent or drug). The methods
of the invention are particularly useful for the prevention,
management, treatment or amelioration numerous forms of cancer
including cancers that have the potential to metastasize or have
metastasized to other organs or tissues. The invention also
provides methods for screening for an antibody that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 as
well as methods to manipulate the Fc region and thereby modulate
the ability of said Fc region to mediate ADCC and/or CDC activity
and/or the binding affinity for one or more Fc ligands (e.g.,
Fc.gamma.Rs, C1q). The invention also provides methods for
generating Fc variant fusions that immunospecifically binds to
Integrin .alpha..sub.v.beta..sub.3. Further, the invention provides
pharmaceutical formulations and kits for use in preventing,
managing, treating or ameliorating cancer or one or more symptoms
thereof.
3. BACKGROUND OF THE INVENTION
3.1 Cancer
[0003] A neoplasm, or tumor, is a neoplastic mass resulting from
abnormal uncontrolled cell growth, which can be benign or
malignant. Benign tumors generally remain localized. Malignant
tumors are collectively termed cancers. The term "malignant"
generally means that the tumor can invade and destroy neighboring
body structures and spread to distant sites to cause death (for
review, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B.
Saunders Co., Philadelphia, pp. 68-122). Cancer can arise in many
sites of the body and behave differently depending upon its origin.
Cancerous cells destroy the part of the body in which they
originate and then spread to other part(s) of the body where they
start new growth and cause more destruction. The progressive growth
and metastasis of tumor cells is dependent on the ability of tumor
cells to stimulate the formation of new blood vessels in a process
called angiogenesis.
[0004] More than 1.2 million Americans develop cancer each year.
Cancer is the second leading case of death in the United States and
if current trends continue, cancer is expected to be the leading
cause of the death by the year 2010. Lung and prostate cancer are
the top cancer killers for men in the United States. Lung and
breast cancer are the top cancer killers for women in the United
States. One in two men in the United States will be diagnosed with
cancer at some time during his lifetime. One in three women in the
United States will be diagnosed with cancer at some time during her
lifetime.
[0005] A cure for cancer has yet to be found. Current treatment
options, such as surgery, chemotherapy and radiation treatment, are
oftentimes either ineffective or present serious side effects.
3.2 Integrins
[0006] Integrins are a class of cell adhesion receptors that
mediate both cell-cell and cell-extracellular matrix adhesion
events. Integrins consist of heterodimeric polypeptides where a
single .alpha. chain polypeptide noncovalently associates with a
single .beta. chain. There are now about 16 distinct .alpha. chain
polypeptides and at least about 8 different .beta. chain
polypeptides that constitute the integrin family of cell adhesion
receptors. In general, different binding specificities and tissue
distributions are derived from unique combinations of the .alpha.
and .beta. chain polypeptides or integrin subunits. The family to
which a particular integrin is associated with is usually
characterized by the .beta. subunit. However, the ligand binding
activity of the integrin is largely influenced by the .alpha.
subunit.
[0007] As cell adhesion receptors, integrins are involved in a
variety of physiological processes including, for example, cell
attachment, cell migration and cell proliferation. Different
integrins play different roles in each of these biological
processes and the inappropriate regulation of their function or
activity can lead to various pathological conditions. For example,
inappropriate endothelial cell proliferation during angiogenesis,
also called neovascularization, of a tumor was found to be mediated
by cells expressing vitronectin binding integrins. In this regard,
the inhibition of the vitronectin-binding Integrin
.alpha..sub.v.beta..sub.3 also inhibits this process of tumor
angiogenesis. By this same criteria, Integrin
.alpha..sub.v.beta..sub.3 has also been shown to mediate the
abnormal cell proliferation associated with restenosis and
granulation tissue development in cutaneous wounds, for example.
Additional disease or pathological states mediated or influenced by
Integrin .alpha..sub.v.beta..sub.3 include, for example,
metastasis, osteoporosis, age-related macular degeneration,
diabetic retinopathy and inflammatory diseases such as rheumatoid
arthritis and psoriasis. There is now considerable evidence that
progressive tumor growth is dependent upon angiogenesis (Gastl et
al., 1997, Oncol 54:177-184). Thus, agents which can specifically
inhibit Integrin .alpha..sub.v.beta..sub.3, thus preventing or
inhibiting angiogenesis, would be valuable for the therapeutic
treatment of diseases including cancer.
3.3 Cancer Therapy
[0008] Currently, cancer therapy may involve surgery, chemotherapy,
hormonal therapy and/or radiation treatment to eradicate neoplastic
cells in a patient (see, for example, Stockdale, 1998, "Principles
of Cancer Patient Management", in Scientific American: Medicine,
vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). All
of these approaches pose significant drawbacks for the patient.
Surgery, for example, may be contraindicated due to the health of
the patient or may be unacceptable to the patient. Additionally,
surgery may not completely remove the neoplastic tissue. Radiation
therapy is only effective when the neoplastic tissue exhibits a
higher sensitivity to radiation than normal tissue, and radiation
therapy can also often elicit serious side effects. Hormonal
therapy is rarely given as a single agent and although can be
effective, is often used to prevent or delay recurrence of cancer
after other treatments have removed the majority of the cancer
cells.
[0009] With respect to chemotherapy, there are a variety of
chemotherapeutic agents available for treatment of cancer. A
significant majority of cancer chemotherapeutics act by inhibiting
DNA synthesis (see, for example, Gilman et al., Goodman and
Gilman's: The Pharmacological Basis of Therapeutics, Eighth Ed.
(Pergamom Press, New York, 1990)). As such chemotherapy agents are
inherently nonspecific. In addition almost all chemotherapeutic
agents are toxic, and chemotherapy causes significant, and often
dangerous, side effects, including severe nausea, bone marrow
depression, immunosuppression, etc. (see, for example, Stockdale,
1998, "Principles Of Cancer Patient Management" in Scientific
American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12,
sect. 10). Furthermore, even with administration of combinations of
chemotherapeutic agents, many tumor cells are resistant or develop
resistance to the chemotherapeutic agents.
[0010] Recently, cancer therapy could also involve biological
therapy or immunotherapy. Biological therapies/immunotherapies are
limited in number and although more specific then chemotherapeutic
agents many still target both health and cancerous cells. In
addition, such therapies may produce side effects such as rashes or
swellings, flu-like symptoms, including fever, chills and fatigue,
digestive tract problems or allergic reactions.
[0011] Thus, there is a significant need for alternative cancer
treatments, particularly for treatments that more specifically
target cancer cells. Integrin .alpha..sub.v.beta..sub.3 is one of
the best characterized integrins implicated in tumor induced
angiogenesis. Integrin .alpha..sub.v.beta..sub.3 is highly
expressed on some human tumors (e.g., breast tumors), but not
readily detected in benign breast tissue. Thus a cancer treatment
that would specifically inhibit Integrin .alpha..sub.v.beta..sub.3
would be a powerful tool for the treatment and prevention of
cancers.
3.4 Antibodies for the Treatment of Cancer
[0012] Antibodies are immunological proteins that bind a specific
antigen. In most mammals, including humans and mice, antibodies are
constructed from paired heavy and light polypeptide chains. Each
chain is made up of two distinct regions, referred to as the
variable (Fv) and constant (Fc) regions. The light and heavy chain
Fv regions contain the antigen binding determinants of the molecule
and are responsible for binding the target antigen. The Fc regions
define the class (or isotype) of antibody (IgG for example) and are
responsible for binding a number of natural proteins to elicit
important biochemical events.
[0013] The Fc region of an antibody interacts with a number of
ligands including Fc receptors and other ligands, imparting an
array of important functional capabilities referred to as effector
functions. An important family of Fc receptors for the IgG class
are the Fc gamma receptors (Fc.gamma.Rs). These receptors mediate
communication between antibodies and the cellular arm of the immune
system (Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220;
Ravetch et al., 2001, Annu Rev Immunol 19:275-290). In humans this
protein family includes Fc.gamma.RI (CID64), including isoforms
Fc.gamma.RIA, Fc.gamma.RIB, and Fc.gamma.RIC; Fc.gamma.RII (CD32),
including isoforms Fc.gamma.RIIA, Fc.gamma.RIIB, and Fc.gamma.RIIC;
and Fc.gamma.RIII (CID16), including isoforms Fc.gamma.RIIIA and
Fc.gamma.RIIB (Jefferis et al., 2002, Immunol Lett 82:57-65). These
receptors typically have an extracellular domain that mediates
binding to Fc, a membrane spanning region, and an intracellular
domain that may mediate some signaling event within the cell. These
different Fc.gamma.R subtypes are expressed on different cell types
(reviewed in Ravetch et al., 1991, Annu Rev Immunol 9:457-492). For
example, in humans, Fc.gamma.RIIIB is found only on neutrophils,
whereas Fc.gamma.RIIIA is found on macrophages, monocytes, natural
killer (NK) cells, and a subpopulation of T-cells.
[0014] Formation of the Fc/Fc.gamma.R complex recruits effector
cells to sites of bound antigen, typically resulting in signaling
events within the cells and important subsequent immune responses
such as release of inflammation mediators, B cell activation,
endocytosis, phagocytosis, and cytotoxic attack. The ability to
mediate cytotoxic and phagocytic effector functions is a potential
mechanism by which antibodies destroy targeted cells. The
cell-mediated reaction wherein nonspecific cytotoxic cells that
express Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell is referred to as
antibody dependent cell-mediated cytotoxicity (ADCC) (Raghavan et
al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000,
Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu Rev Immunol
19:275-290). Notably, the primary cells for mediating ADCC, NK
cells, express only Fc.gamma.RIIIA only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII (Ravetch et al., 1991,
supra).
[0015] Another important Fc ligand is the complement protein C1q.
Fc binding to C1q mediates a process called complement dependent
cytotoxicity (CDC) (reviewed in Ward et al., 1995, Ther Immunol
2:77-94). C1q is capable of binding six antibodies, although
binding to two IgGs is sufficient to activate the complement
cascade. C1q forms a complex with the C1r and C1s serine proteases
to form the C1 complex of the complement pathway.
[0016] Several key features of antibodies including but not limited
to, specificity for target, ability to mediate immune effector
mechanisms, and long half-life in serum, make antibodies powerful
therapeutics. Numerous monoclonal antibodies are currently in
development or are being used therapeutically for the treatment of
a variety of conditions including cancer. For example Vitaxin.RTM.
(MedImmune), a humanized Integrin .alpha..sub.v.beta..sub.3
antibody (e.g., PCT publication WO 2003/075957), Herceptin.RTM.
(Genentech), a humanized anti-Her2/neu antibody approved to treat
breast cancer (e.g., U.S. Pat. No. 5,677,171), CNTO 95 (Centocor),
a human Integrin .alpha..sub.v antibody (PCT publication WO
02/12501), Rituxan.RTM. (IDEC/Genentech/Roche), a chimeric
anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma (e.g.,
U.S. Pat. No. 5,736,137) and Erbitux.RTM. (ImClone), a chimeric
anti-EGFR antibody (e.g., U.S. Pat. No. 4,943,533).
[0017] There are a number of possible mechanisms by which
antibodies destroy tumor cells, including anti-proliferation via
blockage of needed growth pathways, intracellular signaling leading
to apoptosis, enhanced down regulation and/or turnover of
receptors, ADCC, CDC, and promotion of an adaptive immune response
(Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie et al.,
2000, Immunol Today 21:403-410). However, despite widespread use,
antibodies are not optimized for clinic use and many have
suboptimal anticancer potency. Thus, there is a significant need to
enhance the capacity of antibodies to destroy targeted cancer
cells. Methods for enhancing the anti-tumor-potency of antibodies
via enhancement of their ability to mediate cytotoxic effector
functions such as ADCC and CDC are particularly promising. The
importance of Fc.gamma.R-mediated effector functions for the
anti-cancer activity of antibodies has been demonstrated in mice
(Clynes et al., 1998, Proc Natl Acad Sci USA 95:652-656; Clynes et
al., 2000, Nat Med 6:443-446), and the affinity of the interaction
between Fc and certain Fc.gamma.Rs correlates with targeted
cytotoxicity in cell-based assays (Shields et al., 2001, J Biol
Chem 276:6591-6604; Presta et al., 2002, Biochem Soc Trans
30:487-490; Shields et al., 2002, J Biol Chem 277:26733-26740).
Together these data suggest that manipulating the binding ability
of the Fc region of an IgG1 antibody to certain Fc.gamma.Rs may
enhance effector functions resulting in more effective destruction
of cancer cells in patients. Furthermore, because Fc.gamma.Rs can
mediate antigen uptake and processing by antigen presenting cells,
enhanced Fc/Fc.gamma.R affinity may also improve the capacity of
antibody therapeutics to elicit an adaptive immune response.
[0018] While enhancing effector function can increase the capacity
of antibodies to destroy target cells, for some antibody therapies
reduced or eliminated effector function may be more desirable. This
is particularly true for those antibodies designed to deliver a
drug (e.g., toxins and isotopes) to the target cell where the
Fc/Fc.gamma.R mediated effector functions bring healthy immune
cells into the proximity of the deadly payload, resulting in
depletion of normal lymphoid tissue along with the target cells
(Hutchins et al., 1995, PNAS USA 92:11980-11984; White et al.,
2001, Annu Rev Med 52:125-145). In these cases the use of Fc
variants that poorly recruit complement or effector cells would be
of tremendous benefit (see for example, Wu et al., 2000, Cell
Immunol 200:16-26; Shields et al., 2001, J. Biol Chem
276:6591-6604; U.S. Pat. No. 6,194,551; U.S. Pat. No. 5,885,573 and
PCT publication WO 04/029207).
[0019] All Fc.gamma.Rs bind the same region on the Fc of the IgG
subclass, but with different affinities (e.g., Fc.gamma.RI is a
high affinity while Fc.gamma.RII and Fc.gamma.RIII are low affinity
binders). Other differences between the Fc.gamma.Rs are
mechanistic. For example, Fc.gamma.RI, Fc.gamma.RIIA/C, and
Fc.gamma.RIIIA are positive regulators of immune complex triggered
activation, characterized by having an immunoreceptor
tyrosine-based activation motif (ITAM) while Fc.gamma.RIIB has an
immunoreceptor tyrosine-based inhibition motif (ITIM) and is
therefore inhibitory. Thus, the balance between activating and
inhibiting receptors is an important consideration. For example,
enhancing Fc binding to the positive regulators (e.g.,
Fc.gamma.RIIIA) while leaving unchanged or even reducing Fc binding
to the negative regulator Fc.gamma.RIIB could result in optimized
effector function such as enhanced ADCC mediated destruction of
tumor cells. Another critical consideration is that Fc variants
should be engineered such that the binding to Fc.gamma.Rs and/or
C1q is modulated in the desired manner but so that they maintain
their stability, solubility, structural integrity as well as their
ability to interact with other important Fc ligands such as FcRn
and proteins A and G.
[0020] Numerous mutagenesis studies have been carried out on the Fc
domain (See for example, Duncan et al., 1988, Nature 332:563-564;
Lund et al., 1995, Faseb J 9:115-119; Lund et al., 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al.,
2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans
30:487-490; U.S. Pat. Nos. 5,624,821, 5,885,573 and PCT publication
Nos. WO 00/42072, WO 99/58572 and WO 04/029207). While the vast
majority of substitutions reduce or ablate Fc binding with
Fc.gamma.Rs some have resulted in higher Fc.gamma.R affinity.
However, most of the methods disclosed resulted in only modest
improvements in FcR.gamma.IIIA binding and ADCC activity. The
present invention provides for the first time a modified Fc of
antibody that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 that has increased binding to
FcR.gamma.IIIA binding, significant enhancement in ADCC and does
not show an increase in FcR.gamma.IIB binding.
[0021] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
4. SUMMARY OF THE INVENTION
[0022] The present invention provides novel antibodies comprising
immunologically active fragments of immunoglobulin molecules and an
Fc region that further comprises at least one novel amino acid
residue of the invention (also referred to herein as "high effector
function amino acid residue(s))". Said novel antibodies are
referred to herein as "Fc variants of the invention" or "Fc
variants." Fc binding interactions are essential for a variety of
effector functions and downstream signaling events including, but
not limited to, antibody dependent cell-mediated cytotoxicity
(ADCC) and complement dependent cytotoxicity (CDC). Accordingly,
the invention provides Fc variants that exhibit altered binding
affinity for at least one or more Fc ligands (e.g., Fc.gamma.Rs,
C1q) relative to an antibody having the same amino acid sequence as
the molecule of the invention but not comprising the novel amino
acids residues of the invention (referred to herein as a
"comparable molecule") such as, for example, an antibody comprising
an unmodified Fc region containing naturally occurring amino acid
residues at the corresponding position in the Fc domain. In
addition, the present invention provides novel Fc variants
comprising a variable region, or fragment thereof, that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3 and
at least one high effector function amino acid residue (e.g., 239D,
330L, 332E).
[0023] The present invention further provides Fc variants of
antibodies that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3, said Fc variants comprising an Fc region
in which at least one amino acid residue has been substituted. It
is specifically contemplated that said Fc variants may be generated
by methods well known to one skilled in the art. Briefly, such
methods include but are not limited to, combining a variable region
with the desired specificity (e.g., a variable region isolated from
a phage display or expression library or derived from a human or
non-human antibody) with an Fc region containing at least one high
effector function amino acid residue. Alternatively, one skilled in
the art may generate an Fc variant by substituting at least one
amino acid residue in the Fc region of an antibody.
[0024] The present invention also provides Fc variants that have
altered binding affinity for one or more Fc ligands (e.g.,
Fc.gamma.Rs, C1q) relative to a comparable molecule (e.g., an
antibody having an original unmodified Fc region). In one
embodiment, the Fc variants have higher binding affinity to
activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA) and/or unchanged or
lower binding affinity to inhibitory Fc.gamma.Rs (e.g.,
Fc.gamma.RIIB) relative to a comparable molecule (e.g., an antibody
having an original unmodified Fc region)). The present invention
further provides Fc variants with enhanced ADCC function relative
to a comparable molecule (e.g., an antibody having an original
unmodified Fc region)). In another embodiment, the Fc variants of
the invention have enhanced ability to mediate ADCC ("referred to
herein as ADCC activity") in addition to the above changes in
Fc.gamma.R affinities relative to a comparable molecule (e.g., an
antibody having an original unmodified Fc region)). In still
another embodiment, the Fc variants of the invention are variants
of an antibody that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3. Furthermore, the Fc variants of the
invention do not have significantly altered antigen binding
specificity.
[0025] The present invention also provides Fc variants have lower
binding affinity to activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA)
and/or increased binding affinity to inhibitory Fc.gamma.Rs (e.g.,
Fc.gamma.RIIB) relative to a comparable molecule (e.g., an antibody
having an original unmodified Fc region). The present invention
further provides Fc variants with decreased ADCC activity relative
to a comparable molecule (e.g., an antibody having an original
unmodified Fc region). In one embodiment, the Fc variants of the
invention exhibit decreased ADCC activity in addition to the above
changes in Fc.gamma.R affinities relative to a comparable molecule
(e.g., an antibody having an original unmodified Fc region). In
another embodiment, the Fc variants of the invention are variants
of an antibody that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3. Furthermore, the Fc variants of the
invention do not have significantly altered antigen binding
specificity.
[0026] The present invention additionally provides Fc variants that
have altered binding affinity to the complement protein C1q
relative to a comparable molecule (e.g., an antibody having an
original unmodified Fc region). In one embodiment, the Fc variants
have enhanced binding affinity to C1q and enhanced ability to
mediate CDC (referred to herein as "CDC activity"). In another
embodiment, the Fc variants have reduced binding affinity to C1q
and reduced CDC activity relative to a comparable molecule (e.g.,
an antibody having an original unmodified Fc region). In still
another embodiment, the Fc variants of the invention are variants
of an antibody that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3.
[0027] In a specific embodiment, Fc variants of the invention
comprise an Fc region comprising at least one high effector
function amino acid reside selected from the group consisting of:
234E, 235R, 235A, 235W, 235P, 235V, 235Y, 236E, 239D, 265L, 269S,
269G, 298I, 298T, 298F, 327N, 327G, 327W, 328S, 328V, 329H, 329Q,
330K, 330V, 330G, 330Y, 330T, 330L, 3301, 330R, 330C, 332E, 332H,
332S, 332W, 332F, 332D, and 332Y, wherein the numbering system is
that of the EU index as set forth in Kabat et al. (1991, NIH
Publication 91-3242, National Technical Information Service,
Springfield, Va.).
[0028] In another specific embodiment, Fc variants of the invention
comprise an Fc region comprising at least one high effector
function amino acid residue selected from the group consisting of:
239D, 330K, 330V, 330G, 330Y, 330T, 330L, 3301, 330R, 330C, 332E,
332H, 332S, 332W, 332F, 332D, and 332Y wherein the numbering system
is that of the EU index as set forth in Kabat.
[0029] In still another specific embodiment, Fc variants of the
invention comprise an Fc region comprising at least one high
effector function amino acid residue selected from the group
consisting of: 239D, 330L and 332E. In yet another embodiment, Fc
variants of the invention comprise an Fc region comprising at least
the high effector function amino acid residue 332E. In a specific
embodiment, Fc variants of the invention comprise an Fc region
comprising the high effector function amino acid residues 239D,
330L and 332E.
[0030] In one embodiment, the Fc variants comprise at least one
amino acid substitution at a position selected from the group
consisting of: 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226,227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 239, 242, 246, 250, 251,
257, 259, 260, 261, 265, 269, 273, 274, 275, 277, 281, 282, 284,
287, 291, 298, 300, 302, 304, 306, 308, 310, 314, 316, 318, 319,
321, 323, 327, 328, 329, 330, 332 and 336, wherein the numbering of
the residues in the Fc region is that of the EU index as set forth
in Kabat.
[0031] In a specific embodiment, the Fc variants comprise at least
one substitution selected from the group consisting of: L234E,
L235R, L235A, L235W, L235P, L235V, L235Y, G236E, S239D, D265L,
E269S, E269G, S2981, S298T, S298F, A327N, A327G, A327W, L328S,
L328V, P329H, P329Q, A330K, A330V, A330G, A330Y, A330T, A330L,
A3301, A330R, A330C, I332E, I332H, I332S, I332W, I332F, I332D, and
I332Y, wherein the numbering system is that of the EU index as set
forth in Kabat. In another embodiment, the Fc variants comprise at
least one substitution selected from the group consisting of S239D,
A330L and I332E. In still another embodiment, the Fc variants
comprise at least each of the following substitutions, S239D, A330L
and I332E. In yet another embodiment, the Fc variants have at least
the amino acid substitution I332E.
[0032] It is an object of the present invention to provide a Fc
variants that bind with greater affinity to one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q). In one embodiment, said variants have an
affinity for one or more Fc ligand (e.g., Fc.gamma.Rs, C1q) that is
at least 2 fold greater than that of a comparable molecule (e.g.,
an antibody prior to Fc modification). In another embodiment, the
Fc variants of the invention have affinity for an Fc ligand (e.g.,
Fc.gamma.R, C1q) that is between about 2 fold and about 500 fold
greater than that of a comparable molecule (e.g., an antibody prior
to Fc modification). In still another embodiment, the Fc variants
of the invention have affinity for an Fc ligand (e.g., Fc.gamma.R,
C1q) that is between 2 fold and 500 fold greater than that of a
comparable molecule (e.g., an antibody prior to Fc modification).
In one specific embodiment, an Fc variant of the invention has a
greater affinity for Fc.gamma.RIIIA. In another specific
embodiment, an Fc variant of the invention has a greater affinity
for Fc.gamma.RIIB. In yet another specific embodiment, an Fc
variant of the invention has a greater affinity for C1q.
[0033] It is a further object of the present invention to provide
Fc variants that bind with reduced affinity to one or more Fc
ligand (e.g., Fc.gamma.Rs, C1q). In one embodiment, the Fc variants
of the invention have an affinity for one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) that is between 2 fold and 500 fold lower than
that of a comparable molecule (e.g., an antibody prior to Fc
modification). In another embodiment, the Fc variants of the
invention have an affinity for one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) that is between about 2 fold and about 500 fold
lower than that of a comparable molecule (e.g., an antibody prior
to Fc modification). In a specific embodiment, the Fc variants of
the invention have an affinity for Fc.gamma.RIIB that is either
unchanged, or reduced. In another specific embodiment, the Fc
variants of the invention have an affinity for Fc.gamma.RIIIA that
is reduced. In yet another embodiment, the Fc variants of the
invention have an affinity for C1q that is reduced.
[0034] It is a further object of the present invention to provide
Fc variants that have enhanced ADCC and/or CDC activity. In one
embodiment, Fc variants of the invention have ADCC and/or CDC
activity that is at least 2 fold greater then that of a comparable
molecule (e.g., an antibody prior to Fc modification). In another
embodiment, the Fc variants of the invention have ADCC and/or CDC
activity that is between about 2 fold and about 100 fold greater
then that of a comparable molecule. In yet another embodiment, the
Fc variants of the invention have ADCC and/or CDC activity that is
between 2 fold and 100 fold greater then that of a comparable
molecule.
[0035] It is a further object of the present invention to provide
Fc variants that have reduced ADCC and/or CDC activity. In one
embodiment, Fc variants of the invention have ADCC and/or CDC
activity that is at least 2 fold lower then that of a comparable
molecule (e.g., an antibody prior to Fc modification). In another
embodiment, the Fc variants of the invention have ADCC and/or CDC
activity that is between about 2 fold and about 100 fold lower then
that of a comparable molecule. In another embodiment, the Fc
variants of the invention have ADCC and/or CDC activity that is
between 2 fold and 100 fold lower then that of a comparable
molecule.
[0036] In one specific embodiment, an Fc variant of the invention
has an increased affinity for Fc.gamma.RIIIA and an affinity for
Fc.gamma.RIIB that is unchanged or reduced and enhanced ADCC
activity relative to a comparable molecule (e.g., an antibody prior
to Fc modification). In another specific embodiment, an Fc variant
of the invention has an equilibrium dissociation constant (K.sub.D)
that is decreased between about 2 fold and about 10 fold, or
between about 5 fold and about 50 fold, or between about 25 fold
and about 250 fold, or between about 100 fold and about 500 fold,
relative to a comparable molecule. In another specific embodiment,
an Fc variant of the invention has an equilibrium dissociation
constant (K.sub.D) that is decreased between 2 fold and 10 fold, or
between 5 fold and 50 fold, or between 25 fold and 250 fold, or
between 100 fold and 500 fold, relative to a comparable molecule.
In another specific embodiment, an Fc variant of the invention has
a ratio of Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium dissociation
constants (K.sub.D) that is decreased and enhanced ADCC activity
relative to a comparable molecule.
[0037] In one embodiment, an Fc variant of the invention has an
increased affinity for Fc.gamma.RIIIA and an affinity for
Fc.gamma.RIIB that is unchanged or reduced, an affinity for C1q
that is reduced and enhanced ADCC activity relative to a comparable
molecule (e.g., an antibody prior to Fc modification).
[0038] In another embodiment, an Fc variant of the invention has a
decreased affinity for Fc.gamma.RIIIA, an affinity for
Fc.gamma.RIIB that is increased and reduced ADCC activity relative
to a comparable molecule (e.g., an antibody prior to Fc
modification). In still another embodiment, an Fc variant of the
invention has a ratio of Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium
dissociation constants (K.sub.D) that is increased and reduced ADCC
activity relative to a comparable molecule.
[0039] The binding properties of a receptor for its ligand, may be
determined by a variety of methods well-known in the art, including
but not limited to, equilibrium methods (e.g., enzyme-linked
immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or
kinetics (e.g., BIACORE.RTM. analysis), and other methods such as
indirect binding assays, competitive inhibition assays,
fluorescence resonance energy transfer (FRET), gel electrophoresis
and chromatography (e.g., gel filtration). These and other
well-known methods may utilize a label on one or more of the
components being examined and/or employ a variety of detection
methods including but not limited to chromogenic, fluorescent,
luminescent, or isotopic labels. A detailed description of binding
affinities and kinetics can be found in Paul, W. E., ed.,
Fundamental Immunology, 4.sup.th Ed., Lippincott-Raven,
Philadelphia (1999), which focuses on antibody-immunogen
interactions.
[0040] The Fc variants of the present invention may be combined
with other Fc modifications (e.g., other amino acid substitutions,
altered glycosylation, etc.), including but not limited to
modifications that alter Fc ligand binding and/or effector
function. The invention encompasses combining an Fc variant of the
invention with other Fc modifications to provide additive,
synergistic, or novel properties in antibodies or Fc fusions. In
one embodiment, the other Fc modifications enhance the phenotype of
the Fc variants of the present invention (e.g., Fc variant
comprising at least one high effector function amino acid) with
which they are combined. For example, if an Fc variant (i.e.,
incorporating a hinge modification of the invention) is combined
with a mutant known to bind Fc.gamma.RIIIA with a higher affinity
than a comparable molecule comprising a wild type Fc region; the
combination results in a greater fold enhancement in Fc.gamma.RIIIA
affinity.
[0041] The invention encompasses molecules that comprise homodimers
or heterodimers of Fc regions wherein at least one Fc region
incorporates at least one high effector function amino acid of the
invention. Heterodimers comprising Fc regions refer to molecules
where the two Fc chains have different sequences. In some
embodiments, in the heterodimeric molecules comprising an Fc region
incorporating at least one high effector function amino acid and/or
other Fc modification, each chain has one or more different
modifications from the other chain. In other embodiments, in the
heterodimeric molecules comprising an Fc region incorporating a
hinge modification, one chain contains the wild-type Fc region and
the other chains comprises one or more modifications. Methods of
engineering heterodimeric Fc containing molecules are known in the
art and encompassed within the invention.
[0042] In one embodiment, an Fc variant of the invention with
modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3. In
another embodiment, said Fc variants are antagonists of Integrin
.alpha..sub.v.beta..sub.3. An antagonist of Integrin
.alpha..sub.v.beta..sub.3 is any molecule that blocks, inhibits,
reduces or neutralizes the function, activity and/or expression of
Integrin .alpha..sub.v.beta..sub.3. Thus, an antagonist of Integrin
can block angiogenesis (also commonly referred to as
neovascularization) and/or tumor cell growth resulting in, for
example, tumor regression. In another embodiment, an Fc variant of
the invention is a variant of an LM609 antibody or an antibody
derived therefrom that immunospecifically binds Integrin
.alpha..sub.v.beta..sub.3, such as chimerized and/or humanized
versions of LM609, such as, for example, the antibody Vitaxin.RTM..
Such antibodies have been described in PCT Publication Nos. WO
89/05155, WO 98/33919 and WO 00/78815 as well as U.S. Pat. No.
5,753,230, which are incorporated by reference herein in their
entireties. In a particular embodiment, said Fc variant is an
antibody that competes with LM609 or Vitaxin.RTM., or an
antigen-binding fragment thereof for binding to Integrin
.alpha..sub.v.beta..sub.3.
[0043] In one embodiment, an Fc variant of the invention with
modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity
preferentially binds Integrin .alpha..sub.v.beta..sub.3 over other
integrins. In another embodiment, said Fc variant of the invention
does not immunoreact with an .alpha..sub.v subunit. In another
embodiment, said Fc variant of the invention does immunoreact with
an .alpha..sub.v subunit. In another embodiment, the Fc variant of
the invention does not immunoreact with a .beta..sub.3 subunit. In
yet another embodiment, the Fc variant of the invention does
immunoreact with a .beta..sub.3 subunit. In still another
embodiment, the Fc variant of the invention does not immunoreact
with integrins other than .alpha..sub.v.beta..sub.3. In still
another embodiment, the Fc variant of the invention immunoreacts
with both Integrin .alpha..sub.v.beta..sub.3 and Integrin
.alpha..sub.v.beta..sub.5 or with more than one Integrin
.alpha..beta. complex. The Fc variant may have the same
immunoreactivity for both Integrin .alpha..sub.v.beta..sub.3 and
Integrin .alpha..sub.v.beta..sub.5 or alternatively, the Fc variant
may immunoreact more strongly with Integrin
.alpha..sub.v.beta..sub.3 than with Integrin
.alpha..sub.v.beta..sub.5, or more strongly with Integrin
.alpha..sub.v.beta..sub.5 than with Integrin
.alpha..sub.v.beta..sub.3. In another embodiment the Fc variant
binds an integrin other than Integrin .alpha..sub.v.beta..sub.3
(e.g., .alpha..sub.v.beta..sub.1, .alpha..sub.1.beta..sub.1,
.alpha..sub.2.beta..sub.1, .alpha..sub.5.beta..sub.1,
.alpha..sub.D.beta..sub.2, .alpha..sub.IIb.beta..sub.2).
[0044] The present invention also encompasses Fc variants with
modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
conjugated or fused to a moiety (e.g., therapeutic agent or
drug).
[0045] The present invention encompasses the use of Fc variants
with modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 to
inhibit or reduce angiogenesis.
[0046] The invention also encompasses the use of Fc variants with
modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
conjugated or fused to a moiety (e.g., therapeutic agent or drug)
to inhibit or reduce angiogenesis.
[0047] The present invention also encompasses the use of Fc
variants with modified binding affinity to one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3 for
the prevention, treatment, management or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated diseases and disorders or one or
more symptoms thereof, including but not limited to cancer,
inflammatory and autoimmune diseases either alone or in combination
with other therapies.
[0048] The invention also encompasses the use of Fc variants with
modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immuno-specifically bind to Integrin .alpha..sub.v.beta..sub.3
conjugated or fused to a moiety (e.g., therapeutic agent or drug)
for the prevention, treatment, management or amelioration of
Integrin .alpha..sub.v.beta..sub.3-mediated diseases and disorders
or one or more symptoms thereof, including but not limited to
cancer, inflammatory and autoimmune diseases either alone or in
combination with other therapies.
[0049] The invention further encompasses treatment protocols that
enhance the prophylactic or therapeutic effect of Fc variants with
altered binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3.
[0050] The invention also provides methods for screening for
antibody antagonists of Integrin .alpha..sub.v.beta..sub.3
including but not limited to assays that monitor Integrin
.alpha..sub.v.beta..sub.3 activity (e.g., cell adhesion,
angiogenesis, tumor cell growth and tumor progression) and/or
plasma concentration. In addition, the invention provides methods
for identifying monoclonal antibodies that bind to the
heterodimerized .alpha..sub.v.beta..sub.3 but not the .alpha..sub.v
or the .beta..sub.3 chains when not included in a heterodimer.
Further, the invention provides for a method to manipulate both the
ADCC and or CDC activity as well as the binding affinities for
Fc.gamma.R and/or C1q of antibodies identified using such screening
methods. The antibodies identified and manipulated utilizing such
methods can be used for the prevention, treatment, management or
amelioration of Integrin .alpha..sub.v.beta..sub.3-mediated
diseases and disorders or one or more symptoms thereof, including
but not limited to cancer, inflammatory and autoimmune diseases
either alone or in combination with other therapies.
[0051] The present invention provides kits comprising one or more
Fc variants with modified binding affinity to one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
conjugated or fused to a detectable agent, therapeutic agent or
drug, in one or more containers, can be used for the prevention,
treatment, management or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated diseases and disorders or one or
more symptoms thereof, including but not limited to cancer,
inflammatory and autoimmune diseases either alone or in combination
with other therapies.
5. BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1. The nucleotide and deduced amino acid sequence of
the variable region of the antibody Vitaxin.RTM., (A) heavy chain
variable region (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) (B)
light chain variable region (SEQ ID NO: 2 and SEQ ID NO: 4,
respectively). The CDRs are underlined.
[0053] FIG. 2. The nucleotide and deduced amino acid sequence of
the variable region of the antibody 12G3H11 (abbreviated "12G3) (A)
heavy chain variable region (SEQ ID NO: 62 and SEQ ID NO: 64,
respectively) (B) light chain variable region (SEQ ID NO: 63 and
SEQ ID NO: 65, respectively). The CDRs are underlined.
[0054] FIG. 3. The nucleotide and deduced amino acid sequence of
the variable region of the antibody 3F2 (A) heavy chain variable
region (SEQ ID NO: 66 and SEQ ID NO: 68, respectively) (B) light
chain variable region (SEQ ID NO: 67 and SEQ ID NO: 69,
respectively). The CDRs are underlined.
[0055] FIG. 4. Map of the expression plasmid used for the
production of full length IgGs. SmaI/BsiWI restriction sites used
to clone the light chain variable region, XbaI/ApaI restriction
sites used to clone variable region of heavy chain and ApaI/NotI
restriction sites were used to replace the constant region of the
heavy chain.
[0056] FIG. 5. Screening of Vitaxin Fc variant clones by
characterizing their relative binding to Fc.gamma.RIIIA compared to
parental scFv-Fc as determined by ELISA. Numerous clones were seen
to have improved binding.
[0057] FIG. 6. Relative ADCC activity of several Vitaxin Fc variant
clones against M21 cells as determined by a cell-based assay.
Several Fc variants, including I332E, showed improved ADCC activity
relative to the parental scFv-Fc.
[0058] FIG. 7. All 20 amino acids were substituted at position 332
of Vitaxin. The relative binding affinities of each position 332 Fc
variant to Fc.gamma.RIIIA was determined by ELISA (panel A). The
relative ADCC activity of each position 332 Fc variant was
determined by a cell-based assay (panel B). The I322E Fc variant
was seen to provide the greatest improvement in both binding and in
ADCC activity.
[0059] FIG. 8. Binding of Vitaxin.RTM. and the I332E (Vitaxin-1M)
Fc variant to Fc.gamma.RIIIA (A) and Fc.gamma.RIIB (B) as
determined by ELISA. The binding of Vitaxin-1M Fc variant to Fc
Fc.gamma.RIIIA is improved while the binding to Fc.gamma.RIIB
appears unchanged.
[0060] FIG. 9. Cell-based ADCC assay of Vitaxin.RTM. and the I332E
(Vitaxin-1M) Fc variant using 50:1 ratio of effector to target
cells at a variety of antibody concentrations from 0.4 to 1000
ng/ml. The I332E Fc variant shows higher ADCC activity over a wide
range of antibody concentrations.
[0061] FIG. 10. Cell-based ADCC assay of Vitaxin.RTM. and the I332E
(Vitaxin-1M) Fc variant using different ratios of effector to
target cells and different amounts of antibody ranging from 2.5 ng
to 200 ng per well. The I332E Fc variant shows higher ADCC activity
over a wide range of antibody concentrations at all E:T ratios.
[0062] FIG. 11. Cell-based ADCC assay of Vitaxin.RTM. and the
Vitaxin S239D/A330L/I332E (Vitaxin-3M) Fc variant against several
target cell lines expressing different levels of Integrin
.alpha.V.beta.3, A498 (moderate), DU145 (low), M21 (high) and ACHN
(moderate), using two different E:T ratios and antibody amounts
ranging from 4 ng to 400 ng per well. In all cases the
S239D/A330L/I332E (Vitaxin-3M) Vitaxin Fc variant shows higher ADCC
activity.
[0063] FIG. 12. ELISA analysis of the wild type anti-EphA2 antibody
3F2 and the 3F2 I332E (3F2-1M) and 3F2 S239D/A330L/I332E (3F2-3M)
Fc variants binding to Fc.gamma.RIIIA tetramer (panel A),
Fc.gamma.RIIIA monomer (panel B) and C1q (panel C). Both the 3F2-1M
and 3F2-3M Fc variants bind better to Fc.gamma.RIIIA monomers and
tetramers, although the 3F2-3M Fc variant binds the monomer
significantly better then either the wild type antibody or 3F2-1M
Fc variant. In contrast both the 3F2-1M and 3F2-3M Fc variants did
not bind C1q to the same degree as the wild type antibody with the
3M Fc variant showing the largest decrease in binding.
[0064] FIG. 13. FACS analysis of anti-EphA2 antibody 3F2-WT, 3F2-1M
and 3F2-3M binding to cells via Fc-domain interactions. THP-1 and
NK cells were stained with antibodies to Fc.gamma.RI, Fc.gamma.RII
and Fc.gamma.RIII (also commonly referred to CD64, CD32 and CD16,
respectively). THP-1 cells have high levels of CD32 on their cell
surface, moderate levels of CD64 and very low levels of CD16 (panel
A). NK cells however show the opposite profile, high levels of CD16
and low levels of CD32 and CD64 (panel B). All three versions of
3F2 (wt, 1M and 3M) bound to a similar degree to THP-1 cells (panel
C). However, the variants were seen to bind to a greater extent to
NK cells, with the 3F2-3M Fc variant showing the largest increase
in binding (panel D).
[0065] FIG. 14. Cell-based ADCC assay of 12G3H11 (anti-EphA2
antibody) and its I332E Fc variant using 50:1 ratio of effector to
A549 target cells (panel A) and a similar study using two different
E:T ratios from (panel B). In both studies the amount of antibody
ranged from 4 ng to 400 ng per well. The I332E Fc variant shows
higher ADCC activity over a wide range of antibody concentrations
at all E:T ratios.
[0066] FIG. 15. Cell-based ADCC assay of anti-EphA2 antibody 3F2
and the 3F2-1M and 3F2-3M Fc variants to target cells expressing
high (T231, A549) levels of EphA2. In each assay the antibody
concentration ranged from 0.02 ug/ml to 2 .mu.g/ml. E:T ratios
varied from 12.5:1 to 100:1 depending on the assay. The 3F2-3M Fc
variant was seen to have the highest ADCC activity against all cell
types. Although the 3F2-1M Fc variant showed higher ADCC activity
against most cell types than the 3F2 wild type, it was generally
not as active as the 3F2-3M Fc variant.
[0067] FIG. 16. Cell-based ADCC assay of anti-EphA2 antibody
3F2-WT, 3F2-1M and 3F2-3M Fc variants to target cells expressing
high (Hey8) and moderate (SKOV3) levels of EphA2. The antibody
concentration and E:T ratios are the same as for FIG. 15. The
3F2-3M Fc variant was seen to have the highest ADCC activity
against all cell types. Although the 3F2-1M Fc variant showed
higher ADCC activity against most cell types than the 3F-WT, it was
generally not as active as the 3F2-3M Fc variant.
[0068] FIG. 17. Cell-based ADCC assay of anti-EphA2 antibody 3F2,
3F2-1M and 3F2-3M Fc variants to target cells expressing low (A498,
SKMEL28) levels of EphA2. The SKMEL28 cells express Integrin
.alpha.V.beta.5 as were also used as target cells for the Vitaxin
and Vitaxin-3M antibodies. The antibody concentration and E:T
ratios are the same as for FIG. 15. None of the 3F2 antibodies were
seen to have activity against SKMEL28 cells although both Vitaxin
and the Vitaxin-3M antibodies had activity the Vitaxin-3M Fc
variant was significantly more active.
6. DETAILED DESCRIPTION OF THE INVENTION
[0069] The present invention provides certain amino acid residues
in the Fc region of an IgG antibody that correlate with high
effector function. Further, the invention provides high effector
function residues in the Fc region of an antibody which exhibit
high binding affinity for the Fc receptor, Fc.gamma.RIIIA. In
further embodiments, the invention encompasses the introduction of
at least one of the high effector amino acid residues of the
invention that does not result in a concomitant increase in binding
the Fc.gamma.RIIB receptor. In another embodiment, the invention
encompasses the introduction of at least one of the high effector
amino acid residues of the invention that results in a concomitant
decrease in binding the Fc.gamma.RIIB receptor and/or C1q. In still
another embodiment, the introduction of at least one of the high
effector amino acid residues of the invention that results in a
concomitant increase in binding to both the Fc.gamma.RIIIA and
Fc.gamma.RIIB receptors. In yet another embodiment, the ratio of
Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium dissociation constants
(K.sub.D), is decreased. Furthermore, the presence of at least one
of the high effector amino acid residue of the invention results in
antibodies with an enhanced antibody dependent cell-mediated
cytotoxicity (ADCC) activity. Accordingly, the invention provides
Fc variants that exhibit altered effector function (e.g., ADCC,
CDC, etc.) and/or altered binding affinity for at least one Fc
ligand (e.g., Fc.gamma.RIIIA, Fc.gamma.RIIB, C1q, etc.) relative to
an antibody (or other Fc-domain containing polypeptide) having the
same amino acid sequence as the molecule of the invention but not
comprising the novel amino acids residues of the invention
(referred to herein as a "comparable molecule") such as an antibody
comprising an unmodified Fc region containing naturally occurring
amino acid residues at the corresponding position in the Fc domain.
In particular, the present invention provides Fc variants
comprising a variable region, or fragment thereof, that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 and
a Fc region that further comprises at least one high effector
function amino acid residue (e.g., 239D, 330L, 332E).
[0070] The present invention further provides Fc variants of
antibodies that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3, said Fc variants comprising an Fc region
in which at least one amino acid residue has been substituted. The
present invention also relates to Fc variants with altered binding
affinity to their Fc.gamma.Rs compared to that of a comparable
molecule (e.g., an antibody having an original unmodified Fc
region). In one embodiment, the Fc variants have higher binding
affinity to activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA). In a
specific embodiment, the Fc variants of the invention have
equilibrium dissociation constants (K.sub.D) that are decreased
relative to a comparable molecule. In another embodiment the Fc
variants have higher binding affinity to activating Fc.gamma.Rs and
unchanged or lower binding affinity to inhibitory Fc.gamma.Rs
(e.g., Fc.gamma.RIIB). Also contemplated, are Fc variants which
have a ratio of Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium
dissociation constants (K.sub.D) that are decreased relative to a
comparable molecule. In one embodiment, the Fc variants of the
invention also exhibit increased ADCC activity when compared to a
comparable molecule (e.g., an antibody having an original
unmodified Fc region) in addition to the above changes in
Fc.gamma.R affinities. In another embodiment, the Fc variants of
the invention are variants of an antibody that immunospecifically
binds to Integrin .alpha..sub.v.beta..sub.3. In a specific
embodiment, the Fc variants of the invention immunospecifically
bind Integrin .alpha..sub.v.beta..sub.3 and are Integrin
.alpha..sub.v.beta..sub.3 antagonists.
[0071] The antibodies of the present invention may be produced "de
novo" by combining a variable domain, or fragment thereof, that
immunospecifically binds Integrin .alpha..sub.v.beta..sub.3 with an
Fc domain comprising one or more of the high effector function
residues disclosed herein, or may be produced by modifying an Fc
domain-containing antibody that binds .alpha..sub.v.beta..sub.3
Integrin by introducing one or more high effector function residues
into the Fc domain.
[0072] The present invention also relates to novel Fc variants with
a higher binding affinity to inhibitory Fc.gamma.Rs and a lower
binding affinity to activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA)
when reative to a comparable molecule (e.g., an antibody having an
original unmodified Fc region). It is contemplated that said Fc
variants will also exhibit a reduced ability to mediate ADCC
activity relative to a comparable molecule (e.g., an antibody
having an original unmodified Fc region). In one embodiment, the Fc
variants of the invention are variants of an antibody that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3. In
a specific embodiment, the Fc variants of the invention with a
higher binding affinity to inhibitory Fc.gamma.Rs and a lower
binding affinity to activating Fc.gamma.Rs immunospecifically bind
Integrin .alpha..sub.v.beta..sub.3 and are Integrin
.alpha..sub.v.beta..sub.3 antagonists.
[0073] In addition, the present invention further provides novel Fc
variants with altered binding to C1q relative to a comparable
molecule (e.g., an antibody having an original unmodified Fc
region). Specifically, the Fc variants of the invention may exhibit
a higher binding affinity for C1q and increased CDC activity.
Alternatively, the Fc variants of the invention may exhibit a lower
binding affinity for C1q and reduced CDC activity. In other
situations, the Fc variants of the invention with altered binding
to C1q exhibit CDC activity that is unchanged relative to a
comparable molecule. It is specifically contemplated that Fc
variants with alterations in C1q binding and CDC activity may also
exhibit alterations in binding to one or more Fc.gamma.Rs and/or
ADCC activity. In one embodiment, the Fc variants of the invention
are variants of an antibody that immunospecifically binds to
Integrin .alpha..sub.v.beta..sub.3. In a In another embodiment
embodiment, the Fc variants of the invention altered binding to C1q
immunospecifically bind Integrin .alpha..sub.v.beta..sub.3 and are
Integrin .alpha..sub.v.beta..sub.3 antagonists.
[0074] Also encompassed by the invention are Fc variants that
inhibit the functional activity of Integrin
.alpha..sub.v.beta..sub.3 or inhibit Integrin
.alpha..sub.v.beta..sub.3-mediated pathologies, such molecules are
also referred to herein as Integrin .alpha..sub.v.beta..sub.3
antagonists. Accordingly, the invention provides antibodies useful
for the inhibition of angiogenesis or the inhibition of other
functions mediated or influenced by Integrin
.alpha..sub.v.beta..sub.3, including but not limited to cell
proliferation, cell attachment, cell migration, granulation tissue
development, tumor growth, tumor cell invasion and/or inflammation.
Such antibodies have been described in International Publication
Nos. WO 89/05155, WO 98/33919 and WO 00/78815 as well as U.S. Pat.
No. 5,753,230, which are incorporated by reference herein in their
entireties.
[0075] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, camelised antibodies, chimeric
antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv),
Fab fragments, F (ab') fragments, and anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above.
In particular, antibodies include immunoglobulin molecules and
immunologically active fragments of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site, these fragments may
or may not be fused to another immunoglobulin domain including but
not limited to, an Fc region or fragment thereof. As outlined
herein, the terms "antibody" and "antibodies" specifically include
the Fc variants described herein, full length antibodies and
variant Fc-Fusions comprising Fc regions, or fragments thereof,
comprising at least one novel amino acid residue described herein
fused to an immunologically active fragment of an immunoglobulin or
to other proteins as described herein. Such variant Fc fusions
include but are not limited to, scFv-Fc fusions, variable region
(e.g., VL and VH)--Fc fusions, scFv-scFv-Fc fusions. Immunoglobulin
molecules 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.
[0076] As used herein, the term "immunospecifically binds to
Integrin .alpha..sub.v.beta..sub.3" and analogous terms refer to
peptides, polypeptides, proteins, fusion proteins and antibodies or
fragments thereof that specifically bind to Integrin
.alpha..sub.v.beta..sub.3 or a fragment thereof. A peptide,
polypeptide, protein, or antibody that immunospecifically binds to
an Integrin .alpha..sub.v.beta..sub.3 or a fragment thereof may
bind to other peptides, polypeptides, or proteins with lower
affinity as determined by, e.g., immunoassays, BIAcore, or other
assays known in the art. Antibodies or fragments that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3 or a
fragment thereof may be cross-reactive with related antigens. It is
contemplated that antibodies or fragments that immuno-specifically
bind to Integrin .alpha..sub.v.beta..sub.3 or a fragment thereof
preferentially bind Integrin .alpha..sub.v.beta..sub.3 over other
antigens. However, the present invention specifically encompasses
antibodies with multiple specificities (e.g., an antibody with
specificity for two or more discrete antigens (reviewed in Cao et
al., 2003, Adv Drug Deliv Rev 55:171-197; Hudson et al., 2003, Nat
Med 1: 129-134)) in the definition of an antibody that
"immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3."
For example, bispecific antibodies contain two different binding
specificities fused together. In the simplest case a bispecific
antibody would bind to two adjacent epitopes on a single target
antigen, such an antibody would not cross-react with other antigens
(as described supra). Alternatively, bispecific antibodies can bind
to two different antigens, such an antibody immunospecifically
binds to two different molecules but not to other unrelated
molecules. In addition, an antibody that immunospecifically binds
Integrin .alpha..sub.v.beta..sub.3 may cross-react with related
integrins. Another class of multispecific antibodies may recognize
a shared subunit of multi-subunit complexes in the context of one
or more specific complexes. For example CNTO 95 (Trikha et al.,
2004, Int J Cancer 110:326-335) recognizes Integrin .alpha..sub.v
in the context of both Integrin .alpha..sub.v.beta..sub.3 and
Integrin .alpha..sub.v.beta..sub.5. Thus, a multispecific antibody
may immunospecifically bind to both Integrin
.alpha..sub.v.beta..sub.3 and one or more additional molecules such
as Integrin .alpha..sub.v.beta..sub.5.
[0077] Antibodies or fragments that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3 or a fragment thereof can be
identified, for example, by immunoassays, BIAcore, or other
techniques known to those of skill in the art. An antibody or
fragment thereof binds specifically to Integrin
.alpha..sub.v.beta..sub.3 or a fragment thereof when it binds to
Integrin .alpha..sub.v.beta..sub.3 or a fragment thereof with
higher affinity than to any cross-reactive antigen as determined
using experimental techniques, such as radioimmunoassays (RIA) and
enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul, ed.,
1989, Fundamental Immunology Second Edition, Raven Press, New York
at pages 332-336 for a discussion regarding antibody
specificity.
[0078] Without wishing to be bound by any particular theory, the
amino acid substitutions of the invention alter the affinity of an
antibody for its Fc.gamma.Rs and/or the complement protein C1q by
modulating one or more of the factors that regulate protein-protein
interactions (e.g., receptor-ligand and antibody-antigen
interactions). Such factors include but are not limited to, factors
affecting protein folding or three dimensional configuration such
as hydrogen bonds, hydrophobic interactions, ionic interactions,
Von der Waals forces and/or disulfide bonds as well as factors
affecting allosteric interactions, solubility and covalent
modifications.
[0079] Without wishing to be bound by any particular theory, the
amino acid substitutions of the invention modulate the ADCC and/or
CDC activity of an antibody by altering one more of the factors
that influence downstream effector function including but not
limited to, the affinity of the antibody for its Fc.gamma.Rs and/or
to C1q, ability to mediate cytotoxic effector and/or complement
cascade functions, protein stability, antibody half life and
recruitment of effector cells and/or molecules.
[0080] It will be understood that Fc region (also referred to
herein as "Fc" and "Fc polypeptide") as used herein includes the
polypeptides comprising the constant region of an antibody
excluding the first constant region immunoglobulin domain. Thus Fc
refers to the last two constant region immunoglobulin domains of
IgA, IgD, and IgG, and the last three constant region
immunoglobulin domains of IgE and IgM, and the flexible hinge
N-terminal to these domains. For IgA and IgM Fc may include the J
chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and
Cgamma3 (C.gamma.2 and C.gamma.3) and the hinge between Cgamma1
(C.gamma.1) and Cgamma2 (C.gamma.2). Although the boundaries of the
Fc region may vary, the human IgG heavy chain Fc region is usually
defined to comprise residues C226 or P230 to its carboxyl-terminus,
wherein the numbering is according to the EU index as in Kabat et
al. (1991, NIH Publication 91-3242, National Technical Information
Service, Springfield, Va.). The "EU index as set forth in Kabat"
refers to the residue numbering of the human IgG1 EU antibody as
described in Kabat et al. supra. Fc may refer to this region in
isolation, or this region in the context of an antibody, antibody
fragment, or Fc fusion protein. Note: Polymorphisms have been
observed at a number of Fc positions, including but not limited to
Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences
between the presented sequence and sequences in the prior art may
exist.
[0081] It will be understood that the complementarity determining
regions (CDRs) residue numbers referred to herein are those of
Kabat et al. (1991, NIH Publication 91-3242, National Technical
Information Service, Springfield, Va.). Specifically, residues
24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain
variable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in
the heavy chain variable domain. Note that CDRs vary considerably
from antibody to antibody (and by definition will not exhibit
homology with the Kabat consensus sequences). Maximal alignment of
framework residues frequently requires the insertion of "spacer"
residues in the numbering system, to be used for the Fv region. It
will be understood that the CDRs referred to herein are those of
Kabat et al. supra. In addition, the identity of certain individual
residues at any given Kabat site number may vary from antibody
chain to antibody chain due to interspecies or allelic
divergence.
[0082] In one embodiment, Fc variants of the invention will have at
least one amino acid substitution of the Fc region wherein said
antibody variant has a modified binding affinity for its
Fc.gamma.Rs and/or for C1q relative to a comparable molecule (e.g.,
the original antibody without said substitution).
[0083] In a specific embodiment, Fc variants comprise an Fc region
comprising at least one high effector function amino acid reside
selected from the group consisting of: 234E, 235R, 235A, 235W,
235P, 235V, 235Y, 236E, 239D, 265L, 269S, 269G, 2981, 298T, 298F,
327N, 327G, 327W, 328S, 328V, 329H, 329Q, 330K, 330V, 330G, 330Y,
330T, 330L, 3301, 330R, 330C, 332E, 332H, 332S, 332W, 332F, 332D,
and 332Y, wherein the numbering system is that of the EU index as
set forth in Kabat. Specific high effector function amino acid
residues of the invention are also set forth in Table 1.
[0084] In another embodiment, the Fc variants comprise an Fc region
comprising at least 2, or at least 3, or at least 4, or at least 5,
or at least 6, or at least 7, or at least 8, or at least 9, or at
least 10, or at least 20, or at least 30, or at least 40, or at
least 50, or at least 60, or at least 70, or at least 80, or at
least 90, or at least 100, or at least 200 high effector function
amino acid residues.
[0085] In another specific embodiment, Fc variants of the invention
comprise an Fc region comprising at least one high effector
function amino acid residue selected from the group consisting of:
239D, 330K, 330V, 330G, 330Y, 330T, 330L, 3301, 330R, 330C, 332E,
332H, 332S, 332W, 332F, 332D, and 332Y, wherein the numbering
system is that of the EU index as set forth in Kabat.
[0086] In still another specific embodiment, Fc variants of the
invention comprise an Fc region comprising at least one high
effector function amino acid residue selected from the group
consisting of: 239D, 330L and 332E. In another embodiment, Fc
variants of the invention comprise an Fc region comprising at least
the high effector function amino acid residue 332E. In a specific
embodiment, Fc variants of the invention comprise an Fc region
comprising the high effector function amino acid residues 239D,
330L and 332E.
[0087] In a specific embodiment, Fc variants will have one or more
amino acid substitutions at positions selected from the group
consisting of: 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,
216, 217, 218, 219, 220, 221, 222, 223,224,225,226, 227, 228, 229,
230, 231, 232, 233, 234, 235, 236, 237, 239, 242, 246, 250, 251,
257, 259, 260, 261, 265, 269, 273, 274, 275, 277, 281, 282, 284,
287, 291, 298, 300, 302, 304, 306, 308, 310, 314, 316, 318, 319,
321, 323, 327, 328, 329, 330, 332 and 336, of the Fc region wherein
the numbering of the residues in the Fc region is that of the EU
index as set forth in Kabat.
[0088] In another specific embodiment, the Fc variants comprise at
least one substitution selected from the group consisting of:
L234E, L235R, L235A, L235W, L235P, L235V, L235Y, G236E, S239D,
D265L, E269S, E269G, S298I, S298T, S298F, A327N, A327G, A327W,
L328S, L328V, P329H, P329Q, A330K, A330V, A330G, A330Y, A330T,
A330L, A330I, A330R, A330C, I332E, I332H, I332S, I332W, I332F,
I332D, and I332Y, wherein the numbering system is that of the EU
index as set forth in Kabat. Specific amino acid substitutions of
the invention are also set forth in Table 1.
[0089] In another embodiment, the Fc variants comprise at least 2,
or at least 3, or at least 4, or at least 5, or at least 6, or at
least 7, or at least 8, or at least 9, or at least 10, or at least
20, or at least 30, or at least 40, or at least 50, or at least 60,
or at least 70, or at least 80, or at least 90, or at least 100, or
at least 200 amino acid substitutions of the Fc region.
TABLE-US-00001 TABLE 1 Specific Amino Acid Residues with High
Effector Function (HEF) Position.sup.a Amino Acid.sup.b HEF
Residue(s).sup.c 234 L E 235 L R, A, W, P, V, Y 236 G E 239 S D 265
D L 269 E S, G 298 S I, T, F 327 A N, G, W 328 L S, V 329 P H, Q
330 A K, V, G, Y, T, L, I, R, C 332 I E, H, S, W, F, Y, D
.sup.aheavy chain position number and amino acid residue
.sup.bamino acid residue present in naturally occurring antibody
.sup.cresidues that can be engineered into corresponding position
to generate an Fc region with high effector function.
[0090] In one embodiment, the Fc variants comprise at least one
substitution selected from the group consisting of S239D, A330L and
I332E. In another preferred embodiment, the Fc variants comprise at
least each of the following substitutions, S239D, A330L and I332E.
In another embodiment, the Fc variants of the invention have at
least the amino acid substitution I332E.
[0091] It is specifically contemplated that conservative amino acid
substitutions may be made for said amino acid substitutions in the
Fc of the antibody of interest, described supra (see Table 1). It
is well known in the art that "conservative amino acid
substitution" refers to amino acid substitutions that substitute
functionally-equivalent amino acids. Conservative amino acid
changes result in silent changes in the amino acid sequence of the
resulting peptide. For example, one or more amino acids of a
similar polarity act as functional equivalents and result in a
silent alteration within the amino acid sequence of the peptide.
Substitutions that are charge neutral and which replace a residue
with a smaller residue may also be considered "conservative
substitutions" even if the residues are in different groups (e.g.,
replacement of phenylalanine with the smaller isoleucine). Families
of amino acid residues having similar side chains have been defined
in the art. Several families of conservative amino acid
substitutions are shown in Table 2. TABLE-US-00002 TABLE 2 Families
of Conservative Amino Acid Substitutions Family Amino Acids
non-polar Trp, Phe, Met, Leu, Ile, Val, Ala, Pro uncharged polar
Gly, Ser, Thr, Asn, Gln, Tyr, Cys acidic/negatively charged Asp,
Glu basic/positively charged Arg, Lys, His Beta-branched Thr, Val,
Ile residues that influence chain orientation Gly, Pro aromatic
Trp, Tyr, Phe, His
[0092] The term "conservative amino acid substitution" also refers
to the use of amino acid analogs or variants. Guidance concerning
how to make phenotypically silent amino acid substitutions is
provided in Bowie et al., "Deciphering the Message in Protein
Sequences: Tolerance to Amino Acid Substitutions," (1990, Science
247:1306-1310).
[0093] In another embodiment, the Fc variants have at least the
amino acid substitution I332D.
[0094] One skilled in the art will understand that that the Fc
variants of the invention may have altered Fc.gamma.R and/or C1 q
binding properties (examples of binding properties include but are
not limited to, binding specificity, equilibrium dissociation
constant (K.sub.D), dissociation and association rates (K.sub.off
and K.sub.on respectively), binding affinity and/or avidity) and
that certain alterations are more or less desirable. It is well
known in the art that the equilibrium dissociation constant
(K.sub.D) is defined as k.sub.off/k.sub.on. It is generally
understood that a binding molecule (e.g., and antibody) with a low
K.sub.D is preferable to a binding molecule (e.g., and antibody)
with a high K.sub.D. However, in some instances the value of the
k.sub.on or k.sub.off may be more relevant than the value of the
K.sub.D. One skilled in the art can determine which kinetic
parameter is most important for a given antibody application. For
example a modification that enhances Fc binding to one or more
positive regulators (e.g., Fc.gamma.RIIIA) while leaving unchanged
or even reducing Fc binding to the negative regulator Fc.gamma.RIIB
would be more preferable for enhancing ADCC activity.
Alternatively, a modification that reduced binding to one or more
positive regulator and/or enhanced binding to Fc.gamma.RIIB would
be preferable for reducing ADCC activity. Accordingly, the ratio of
binding affinities (e.g., equilibrium dissociation constants
(K.sub.D)) can indicate if the ADCC activity of an Fc variant is
enhanced or decreased. For example a decrease in the ratio of
Fc.gamma.RIILA/Fc.gamma.RIIB equilibrium dissociation constants
(K.sub.D), will correlate with improved ADCC activity, while an
increase in the ratio will correlate with a decrease in ADCC
activity. Additionally, modifications that enhanced binding to C1q
would be preferable for enhancing CDC activity while modification
that reduced binding to C1q would be preferable for reducing or
eliminating CDC activity.
[0095] The affinities and binding properties of an Fc domain for
its ligand, may be determined by a variety of in vitro assay
methods (biochemical or immunological based assays) known in the
art for determining Fc-Fc.gamma.R interactions, i.e., specific
binding of an Fc region to an Fc.gamma.R including but not limited
to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay
(ELISA); see Example 3, or radioimmunoassay (RIA)), or kinetics
(e.g., BIACORE.RTM. analysis), and other methods such as indirect
binding assays, competitive inhibition assays, fluorescence
resonance energy transfer (FRET), gel electrophoresis and
chromatography (e.g., gel filtration). These and other methods may
utilize a label on one or more of the components being examined
and/or employ a variety of detection methods including but not
limited to chromogenic, fluorescent, luminescent, or isotopic
labels. A detailed description of binding affinities and kinetics
can be found in Paul, W. E., ed., Fundamental Immunology, 4.sup.th
Ed., Lippincott-Raven, Philadelphia (1999), which focuses on
antibody-immunogen interactions.
[0096] In a one embodiment, the Fc variants of the invention bind
Fc.gamma.RIIIA with increased affinity relative to a comparable
molecule. In another embodiment, the Fc variants of the invention
bind Fc.gamma.RIIIA with increased affinity and bind Fc.gamma.RIIB
with a binding affinity that is unchanged relative to a comparable
molecule. In still another embodiment, the Fc variants of the
invention bind Fc.gamma.RIIIA with increased affinity and bind
Fc.gamma.RIIB with a decreased affinity relative to a comparable
molecule. In yet another embodiment, the Fc variants of the
invention have a ratio of Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium
dissociation constants (K.sub.D) that is decreased relative to a
comparable molecule.
[0097] In one embodiment, the Fc variants of the invention bind
Fc.gamma.RIIIA with increased affinity and bind Fc.gamma.RIIB with
a decreased affinity when relative to a comparable molecule and
immunospecifically bind Integrin .alpha.V.beta.3.
[0098] In one embodiment, said Fc variants bind with increased
affinity to Fc.gamma.RIIIA. In one embodiment, said Fc variants
have affinity for Fc.gamma.RIIIA that is at least 2 fold, or at
least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10
fold, or at least 20 fold, or at least 30 fold, or at least 40
fold, or at least 50 fold, or at least 60 fold, or at least 70
fold, or at least 80 fold, or at least 90 fold, or at least 100
fold, or at least 200 fold greater than that of a comparable
molecule.
[0099] In another embodiment, an Fc variant of the invention has an
equilibrium dissociation constant (K.sub.D) that is decreased
between about 2 fold and about 10 fold, or between about 5 fold and
about 50 fold, or between about 25 fold and about 250 fold, or
between about 100 fold and about 500 fold, or between about 250
fold and about 1000 fold relative to a comparable molecule. In
another embodiment, an Fc variant of the invention has an
equilibrium dissociation constant (K.sub.D) that is decreased
between 2 fold and 10 fold, or between 5 fold and 50 fold, or
between 25 fold and 250 fold, or between 100 fold and 500 fold, or
between 250 fold and 1000 fold relative to a comparable molecule.
In a specific embodiment, said Fc variants have an equilibrium
dissociation constants (K.sub.D) for Fc.gamma.RIIIA that is reduced
by at least 2 fold, or at least 3 fold, or at least 5 fold, or at
least 7 fold, or a least 10 fold, or at least 20 fold, or at least
30 fold, or at least 40 fold, or at least 50 fold, or at least 60
fold, or at least 70 fold, or at least 80 fold, or at least 90
fold, or at least 100 fold, or at least 200 fold, or at least 400
fold, or at least 600 fold, relative to a comparable molecule.
[0100] In one embodiment, said Fc variant binds to Fc.gamma.RIIB
with an affinity that is unchanged or reduced. In another
embodiment said Fc variants have affinity for Fc.gamma.RIIB that is
unchanged or reduced by at least 1 fold, or by at least 3 fold, or
by at least 5 fold or by at least 10 or by at least 20 fold, or by
at least 50 fold relative to a comparable molecule.
[0101] In another embodiment, said Fc variants have an equilibrium
dissociation constants (K.sub.D) for Fc.gamma.RIIB that is
unchanged or increased by at least at least 2 fold, or at least 3
fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 30 fold, or at least 40 fold, or
at least 50 fold, or at least 60 fold, or at least 70 fold, or at
least 80 fold, or at least 90 fold, or at least 100 fold, or at
least 200 fold relative to a comparable molecule.
[0102] In another embodiment, the Fc variants of the invention bind
Fc.gamma.RIIIA with decreased affinity and bind Fc.gamma.RIIB with
increased affinity when compared to the original antibodies without
the substituted Fc. In still another embodiment said Fc variants
have affinity for Fc.gamma.RIIIA that is reduced by at least 1
fold, or by at least 3 fold, or by at least 5 fold or by at least
10 or by at least 20 fold, or by at least 50 fold when compared to
that of the original antibody without the substituted Fc. In yet
another embodiment said Fc variants have affinity for Fc.gamma.RIIB
that is at least 2 fold, or at least 3 fold, or at least 5 fold, or
at least 7 fold, or a least 10 fold, or at least 20 fold, or at
least 50 fold or at least 100 fold, greater than that of a
comparable molecule.
[0103] In still another embodiment, the Fc variants have an
equilibrium dissociation constants (K.sub.D) for Fc.gamma.RIIIA
that are increased by at least 1 fold, or by at least 3 fold, or by
at least 5 fold or by at least 10 or by at least 20 fold, or by at
least 50 fold when compared to that of the original antibody
without the substituted Fc. In yet another embodiment said Fc
variants have equilibrium dissociation constants (K.sub.D) for
Fc.gamma.RIIB that are decreased at least 2 fold, or at least 3
fold, or at least 5 fold, or at least 7 fold, or a least 10 fold,
or at least 20 fold, or at least 50 fold or at least 100 fold,
relative to a comparable molecule.
[0104] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enables these
cytotoxic effector cells to bind specifically 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 absolutely required
for such killing. Lysis of the target cell is extracellular,
requires direct cell-to-cell contact, and does not involve
complement.
[0105] The ability of any particular antibody to mediate lysis of
the target cell by ADCC can be assayed. To assess ADCC activity an
antibody of interest is added to target cells in combination with
immune effector cells, which may 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., 1985 79:277-282;
Bruggemann et al., 1987, J Exp Med 166:1351-1361; Wilkinson et al.,
2001, J Immunol Methods 258:183-191; Patel et al., 1995 J Immunol
Methods 184:29-38 and herein (see Example 3). Alternatively, or
additionally, ADCC activity of the antibody of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al., 1998, PNAS USA 95:652-656.
[0106] It is contemplated that the Fc variants of the invention are
also characterized by in vitro functional assays for determining
one or more Fc.gamma.R mediator effector cell functions (See
Example 3). In certain embodiments, the molecules of the invention
have similar binding properties and effector cell functions in in
vivo models (such as those described and disclosed herein) as those
in in vitro based assays However, the present invention does not
exclude molecules of the invention that do not exhibit the desired
phenotype in in vitro based assays but do exhibit the desired
phenotype in vivo.
[0107] The present invention further provides Fc variants with
enhanced ADCC function. In one embodiment, the Fc variants of the
invention have increased ADCC activity. In another embodiment said
Fc variants have 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
a specific embodiment, Fc variants of the invention bind
Fc.gamma.RIIIA with increased affinity, bind Fc.gamma.RIIB with
decreased affinity and have enhanced ADCC activity relative to a
comparable molecule.
[0108] In one embodiment, the Fc variants of the invention have
enhanced ADCC activity and immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3. In one embodiment the Fc variants of the
invention have enhanced ADCC activity and have a ratio of
Fc.gamma.RIIIA/Fc.gamma.RIIB equilibrium dissociation constants
(K.sub.D) that is decreased relative to a comparable molecule and
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3 In
another embodiment, the Fc variants of the invention have enhanced
ADCC activity, bind activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA)
with higher affinity and bind inhibitory Fc.gamma.Rs (e.g.,
Fc.gamma.RIIB) with unchanged or lower affinity and
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3.
[0109] The present invention also provides Fc variants with reduced
ADCC function. In one embodiment, the Fc variants of the invention
have reduced ADCC activity. In one embodiment said Fc variants have
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 less than that of a comparable molecule. In a specific
embodiment, Fc variants of the invention bind Fc.gamma.RIIIA with
decreased affinity, bind Fc.gamma.RIIB with increased affinity and
have reduced ADCC activity.
[0110] In one embodiment, the Fc variants of the invention have
reduced ADCC activity and immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3. In another embodiment, the antibody
variants of the invention have reduced ADCC activity, bind
activating Fc.gamma.Rs (e.g., Fc.gamma.RIIIA) with lower affinity,
bind inhibitory Fc.gamma.Rs (e.g., Fc.gamma.RIIB) with higher
affinity and immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3.
[0111] "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., 1996, J. Immunol. Methods, 202:163, may be
performed.
[0112] The present invention further provides Fc variants with
enhanced CDC function. In one embodiment, the Fc variants of the
invention have increased CDC activity. In another embodiment said
Fc variants have 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
another embodiment, an Fc variant of the invention binds C1q with
an affinity that is at least 2 fold, or at least 3 fold, or at
least 5 fold, or at least 7 fold, or a least 10 fold, or at least
20 fold, or at least 50 fold or at least 100 fold, greater than
that of a comparable molecule. In a specific embodiment, Fc
variants of the invention bind C1q with increased affinity; have
enhanced CDC activity and immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3.
[0113] The present invention also provides Fc variants with reduced
CDC function. In one embodiment, the Fc variants of the invention
have reduced CDC activity. In another embodiment said Fc variants
have 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 less than that of relative to a comparable molecule. In
another embodiment, an Fc variant of the invention binds C1q with
an affinity that is reduced by at least 1 fold, or by at least 3
fold, or by at least 5 fold or by at least 10 or by at least 20
fold, or by at least 50 fold relative to a comparable molecule. In
a specific embodiment, Fc variants of the invention bind to
Integrin .alpha..sub.v.beta..sub.3, bind C1q with decreased
affinity have reduced CDC activity and immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3.
[0114] It is also specifically contemplated that the Fc variants of
the invention may contain inter alia one or more additional amino
acid residue substitutions, mutations and/or modifications which
result in an antibody with desired characteristics including but
not limited to: increased serum half life, increase binding
affinity, reduced immunogenicity, increased production, altered Fc
ligand binding, enhanced or reduced ADCC or CDC activity, altered
glycosylation and/or disulfide bonds and modified binding
specificity (for examples see infra). The invention encompasses
combining an Fc variant of the invention with other Fc
modifications to provide additive, synergistic, or novel properties
in antibodies or Fc fusions. In one embodiment, the other Fc
modifications enhance the phenotype of the Fc variant with which
they are combined. For example, if an Fc variant of the invention
is combined with a mutant known to bind Fc.gamma.RIIIA with a
higher affinity than a comparable molecule comprising a wild type
Fc region; the combination with a mutant of the invention results
in a greater fold enhancement in Fc.gamma.RIIIA affinity.
[0115] In one embodiment, the Fc variants of the present invention
may be combined with other known Fc variants such as those
disclosed in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et
al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol
147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et
al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc
Natl. Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol
Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et
al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol
157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624;
Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J
Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26;
Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al.,
2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett
82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); 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; 6,194,551; 6,737,056;
6,821,505; 6,277,375; U.S. patent application Ser. Nos. 10/370,749
and PCT Publications WO 94/2935; WO 99/58572; WO 00/42072; WO
02/060919, WO 04/029207, each of which is incorporated herein by
reference in its entirety.
[0116] In some embodiments, the Fc variants of the present
invention comprises one or more engineered glycoforms, i.e., a
carbohydrate composition that is covalently attached to a molecule
comprising an Fc region. Engineered glycoforms may be useful for a
variety of purposes, including but not limited to enhancing or
reducing effector function. Engineered glycoforms may be generated
by any method known to one skilled in the art, for example by using
engineered or variant expression strains, by co-expression with one
or more enzymes, for example DI N-acetylglucosaminyltransferase III
(GnTI11), by expressing a molecule comprising an Fc region in
various organisms or cell lines from various organisms, or by
modifying carbohydrate(s) after the molecule comprising Fc region
has been expressed. Methods for generating engineered glycoforms
are known in the art, and include but are not limited to those
described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies
et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J
Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem
278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;
U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1;
PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent.TM. technology
(Biowa, Inc. Princeton, N.J.); GlycoMAb.TM. glycosylation
engineering technology (GLYCART biotechnology AG, Zurich,
Switzerland); each of which is incorporated herein by reference in
its entirety. See, e.g., WO 00061739; EA01229125; U.S. 20030115614;
Okazaki et al., 2004, JMB, 336: 1239-49 each of which is
incorporated herein by reference in its entirety. Additional
methods are described in section 6.2 entitled "Antibodies of the
Invention," infra.
[0117] In another embodiment, the Fc variants of the invention are
variants of Vitaxin.RTM., its derivatives, analogs, and
epitope-binding fragments thereof (such as but not limited to,
those disclosed in U.S. Pat. Nos. 6,531,580; 6,590,079; 6,596,850;
PCT Publications WO 89/05155, WO 98/33919, and WO 00/78815) each of
which is incorporated herein by reference in its entirety.
[0118] Integrins are receptor proteins which are of crucial
importance. They are the main way that cells both bind to and
respond to the extracellular matrix and are involved in a variety
of cellular functions such as wound healing, cell differentiation,
homing of tumor cells and apoptosis. They are part of a large
family of cell adhesion receptors which are involved in
cell-extracellular matrix and cell-cell interactions.
Integrin-ligand interactions are accompanied by clustering and
activation of the integrins on the cell surface, which is also
accompanied by the transduction of signals into intracellular
signal transduction pathways that mediate a number of intracellular
events. Molecules known to be involved in the downstream signaling
events include Focal adhesion kinase (FAK), mitogen activated
protein kinase (MAPK) and phospholipase C-gamma (PLC-gamma) among
others. As cell surface molecules the Integrins are readily
accessible target molecules for antibody directed therapies. In
another embodiment, the Fc variants of the invention are variants
of an antibody that immunospecifically binds to an Integrin other
than Integrin .alpha..sub.v.beta..sub.3. Integrins to which an Fc
variant of the invention immunospecifically binds to include but
are not limited to, Integrin .alpha..sub.v.beta..sub.1, Integrin
.alpha..sub.1.beta..sub.1, Integrin .alpha..sub.2.beta..sub.1,
Integrin .alpha..sub.3.beta..sub.1, Integrin
.alpha..sub.4.beta..sub.1, Integrin .alpha..sub.4.beta..sub.7
Integrin .alpha..sub.5.beta..sub.1, Integrin
.alpha..sub.6.beta..sub.1, Integrin .alpha..sub.6.beta..sub.4,
Integrin .alpha..sub.7.beta..sub.1, Integrin .alpha..sub.8
.beta..sub.1, Integrin .alpha..sub.9.beta..sub.1, Integrin
.alpha..sub.D.beta..sub.2, Integrin .alpha..sub.L.beta..sub.2,
Integrin .alpha..sub.M.beta..sub.2, Integrin
.alpha..sub.v.beta..sub.1, Integrin .alpha..sub.v.beta..sub.5,
Integrin .alpha..sub.v.beta..sub.6, Integrin
.alpha..sub.v.beta..sub.8, Integrin .alpha..sub.X.beta..sub.2,
Integrin .alpha..sub.v.beta..sub.1, Integrin
.alpha..sub.IIb.beta..sub.5, Integrin .alpha..sub.IIb.beta..sub.3,
Integrin .alpha..sub.IELb.beta..sub.7.
[0119] In a particular embodiment, the Fc variants of the invention
are antibodies or fragments thereof that compete with Vitaxin.RTM.
or an antigen-binding fragment thereof for binding to Integrin
.alpha..sub.v.beta..sub.3.
[0120] The present invention further encompasses the use of Fc
variants of the invention that have a high binding affinity for
integrin .alpha..sub.v.beta..sub.3. In a specific embodiment, an Fc
variant of the invention that immunospecifically binds to integrin
.alpha..sub.v.beta..sub.3 has an association rate constant or
k.sub.on rate (Fc variant (Ab)+antigen
(Ag).sup.k.sub.on.rarw.Ab-Ag) of at least 10.sup.5M.sup.-1s.sup.-1,
at least 5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least
5.times.10.sup.6M.sup.-1s.sup.-1, at least
10.sup.7M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least
10.sup.8M.sup.-1s.sup.-1. In another embodiment, an Fc variant that
immunospecifically binds to integrin .alpha..sub.v.beta..sub.3 has
a k.sub.on of at least 2.times.10.sup.5M.sup.-1s.sup.-1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least
5.times.10.sup.6M.sup.-1s.sup.-1, at least
10.sup.7M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least
10.sup.8M.sup.-1s.sup.-1.
[0121] In another embodiment, an Fc variant of the invention that
immunospecifically binds to integrin .alpha..sub.v.beta.3 has a
k.sub.off rate (Fc variant (Ab)+antigen
(Ag).sup.k.sub.off.rarw.Ab-Ag) of less than 10.sup.-1s.sup.-1, less
than 5.times.10.sup.-1s.sup.-1, less than 10.sup.-2s.sup.-1, less
than 5.times.10.sup.-2s.sup.-1, less than 10.sup.-3s.sup.-1, less
than 5.times.10.sup.-3s.sup.-1, less than 10.sup.-4s.sup.-1, less
than 5.times.10.sup.-4s.sup.-1, less than 10.sup.-5s.sup.-1, less
than 5.times.10.sup.-5s.sup.-1, less than 10.sup.-6s.sup.-1, less
than 5.times.10.sup.-6s.sup.-1, less than 10.sup.-7s.sup.-1, less
than 5.times.10.sup.-7s.sup.-1, less than 10.sup.-8s.sup.-1, less
than 5.times.10 .sup.-8s.sup.-1, less than 10.sup.-9s.sup.-1, less
than 5.times.10.sup.-9s.sup.-1, or less than 10.sup.-10-1s.sup.-1.
In another embodiment, an Fc variant that immunospecifically binds
to integrin .alpha..sub.v.beta..sub.3 has a k.sub.off, of less than
5.times.10.sup.-4s.sup.-1, less than 10.sup.-5s.sup.-1, less than
5.times.10.sup.-5s.sup.-1, less than 10.sup.-6s.sup.-1, less than
5.times.10.sup.-6s .sup.1, less than 10.sup.-7s.sup.-1, less than
5.times.10.sup.-7s.sup.-1, less than 10.sup.-8s.sup.-1, less than
5.times.10.sup.-8s.sup.-1, less than 10.sup.-9s.sup.-1, less than
5.times.10.sup.-9s.sup.-1, or less than 10.sup.-10s.sup.-1.
[0122] In another embodiment, an Fc variant of the invention that
immunospecifically binds to integrin .alpha..sub.v.beta..sub.3 has
an affinity constant or K.sub.a (k.sub.on/k.sub.off) of at least
10.sup.2M.sup.-1, at least 5.times.10.sup.2M.sup.-1, at least
10.sup.3M.sup.-1, at least 5.times.10.sup.3M.sup.-1, at least
10.sup.4M.sup.-1, at least 5.times.10.sup.4M.sup.-1, at least
10.sup.5M.sup.-1, at least 5.times.10.sup.5M.sup.-1, at least
10.sup.6M.sup.-1, at least 5.times.10.sup.6M.sup.-1, at least
10.sup.7M.sup.-1, at least 5.times.10.sup.7M.sup.-1, at least
10.sup.8M.sup.-1, at least 5.times.10.sup.8M.sup.-1, at least
10.sup.9M.sup.-1, at least 5.times.10.sup.9M.sup.-1, at least
10.sup.10M.sup.-1, at least 5.times.10.sup.1M.sup.-1, at least
10.sup.11M.sup.-1, at least 5.times.10.sup.11M.sup.-1, at least
10.sup.12M.sup.-1, at least 5.times.10.sup.12M, at least
10.sup.13M.sup.-1, at least 5.times.10.sup.13M.sup.-1, at least
10.sup.14M.sup.-1, at least 5.times.10.sup.14M.sup.-1, at least
10.sup.15M.sup.-1, or at least 5.times.10.sup.15M.sup.-1.
[0123] In yet another embodiment, an Fc variant that
immunospecifically binds to integrin .alpha..sub.v.beta..sub.3 has
a dissociation constant or K.sub.d (k.sub.off/k.sub.on) of less
than 10.sup.-2M, less than 5.times.10.sup.-2M, less than
10.sup.-3M, less than 5.times.10.sup.-3M, less than 10.sup.-4M,
less than 5.times.10.sup.-4M, less than 10.sup.-5M, less than
5.times.10 .sup.-5M, less than 10.sup.-6M, less than
5.times.10.sup.-6M, less than 10.sup.-7M, less than
5.times.10.sup.-7M, less than 10.sup.-8M, less than
5.times.10.sup.-8M, less than 10 .sup.-9M, less than
5.times.10.sup.-9M, less than 10.sup.-10M, less than
5.times.10.sup.-10M, less than 10.sup.-11M, less than
5.times.10.sup.-11M, less than 10.sup.-12M, less than
5.times.10.sup.-12M, less than 10.sup.-13M, less than
5.times.10.sup.13M, less than 10.sup.-14M, less than
5.times.10.sup.-14M, less than 10.sup.-15M, or less than
5.times.10.sup.-15M.
6.1 Fc Variants that Immunospecifically Bind to Integrin
.alpha..sub.v.beta..sub.3
[0124] As discussed above, the invention encompasses Fc comprising
a variable region that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 and a Fc region that further comprises at
least one high effector function amino acid residue (e.g., 239D,
330L, 332E wherein the numbering of the residues is that of the EU
index as set forth in Kabat). The invention further encompasses Fc
variants that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3, have altered ADCC and/or CDC activity
and modified binding affinities for one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) relative to a comparable molecule. The invention
encompasses Fc variants of anti-Integrin .alpha..sub.v.beta..sub.3
antibodies including, but not limited to, LM609 (Scripps), the
murine monoclonal LM609 (PCT Publication WO 89/015155 and U.S. Pat.
No. 5,753,230, each of which is incorporated herein by reference in
its entirety); the humanized monoclonal antibody MEDI-522 (a.k.a.
VITAXIN.RTM., MedImmune, Inc., Gaithersburg, Md.; Wu et al., 1998,
PNAS USA 95(11): 6037-6042; PCT Publications WO 90/33919 and WO
00/78815, each of which is incorporated herein by reference in its
entirety); D12 (PCT Publication WO 98/40488); anti-Integrin
.alpha..sub.v.beta..sub.3 antibody PDE 117-706 (ATCC access No.
HB-12224), P112-4C1 (ATCC access No. HB-12225), P113-12A6 (ATCC
access No. HB-12226), P112-11D2 (ATCC access No. HB-12227),
P112-IOD4 (ATCC access No. HB-12228) and P113-IF3 (ATCC access No.
HB-12229). (G.D, Searle & Co., PCT Publication WO 98/46264);
17661-37E and 17661-37E 1-5 (USBiological), MON 2032 and 2033
(CalTag), ab7166 (BV3) and ab 7167 (BV4) (Abcam), WOW-1 (Kiosses et
al., 2001, Nature Cell Biology, 3:316-320), CNTO 95 (Centocor, PCT
publication WO 02/12501 which is incorporated herein by reference
in its entirety) and analogs, derivatives, or fragments
thereof.
[0125] In one embodiment embodiment, the Fc variant is an Fc
variant of Vitaxin.RTM., a humanized blocking monoclonal antibody
that binds Integrin .alpha..sub.v.beta..sub.3. The amino acid
sequence of Vitaxin.RTM. is disclosed, e.g., in PCT Publications WO
98/33919; WO 00/78815; and WO 02/070007; U.S. application Ser. No.
09/339,922, each of which is incorporated herein by reference in
its entirety. The amino acid sequences for the heavy chain variable
region and light chain variable region are provided herein as SEQ
ID NO: 3 and SEQ ID NO: 4, respectively (FIGS. 1A and 1B). The
nucleotide sequence for the heavy chain variable and light chain
variable region are provided herein as SEQ ID NO: 1 and SEQ ID NO:
2, respectively (FIGS. 1A and 1B). In another embodiment, Fc
variant of the present invention binds to the same epitope as
Vitaxin.RTM. or competes with Vitaxin.RTM. for binding to Integrin
.alpha..sub.v.beta..sub.3. In an alternative embodiment, the Fc
variant of the invention that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 is not an Fc variant of Vitaxin.RTM..
[0126] In a specific embodiment, an Fc of the invention is
generated by combining a antigen binding domain (e.g., variable
region) or fragment thereof of an antibody or fragment thereof that
immunospecifically binds Integrin .alpha..sub.v.beta..sub.3
(examples supra) with an Fc region comprising at least one high
effector function amino acid residue. Methods for generating such a
recombinant antibody are well know to one skilled in the art and
are further described infra.
[0127] In one embodiment, the Fc variant of the invention
preferentially binds Integrin .alpha..sub.v.beta..sub.3 over other
integrins. In another embodiment, the Fc variant of the invention
does not immunoreact with an .alpha..sub.v subunit. In another
embodiment, said Fc variant of the invention does immunoreact with
an .alpha..sub.v subunit. In still another embodiment, the Fc
variant of the invention does not immunoreact with an P3 subunit.
In yet another embodiment, the Fc variant of the invention does not
immunoreact with integrins other than .alpha..sub.v.beta..sub.3. In
yet another embodiment, the Fc variant of the invention does
immunoreact with a .beta..sub.3 subunit. In still another
embodiment, the Fc variant of the invention immunoreacts with both
Integrin .alpha..sub.v.beta..sub.3 and Integrin
.alpha..sub.v.beta..sub.5 or with more then one Integrin
.alpha..beta. complex. The variant may have the same
immunoreactivity for both Integrin .alpha..sub.v.beta..sub.3 and
Integrin .alpha..sub.v.beta..sub.5 or alternatively, the Fc variant
may immunoreact more strongly with Integrin
.alpha..sub.v.beta..sub.3 than with Integrin
.alpha..sub.v.beta..sub.5, or more strongly with Integrin
.alpha..sub.v.beta..sub.5 than with Integrin
.alpha..sub.v.beta..sub.3. In another embodiment the Fc variant
binds an integrin other then Integrin .alpha..sub.v.beta..sub.3
(e.g., .alpha..sub.v.beta..sub.1, .alpha..sub.1.beta..sub.1,
.alpha..sub.2.beta..sub.1, .alpha..sub.2.beta..sub.1,
.alpha..sub.5.beta..sub.1, .alpha..sub.D.beta..sub.2,
.alpha..sub.IIb.beta..sub.3).
[0128] The present invention encompasses Fc variants that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3, said
antibodies comprising a variable heavy ("VH") domain having an
amino acid sequence of the VH domain for LM609 or VITAXIN.RTM.. The
present invention also encompasses Fc variants that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3, said
antibodies comprising a variable light ("VL") domain having an
amino acid sequence of the VL domain for LM609 or VITAXIN.RTM.. The
invention further encompasses Fc variants that immuno-specifically
bind to Integrin .alpha..sub.v.beta..sub.3, said antibodies
comprising a VH domain disclosed herein combined with a VL domain
disclosed herein, or other VL domain. The present invention further
encompasses Fc variants Fc variants that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3, said Fc variants comprising a
VL domain disclosed herein combined with a VH domain disclosed
herein, or other VH domain.
[0129] The present invention encompasses Fc variants that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3, said
antibodies comprising a VH CDR having an amino acid sequence of any
one of the VH CDRs listed in Table 3 infra. The present invention
also encompasses Fc variants that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3 said antibodies comprising a VL
CDR having an amino acid sequence of any one of the VL CDRs listed
in Table 3 infra. The present invention also encompasses Fc
variants that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3, said Fc variants comprising one or more
VH CDRs and one or more VL CDRs listed in Table 3. The present
invention further encompasses Fc variants that immunospecifically
binds to Integrin .alpha..sub.v.beta..sub.3 and Fc variants
comprising any combination of some or all of the VH CDRs and VL
CDRs listed in Table 3 infra. TABLE-US-00003 TABLE 3 CDR Sequences
Of LM609 and Vitaxin .RTM. CDR Sequence SEQ ID NO: LM609 VH1 SYDMS
5 LM609 VH2 KVSSGGGSTYYLDTVQG 6 LM609 VH3 HNYGSFAY 7 LM609 VL1
QASQSISNHLH 8 LM609 VL2 YRSQSIS 9 LM609 VL3 QQSGSWPHT 10 Vitaxin
.RTM. VH1 SYDMS 70 Vitaxin .RTM. VH2 KVSSGGGSTYYLDTVQG 71 Vitaxin
.RTM. VH3 HLHGSFAS 72 Vitaxin .RTM. VL1 QASQSISNFLH 73 Vitaxin
.RTM. VL2 TRSQSIS 74 Vitaxin .RTM. VL3 QQSGSYPLT 75
[0130] The present invention also encompasses Fc variants that
compete with Vitaxin.RTM., LM609 or CNTO 95 or an antigen-binding
fragment thereof for binding to Integrin .alpha..sub.v.beta..sub.3.
Competition assays, which can be used to identify such antibodies,
are well known to one skilled in the art. In a particular
embodiment, 1 .mu.g/ml of an antibody of the invention prevents
75%, 80%, 85% or 90% of ORIGEN TAG labeled LM609, Vitaxin.RTM. or
CNTO 95 from binding to biotin-labeled Integrin
.alpha..sub.v.beta..sub.3 as measured by well-known ORIGEN
analysis. In another embodiment, the invention encompasses Fc
variants of antibodies other than those disclosed in WO 98/40488
that compete with Vitaxin.RTM., LM609 or an antigen-binding
fragment thereof for binding to Integrin
.alpha..sub.v.beta..sub.3.
[0131] The present invention also provides Fc variants that
comprise a framework region known to those of skill in the art. In
one embodiment, the fragment region of an antibody of the invention
or fragment thereof is human or humanized. In a specific
embodiment, an Fc variant of the invention comprises the framework
region of Vitaxin.RTM. and/or one or more CDRs from Vitaxin.RTM.
(Table 3 supra).
[0132] The present invention encompasses Fc variants comprising the
amino acid sequence of Vitaxin.RTM. with mutations (e.g., one or
more amino acid substitutions) in the framework or variable regions
in addition to any other substitutions or changes (e.g., Fc
substitution(s) as described supra). In one embodiment, mutations
in these antibodies maintain or enhance the avidity and/or affinity
of the antibodies for the Integrin .alpha..sub.v.beta..sub.3 to
which they immunospecifically bind. Standard techniques known to
those skilled in the art (e.g., immunoassays) can be used to assay
the affinity of an antibody for a particular antigen.
[0133] The present invention encompasses the use of a nucleic acid
molecule(s), generally isolated, encoding an Fc variant that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3. In
a specific embodiment, an isolated nucleic acid molecule encodes an
Fc variant that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3, said Fc variant having the amino acid
sequence of LM609 or Vitaxin.RTM. containing one or more Fc
substitution (e.g. supra). In another embodiment, an isolated
nucleic acid molecule encodes an Fc variant that
immuno-specifically binds to Integrin .alpha..sub.v.beta..sub.3
said Fc variant comprising a VH domain having the amino acid
sequence of the VH domain of LM609 or Vitaxin.RTM.. In another
embodiment, an isolated nucleic acid molecule encodes an Fc variant
that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3, said antibody comprising a VL domain
having the amino acid sequence of the VL domain of LM609 or
Vitaxin.RTM..
[0134] The invention encompasses the use of an isolated nucleic
acid molecule encoding an Fc variant that immunospecifically binds
to Integrin .alpha..sub.v.beta..sub.3, said Fc variant comprising a
VH CDR having the amino acid sequence of any of the VH CDRs listed
in Table 3, supra. In particular, the invention encompasses the use
of an isolated nucleic acid molecule encoding an Fc variant that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 said
antibody comprising one, two, or more VH CDRs having the amino acid
sequence of any of the VH CDRs listed in Table 3, supra.
[0135] The present invention encompasses the use of an isolated
nucleic acid molecule encoding an Fc variant that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3,
said Fc variant comprising a VL CDR having an amino acid sequence
of any of the VL CDRs listed in Table 3, supra. In particular, the
invention encompasses the use of an isolated nucleic acid molecule
encoding an Fc variant that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3, said antibody comprising one, two or
more VL CDRs having the amino acid sequence of any of the VL CDRs
listed in Table 3, supra.
[0136] The present invention encompasses the use of Fc variants
that immuno-specifically bind to Integrin
.alpha..sub.v.beta..sub.3, Fc variants comprising derivatives of
the VH domains, VH CDRs, VL domains, or VL CDRs described herein
that immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3.
Standard techniques known to those of skill in the art can be used
to introduce mutations (e.g., additions, deletions, and/or
substitutions) in the nucleotide sequence encoding an antibody of
the invention, including, for example, site-directed mutagenesis
and PCR-mediated mutagenesis are routinely used to generate amino
acid substitutions. In one embodiment, the VH and/or VL CDRs
derivatives include less than 25 amino acid substitutions, less
than 20 amino acid substitutions, less than 15 amino acid
substitutions, less than 10 amino acid substitutions, less than 5
amino acid substitutions, less than 4 amino acid substitutions,
less than 3 amino acid substitutions, or less than 2 amino acid
substitutions in the relative to the original VH and/or VL CDRs. In
another embodiment, the VH and/or VL CDRs derivatives have
conservative amino acid substitutions (e.g. supra) are made at one
or more predicted non-essential amino acid residues (i.e., amino
acid residues which are not critical for the antibody to
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3).
Alternatively, mutations can be introduced randomly along all or
part of the VH and/or VL CDR coding sequence, such as by saturation
mutagenesis, and the resultant mutants can be screened for
biological activity to identify mutants that retain activity.
Following mutagenesis, the encoded antibody can be expressed and
the activity of the antibody can be determined.
[0137] The present invention encompasses Fc variants of LM609 or
Vitaxin.RTM. with one or more additional amino acid residue
substitutions in the variable light (VL) domain and/or variable
heavy (VH) domain. The present invention also encompasses Fc
variants of LM609 or Vitaxin.RTM. with one or more additional amino
acid residue substitutions in one or more VL CDRs and/or one or
more VH CDRs. The antibody generated by introducing substitutions
in the VH domain, VH CDRs, VL domain and/or VL CDRs of an Fc
variant of LM609 or Vitaxin.RTM. can be tested in vitro and in
vivo, for example, for its ability to bind to Integrin
.alpha..sub.v.beta..sub.3 and/or Fc.gamma.Rs (by, e.g.,
immunoassays including, but not limited to ELISAs and BIAcore), or
for its ability to mediate ADCC, prevent, treat, manage or
ameliorate cancer or one or more symptoms thereof.
[0138] The present invention also encompasses the use of Fc
variants that immuno-specifically bind to Integrin
.alpha..sub.v.beta..sub.3 or a fragment thereof, said Fc variants
comprising an amino acid sequence of a variable heavy chain and/or
variable light chain that is at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least
99% identical to the amino acid sequence of the variable heavy
chain and/or light chain of Vitaxin.RTM. (i.e., SEQ ID NO:3 and/or
SEQ ID NO:4). The present invention further encompasses the use of
Fc variants that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 or a fragment thereof, said antibodies or
antibody fragments comprising an amino acid sequence of one or more
CDRs that is at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical to
the amino acid sequence of one or more CDRs of Vitaxin.RTM.. The
determination of percent identity of two amino acid sequences can
be determined by any method known to one skilled in the art,
including BLAST protein searches.
[0139] The present invention also encompasses the use of Fc
variants that immuno-specifically bind to Integrin
.alpha..sub.v.beta..sub.3 or fragments thereof, where said Fc
variants are encoded by a nucleotide sequence that hybridizes to
the nucleotide sequence of Vitaxin.RTM. (i.e., SEQ ID NO: 1 and/or
SEQ ID NO: 2) under stringent conditions. In another embodiment,
the invention encompasses Fc variants that immunospecifically bind
to Integrin .alpha..sub.v.beta..sub.3 or a fragment thereof, said
Fc variants comprising one or more CDRs encoded by a nucleotide
sequence that hybridizes under stringent conditions to the
nucleotide sequence of one or more CDRs of Vitaxin.RTM.. Stringent
hybridization conditions include, but are not limited to,
hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., highly stringent conditions such as hybridization to
filter-bound DNA in 6.times.SSC at about 45.degree. C. followed by
one or more washes in 0.1.times.SSC/0.2% SDS at about 60.degree.
C., or any other stringent hybridization conditions known to those
skilled in the art (see, for example, Ausubel, F. M. et al., eds.
1989 Current Protocols in Molecular Biology, vol. 1, Green
Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at
pages 6.3.1 to 6.3.6 and 2.10.3).
[0140] Set forth below, is a more detailed description of the
antibodies encompassed within the various aspects of the
invention.
6.2 Antibodies of the Invention
[0141] Fc variants of the invention may include, but are not
limited to, synthetic antibodies, monoclonal antibodies,
oligoclonal antibodies, recombinantly produced antibodies,
intrabodies, multispecific antibodies, bispecific antibodies, human
antibodies, humanized antibodies, chimeric antibodies, synthetic
antibodies, single-chain FvFcs (scFvFc), single-chain Fvs (scFv),
and anti-idiotypic (anti-Id) antibodies. In particular, antibodies
used in the methods of the present invention include immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules. The antibodies of the invention can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA, and IgA.sub.2) or subclass of
immunoglobulin molecule.
[0142] Fc variants of the invention may be from any animal origin
including birds and mammals (e.g., human, murine, donkey, sheep,
rabbit, goat, guinea pig, camel, horse, or chicken). In one
embodiment, the antibodies are human or humanized monoclonal
antibodies. As used herein, "human" antibodies include antibodies
having the amino acid sequence of a human immunoglobulin and
include antibodies isolated from human immunoglobulin libraries or
from mice that express antibodies from human genes.
[0143] Antibodies like all polypeptides have an Isoelectric Point
(pI), which is generally defined as the pH at which a polypeptide
carries no net charge. It is known in the art that protein
solubility is typically lowest when the pH of the solution is equal
to the isoelectric point (pI) of the protein. It is possible to
optimize solubility by altering the number and location of
ionizable residues in the antibody to adjust the pI. For example
the pI of a polypeptide can be manipulated by making the
appropriate amino acid substitutions (e.g., by substituting a
charged amino acid such as a lysine, for an uncharged residue such
as alanine). Without wishing to be bound by any particular theory,
amino acid substitutions of an antibody that result in changes of
the pI of said antibody may improve solubility and/or the stability
of the antibody. One skilled in the art would understand which
amino acid substitutions would be most appropriate for a particular
antibody to achieve a desired pI. The pI of a protein may be
determined by a variety of methods including but not limited to,
isoelectric focusing and various computer algorithms (see for
example Bjellqvist et al., 1993, Electrophoresis 14:1023-1031). In
one embodiment, the pI of the Fc variants of the invention is
higher then about 6.5, about 7.0, about 7.5, about 8.0, about 8.5,
or about 9.0. In one embodiment, substitutions resulting in
alterations in the pI of the Fc variant of the invention will not
significantly diminish its binding affinity for Integrin
.alpha..sub.v.beta..sub.3. In another embodiment, the pI of the Fc
variants of the invention is higher then 6.5, 7.0, 7.5, 8.0, 8.5,
or 9.0. It is specifically contemplated that the substitution(s) of
the Fc region that result in altered binding to Fc.gamma.R
(described supra) may also result in a change in the pI. In another
embodiment, substitution(s) of the Fc region are specifically
chosen to effect both the desired alteration in Fc.gamma.R binding
and any desired change in pI. As used herein the pI value is
defined as the pI of the predominant charge form. The pI of a
protein may be determined by a variety of methods including but not
limited to, isoelectric focusing and various computer algorithms
(see, e.g., Bjellqvist et al., 1993, Electrophoresis 14:1023).
[0144] The Tm of the Fab domain of an antibody, can be a good
indicator of the thermal stability of an antibody and may further
provide an indication of the shelf-life. A lower Tm indicates more
aggregation/less stability, whereas a higher Tm indicates less
aggregation/more stability. Thus, antibodies having higher Tm are
preferable. In one embodiment, the Fab domain of an Fc variant has
a Tm value higher than at least 50.degree. C., 55.degree. C.,
60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C.,
80.degree. C., 85.degree. C., 90.degree. C., 95.degree. C.,
100.degree. C., 105.degree. C., 1101C, 115.degree. C. or
120.degree. C. Thermal melting temperatures (Tm) of a protein
domain (e.g., a Fab domain) can be measured using any standard
method known in the art, for example, by differential scanning
calorimetry (see, e.g., Vermeer et al., 2000, Biophys. J.
78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154).
[0145] Fc variants of the invention may be monospecific,
bispecific, trispecific or have greater multispecificity.
Multispecific antibodies may immunospecifically bind to different
epitopes of desired target molecule or may immunospecifically bind
to both the target molecule as well as a heterologous epitope, such
as a heterologous polypeptide or solid support material. See, e.g.,
International Publication Nos. WO 94/04690; WO 93/17715; WO
92/08802; WO 91/00360; and WO 92/05793; Tutt, et al., 1991, J.
Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648,
5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol.
148:1547-1553). In the present case, one of the binding
specificities is for Integrin .alpha..sub.v.beta..sub.3, the other
one is for any other antigen (i.e., another integrin, a signaling
or effector molecule).
[0146] Multispecific antibodies have binding specificities for at
least two different antigens. While such molecules normally will
only bind two antigens (i.e. bispecific antibodies, BsAbs),
antibodies with additional specificities such as trispecific
antibodies are encompassed by the instant invention. Examples of
BsAbs include without limitation those with one arm directed
against a Integrin .alpha..sub.v.beta..sub.3 and the other arm
directed against any other antigen. Methods for making bispecific
antibodies are known in the art. Traditional production of
full-length bispecific antibodies is based on the coexpression of
two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Millstein et al., 1983,
Nature, 305:537-539 which is incorporated herein by reference in
its entirety). Because of the random assortment of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a
potential mixture of different antibody molecules, of which only
one has the correct bispecific structure. Purification of the
correct molecule, which is usually done by affinity chromatography
steps, is rather cumbersome, and the product yields are low.
Similar procedures are disclosed in WO 93/08829, and in Traunecker
et al., 1991, EMBO J., 10:3655-3659. A more directed approach is
the generation of a Di-diabody a tetravalent bispecific antbodiy.
Methods for producing a Di-diabody are known in the art (see e.g.,
Lu et al., 2003, J Immunol Methods 279:219-32; Marvin et al., 2005,
Acta Pharmacolical Sinica 26:649).
[0147] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when, the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0148] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm (e.g., Integrin
.alpha..sub.v.beta..sub.3), and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. It was found that this asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690 (incorporated herein by reference in its
entirety). For further details of generating bispecific antibodies
see, for example, Suresh et al., 1986, Methods in Enzymology,
121:210 (incorporated herein by reference in its entirety).
According to another approach described in WO96/27011 (incorporated
herein by reference in its entirety), a pair of antibody molecules
can be engineered to maximize the percentage of heterodimers which
are recovered from recombinant cell culture. The preferred
interface comprises at least a part of the CH3 domain of an
antibody constant domain. In this method, one or more small amino
acid side chains from the interface of the first antibody molecule
are replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large
side chain(s) are created on the interface of the second antibody
molecule by replacing large amino acid side chains with smaller
ones (e.g. alanine or threonine). This provides a mechanism for
increasing the yield of the heterodimer over other unwanted
end-products such as homodimers.
[0149] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089) The above referencecs are each incorporated herein by
reference in their entireties. Heteroconjugate antibodies may be
made using any convenient cross-linking methods. Suitable
cross-linking agents are well known in the art, and are disclosed
in U.S. Pat. No. 4,676,980, along with a number of cross-linking
techniques. Each of the above references is incorporated herein by
reference in its entirety.
[0150] Antibodies with more than two valencies incorporating at
least one hinge modification of the invention are contemplated. For
example, trispecific antibodies can be prepared. See, e.g., Tutt et
al. J. Immunol. 147: 60 (1991), which is incorporated herein by
reference.
[0151] The Fc variants of the invention encompass single domain
antibodies, including camelized single domain antibodies (see e.g.,
Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et
al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans,
1999, J. Immunol. Meth. 231:25; International Publication Nos. WO
94/04678 and WO 94/25591; U.S. Pat. No. 6,005,079; which are
incorporated herein by reference in their entireties).
[0152] Other antibodies specifically contemplated are "oligoclonal"
antibodies. As used herein, the term "oligoclonal" antibodies"
refers to a predetermined mixture of distinct monoclonal
antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos.
5,789,208 and 6,335,163 which are incorporated by reference herein.
In one embodiment, oligoclonal antibodies consist of a
predetermined mixture of antibodies against one or more epitopes
are generated in a single cell. In another embodiment, oligoclonal
antibodies comprise a plurality of heavy chains capable of pairing
with a common light chain to generate antibodies with multiple
specificities (e.g., PCT publication WO 04/009618 which is
incorporated by reference herein). Oligoclonal antibodies are
particularly useful when it is desired to target multiple epitopes
on a single target molecule (e.g., Integrin
.alpha..sub.v.beta..sub.3). Those skilled in the art will know or
can determine what type of antibody or mixture of antibodies is
applicable for an intended purpose and desired need.
[0153] Antibodies of the present invention also encompass Fc
variants that have half-lives (e.g., serum half-lives) in a mammal,
(e.g., a human), of greater than 5 days, greater than 10 days,
greater than 15 days, greater than 20 days, greater than 25 days,
greater than 30 days, greater than 35 days, greater than 40 days,
greater than 45 days, greater than 2 months, greater than 3 months,
greater than 4 months, or greater than 5 months. The increased
half-lives of the antibodies of the present invention in a mammal,
(e.g., a human), results in a higher serum titer of said antibodies
or antibody fragments in the mammal, and thus, reduces the
frequency of the administration of said antibodies or antibody
fragments and/or reduces the concentration of said antibodies or
antibody fragments to be administered. Antibodies having increased
in vivo half-lives can be generated by techniques known to those of
skill in the art. For example, antibodies with increased in vivo
half-lives can be generated by modifying (e.g., substituting,
deleting or adding) amino acid residues identified as involved in
the interaction between the Fc domain and the FcRn receptor (see,
e.g., International Publication Nos. WO 97/34631; WO 04/029207;
U.S. Pat. No. 6,737056 and U.S. Patent Publication No.
2003/0190311, each of which are incorporated herein by reference in
their entireties).
[0154] In one embodiment, the Fc variants of the invention may be
chemically modified (e.g., one or more chemical moieties can be
attached to the antibody) or be modified to alter its
glycosylation, again to alter one or more functional properties of
the antibody.
[0155] In still another embodiment, the glycosylation of the Fc
variants of the invention is modified. For example, an aglycoslated
antibody can be made (i.e., the antibody lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity
of the antibody for a target antigen. Such carbohydrate
modifications can be accomplished by, for example, altering one or
more sites of glycosylation within the antibody sequence. For
example, one or more amino acid substitutions can be made that
result in elimination of one or more variable region framework
glycosylation sites to thereby eliminate glycosylation at that
site. Such aglycosylation may increase the affinity of the antibody
for antigen. Such an approach is described in further detail in
U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which is
incorporated herein by reference in its entirety.
[0156] Additionally or alternatively, an Fc variant can be made
that has an altered type of glycosylation, such as a
hypofucosylated antibody having reduced amounts of fucosyl residues
or an antibody having increased bisecting GlcNAc structures. Such
altered glycosylation patterns have been demonstrated to increase
the ADCC ability of antibodies. Such carbohydrate modifications can
be accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. See, for example, Shields, R. L. et al. (2002) J.
Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech.
17:176-1, as well as, European Patent No: EP 1,176,195; PCT
Publications WO 03/035835; WO 99/54342, each of which is
incorporated herein by reference in its entirety.
[0157] In still another embodiment, the glycosylation of an Fc
variant of the invention is modified. For example, an aglycoslated
antibody can be made (i.e., the antibody lacks glycosylation).
Glycosylation can be altered to, for example, increase the affinity
of the antibody for a target antigen. Such carbohydrate
modifications can be accomplished by, for example, altering one or
more sites of glycosylation within the antibody sequence. For
example, one or more amino acid substitutions can be made that
result in elimination of one or more variable region framework
glycosylation sites to thereby eliminate glycosylation at that
site. Such aglycosylation may increase the affinity of the antibody
for antigen. Such an approach is described in further detail in
U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which is
incorporated herein by reference in its entirety.
[0158] Additionally or alternatively, an Fc variant can be made
that has an altered type of glycosylation, such as a
hypofucosylated Fc variant having reduced amounts of fucosyl
residues or an Fc variant having increased bisecting GlcNAc
structures. Such altered glycosylation patterns have been
demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example,
expressing the antibody in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibodies of the invention to thereby produce
an antibody with altered glycosylation. See, for example, Shields,
R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.
(1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP
1,176,195; PCT Publications WO 03/035835; WO 99/54342, each of
which is incorporated herein by reference in its entirety.
6.3 Antibody Conjugates and Derivatives
[0159] Fc variants of the invention include derivatives that are
modified (i.e., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment). 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, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to, specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0160] Antibodies or fragments thereof with increased in vivo
half-lives can be generated by attaching to said antibodies or
antibody fragments polymer molecules such as high molecular weight
polyethyleneglycol (PEG). PEG can be attached to said antibodies or
antibody fragments with or without a multifunctional linker either
through site-specific conjugation of the PEG to the N- or
C-terminus of said antibodies or antibody fragments or via
epsilon-amino groups present on lysine residues. Linear or branched
polymer derivatization that results in minimal loss of biological
activity will be used. The degree of conjugation will be closely
monitored by SDS-PAGE and mass spectrometry to ensure proper
conjugation of PEG molecules to the antibodies. Unreacted PEG can
be separated from antibody-PEG conjugates by, e.g., size exclusion
or ion-exchange chromatography.
[0161] Further, antibodies can be conjugated to albumin in order to
make the antibody or antibody fragment more stable in vivo or have
a longer half life in vivo. The techniques are well known in the
art, see e.g., International Publication Nos. WO 93/15199, WO
93/15200, and WO 01/77137; and European Patent No. EP 413,622, all
of which are incorporated herein by reference. The present
invention encompasses the use of antibodies or fragments thereof
conjugated or fused to one or more moieties, including but not
limited to, peptides, polypeptides, proteins, fusion proteins,
nucleic acid molecules, small molecules, mimetic agents, synthetic
drugs, inorganic molecules, and organic molecules.
[0162] The present invention encompasses the use of antibodies or
fragments thereof recombinantly fused or chemically conjugated
(including both covalent and non-covalent conjugations) to a
heterologous protein or polypeptide (or fragment thereof,
preferably to a polypeptide of at least 10, at least 20, at least
30, at least 40, at least 50, at least 60, at least 70, at least
80, at least 90 or at least 100 amino acids) to generate fusion
proteins. The fusion does not necessarily need to be direct, but
may occur through linker sequences. For example, antibodies may be
used to target heterologous polypeptides to particular cell types,
either in vitro or in vivo, by fusing or conjugating the antibodies
to antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to heterologous polypeptides may
also be used in in vitro immunoassays and purification methods
using methods known in the art. See e.g., International publication
No. WO 93/21232; European Patent No. EP 439,095; Naramura et al.,
1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et
al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol.
146:2446-2452, which are incorporated by reference in their
entireties.
[0163] The present invention further includes formulations
comprising heterologous proteins, peptides or polypeptides fused or
conjugated to antibody fragments. For example, the heterologous
polypeptides may be fused or conjugated to a Fab fragment, Fd
fragment, Fv fragment, F(ab).sub.2 fragment, a VH domain, a VL
domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing
or conjugating polypeptides to antibody portions are well known in
the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP
307,434 and EP 367,166; International publication Nos. WO 96/04388
and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA
88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and
Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (said
references incorporated by reference in their entireties).
[0164] Additional fusion proteins, e.g., of Vitaxin.RTM. or other
anti-integrin .alpha..sub.v.beta..sub.3 antibodies, may be
generated through the techniques of gene-shuffling,
motif-shuffling, exon-shuffling, and/or codon-shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be
employed to alter the activities of antibodies of the invention or
fragments thereof (e.g., antibodies or fragments 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., 1997, Curr. Opinion Biotechnol.
8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82; Hansson,
et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco,
1998, Biotechniques 24(2): 308-313 (each of these patents and
publications are hereby incorporated by reference in its entirety).
Antibodies or fragments thereof, or the encoded antibodies or
fragments thereof, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. One or more portions of a
polynucleotide encoding an antibody or antibody fragment, which
portions immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules.
[0165] Moreover, the antibodies or fragments thereof can be fused
to marker sequences, such as a peptide to facilitate purification.
In specific embodiments, the marker amino acid sequence is a
hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among
others, many of which are commercially available. As described in
Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Other peptide tags useful for purification
include, but are not limited to, the hemagglutinin "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., 1984, Cell 37:767) and the "flag" tag.
[0166] In other embodiments, Fc variants of the present invention
or analogs or derivatives thereof are conjugated to a diagnostic or
detectable agent. Such antibodies can be useful for monitoring or
prognosing the development or progression of a cancer as part of a
clinical testing procedure, such as determining the efficacy of a
particular therapy. Such diagnosis and detection can be
accomplished by coupling the antibody to detectable substances
including, but not limited to various enzymes, such as but not
limited to horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; prosthetic groups,
such as but not limited to streptavidin/biotin and avidin/biotin;
fluorescent materials, such as but not limited to, umbelliferone,
fluorescein, fluorescein isothiocynate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such as
but not limited to iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I,), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115 In, .sup.113In, .sup.112In,
.sup.111In,), and technetium (.sup.99Tc), thallium (.sup.201Ti),
gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149 Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re, .sup.142
Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn,
.sup.75Se, .sup.113Sn, and .sup.117Tin; positron emitting metals
using various positron emission tomographies, noradioactive
paramagnetic metal ions, and molecules that are radiolabelled or
conjugated to specific radioisotopes.
[0167] The present invention further encompasses uses of Fc
variants of the invention or fragments thereof conjugated to a
therapeutic agent.
[0168] In other embodiments, Fc variants of the invention may be
conjugated to a therapeutic moiety such as a cytotoxin, e.g., a
cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent
includes any agent that is detrimental to cells. Examples include
paclitaxel, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide
and analogs or homologs thereof. Therapeutic agents include, but
are not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin), antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),
anti-mitotic agents (e.g., vincristine and vinblastine), and
auristatin E compounds (e.g. monomethyl auristatin E; see for
example U.S. Pat. No. 6,884,869). A more extensive list of
therapeutic moieties can be found in PCT publications WO
03/075957;
[0169] In other embodiments, Fc variants of the invention may be
conjugated to a therapeutic agent or drug moiety that modifies a
given biological response. Therapeutic agents or drug moieties are
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A, Onconase
(or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin,
or diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, an
apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I (see,
International Publication No. WO 97/33899), AIM II (see,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., 1994, J. Immunol., 6:1567), and VEGI (see, International
Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent, e.g., angiostatin or endostatin; or, a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF")), or a growth factor (e.g., growth hormone ("GH")).
[0170] In other embodiments, Fc variants of the invention can be
conjugated to therapeutic moieties such as a radioactive materials
or macrocyclic chelators useful for conjugating radiometal ions
(see above for examples of radioactive materials). In certain
embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA)
which can be attached to the antibody via a linker molecule. Such
linker molecules are commonly known in the art and described in
Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al.,
1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl.
Med. Biol. 26:943-50 each incorporated by reference in their
entireties.
[0171] Techniques for conjugating therapeutic moieties to
antibodies are well known. Moieties can be conjugated to antibodies
by any method known in the art, including, but not limited to
aldehyde/Schiff linkage, sulphydryl linkage, acid-labile linkage,
cis-aconityl linkage, hydrazone linkage, enzymatically degradable
linkage (see generally Garnett, 2002, Adv Drug Deliv Rev
53:171-216). Techniques for conjugating therapeutic moieties to
antibodies are well known, see, 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); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol.
Rev. 62:119-58.
[0172] Methods for fusing or conjugating antibodies to polypeptide
moieties are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;
5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP
307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570;
Ashkenazi et al., 1991, PNAS USA 88:10535-10539; Zheng et al.,
1995, J Immunol 154:5590-5600; and Vil et al., 1992, PNAS USA
89:11337-11341. The fusion of an antibody to a moiety does not
necessarily need to be direct, but may occur through linker
sequences. Such linker molecules are commonly known in the art and
described in Denardo et al., 1998, Clin Cancer Res 4:2483-90;
Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al.,
1999, Nucl Med Biol 26:943-50; Garnett, 2002, Adv Drug Deliv Rev
53:171-216, each of which is incorporated herein by reference in
its entirety.
[0173] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0174] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0175] The therapeutic moiety or drug conjugated to an antibody or
fragment thereof that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 should be chosen to achieve the desired
prophylactic or therapeutic effect(s) for a particular disorder in
a subject. A clinician or other medical personnel should consider
the following when deciding on which therapeutic moiety or drug to
conjugate to an antibody or fragment thereof that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3: the
nature of the disease, the severity of the disease, and the
condition of the subject.
6.4 Methods Of Generating Antibodies
[0176] The Fc variants of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or by recombinant expression
techniques.
[0177] Polyclonal antibodies to Integrin .alpha..sub.v.beta..sub.3
can be produced by various procedures well known in the art. For
example, Integrin .alpha..sub.v.beta..sub.3 or immunogenic
fragments thereof can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce
the production of sera containing polyclonal antibodies specific
for Integrin .alpha..sub.v.beta..sub.3. Various adjuvants may be
used to increase the immunological response, depending on the host
species, and include but are not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants are also well known in the art.
[0178] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0179] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with Integrin
.alpha..sub.v.beta..sub.3 or a domain thereof (e.g., the
extracellular domain) and once an immune response is detected,
e.g., antibodies specific for Integrin .alpha..sub.v.beta..sub.3
are detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Additionally, a RIMMS
(repetitive immunization, multiple sites) technique can be used to
immunize an animal (Kilpatrick et al., 1997, Hybridoma 16:381-9,
incorporated herein by reference in its entirety). Hybridomas are
selected and cloned by limited dilution. The hybridoma clones are
then assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide of the invention.
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0180] Accordingly, monoclonal antibodies can be generated by
culturing a hybridoma cell secreting an antibody wherein,
preferably, the hybridoma is generated by fusing splenocytes
isolated from a mouse immunized with Integrin
.alpha..sub.v.beta..sub.3 or immunogenic fragments thereof, with
myeloma cells and then screening the hybridomas resulting from the
fusion for hybridoma clones that secrete an antibody able to bind
Integrin .alpha..sub.v.beta..sub.3.
[0181] The Fc variants of the invention contain novel amino acid
residues in their Fc regions. Fc variants can be generated by
numerous methods well known to one skilled in the art. Non-limiting
examples include, isolating antibody coding regions (e.g., from
hybridoma) and making one or more desired substitutions in the Fc
region of the isolated antibody coding region. Alternatively, the
variable regions may be subcloned into a vector encoding an Fc
region comprising one or more high effector function amino acid
residues. Additional methods and details are provided below.
[0182] Antibody fragments that recognize specific Integrin
.alpha..sub.v.beta..sub.3 epitopes may be generated by any
technique known to those of skill in the art. For example, Fab and
F(ab')2 fragments of the invention may be produced by proteolytic
cleavage of immunoglobulin molecules, using enzymes such as papain
(to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). F(ab')2 fragments contain the variable region, the
light chain constant region and the CH1 domain of the heavy chain.
Further, the antibodies of the present invention can also be
generated using various phage display methods known in the art.
[0183] In phage display methods, functional antibody domains are
displayed on the surface of phage particles that carry the
polynucleotide sequences encoding them. In particular, DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of lymphoid
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is
electroporated in E. coli and the E. coli is infected with helper
phage. Phage used in these methods are typically filamentous phage
including fd and M13 and the VH and VL domains are usually
recombinantly fused to either the phage gene III or gene VIII.
Phage expressing an antigen binding domain that binds to the
Integrin .alpha..sub.v.beta..sub.3 epitope of interest can be
selected or identified with antigen, e.g., using labeled antigen or
antigen bound or captured to a solid surface or bead. Examples of
phage display methods that can be used to make the antibodies of
the present invention include those disclosed in Brinkman et al.,
1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.
24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,
1994, Advances in Immunology 57:191-280; PCT Publication Nos. WO
90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO
95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.
5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,
5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,
5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
[0184] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
International Publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6): 864-869; Sawai et al., 1995, AJRI 34:26-34;
and Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0185] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma constant, and the PCR amplified VL domains can be
cloned into vectors expressing a VL constant region, e.g., human
kappa or lamba constant regions. In one embodiment, the constant
region is an Fc region containing at least one high effector
function amino acid. In a specific embodiment, the vectors for
expressing the VH or VL domains comprise a promoter, a secretion
signal, a cloning site for both the variable and constant domains,
as well as a selection marker such as neomycin. The VH and VL
domains may also be cloned into one vector expressing the desired
constant regions. The heavy chain conversion vectors and light
chain conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express fill-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art.
[0186] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. Methods for producing chimeric antibodies are known in
the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,8
16397, and 6,311,415, which are incorporated herein by reference in
their entirety.
[0187] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use human or
chimeric antibodies. Completely human antibodies are particularly
desirable for therapeutic treatment of human subjects. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See also
U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO
98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO
96/33735, and WO 91/10741; each of which is incorporated herein by
reference in its entirety.
[0188] A humanized antibody is an antibody or its variant or
fragment thereof which is capable of binding to a predetermined
antigen and which comprises a framework region having substantially
the amino acid sequence of a human immunoglobulin and a CDR having
substantially the amino acid sequence of a non-human
immunoglobulin. A humanized antibody comprises substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, Fabc, Fv) in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin
(i.e., donor antibody) and all or substantially all of the
framework regions are those of a human immunoglobulin consensus
sequence. In one embodiment, a humanized antibody also comprises at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. Ordinarily, the antibody
will contain both the light chain as well as at least the variable
domain of a heavy chain. The antibody also may include the CH1,
hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized
antibody can be selected from any class of immunoglobulins,
including IgM, IgG, IgD, IgA and IgE, and any isotype, including
IgG1, IgG2, IgG3 and IgG4. Usually the constant domain is a
complement fixing constant domain where it is desired that the
humanized antibody exhibit cytotoxic activity, and the class is
typically IgG.sub. 1. Where such cytotoxic activity is not
desirable, the constant domain may be of the IgG.sub.2 class. The
humanized antibody may comprise sequences from more than one class
or isotype, and selecting particular constant domains to optimize
desired effector functions is within the ordinary skill in the art.
The framework and CDR regions of a humanized antibody need not
correspond precisely to the parental sequences, e.g., the donor CDR
or the consensus framework may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or
framework residue at that site does not correspond to either the
consensus or the import antibody. Such mutations, however, will not
be extensive. Usually, at least 75% of the humanized antibody
residues will correspond to those of the parental framework region
(FR) and CDR sequences, more often 90%, and most preferably greater
than 95%. Humanized antibody can be produced using variety of
techniques known in the art, including but not limited to,
CDR-grafting (European Patent No. EP 239,400; International
Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,
5,530,101, and 5,585,089), veneering or resurfacing (European
Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular
Immunology 28(4/5): 489-498; Studnicka et al., 1994, Protein
Engineering 7(6): 805-814; and Roguska et al., 1994, PNAS
91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and
techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat.
No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119-25
(2002), Caldas et al., Protein Eng. 13(5): 353-60 (2000), Morea et
al., Methods 20(3): 267-79 (2000), Baca et al., J. Biol. Chem.
272(16): 10678-84 (1997), Roguska et al., Protein Eng. 9(10):
895-904 (1996), Couto et al., Cancer Res. 55 (23 Supp): 5973s-5977s
(1995), Couto et al., Cancer Res. 55(8): 1717-22 (1995), Sandhu J
S, Gene 150(2): 409-10 (1994), and Pedersen et al., J. Mol. Biol.
235(3): 959-73 (1994). Often, framework residues in the framework
regions will be substituted with the corresponding residue from the
CDR donor antibody to alter, preferably improve, antigen binding.
These framework substitutions are identified by methods well known
in the art, e.g., by modeling of the interactions of the CDR and
framework residues to identify framework residues important for
antigen binding and sequence comparison to identify unusual
framework residues at particular positions. (See, e.g., Queen et
al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature
332:323, which are incorporated herein by reference in their
entireties.)
[0189] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., Integrin .alpha..sub.v.beta..sub.3 or immunogenic fragments
thereof. Monoclonal antibodies directed against the antigen can be
obtained from the immunized, transgenic mice using conventional
hybridoma technology. The human immunoglobulin transgenes harbored
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
International Publication Nos. WO 98/24893, WO 96/34096, and WO
96/33735; and 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, and 5,939,598, which
are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.),
Genpharm (San Jose, Calif.) and Medarex (Princeton, N.J.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0190] Further, the antibodies of the invention can, in turn, be
utilized to generate anti-idiotype antibodies that "mimic" Integrin
.alpha..sub.v.beta..sub.3 using techniques well known to those
skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB
J. 7(5): 437-444; and Nissinoff, 1991, J. Immunol. 147(8):
2429-2438). For example, antibodies of the invention which bind to
and competitively inhibit the binding of Integrin
.alpha..sub.v.beta..sub.3 (as determined by assays well known in
the art and disclosed infra) to its ligands can be used to generate
anti-idiotypes that "mimic" Integrin .alpha..sub.v.beta..sub.3
binding domains and, as a consequence, bind to and neutralize
Integrin .alpha..sub.v.beta..sub.3 and/or its ligands. Such
neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes
can be used in therapeutic regimens to neutralize Integrin
.alpha..sub.v.beta..sub.3. The invention provides methods employing
the use of polynucleotides comprising a nucleotide sequence
encoding an antibody of the invention or a fragment thereof.
[0191] In one embodiment, the nucleotide sequence encoding an
antibody that immunospecifically binds Integrin
.alpha..sub.v.beta..sub.3 is obtained and used to generate the Fc
variants of the invention. The nucleotide sequence can be obtained
from sequencing hybridoma clone DNA. If a clone containing a
nucleic acid encoding a particular antibody or an epitope-binding
fragment thereof is not available, but the sequence of the antibody
molecule or epitope-binding fragment thereof is known, a nucleic
acid encoding the immunoglobulin may be chemically synthesized or
obtained from a suitable source (e.g., an antibody cDNA library, or
a cDNA library generated from, or nucleic acid, preferably poly A+
RNA, isolated from any tissue or cells expressing the antibody,
such as hybridoma cells selected to express an antibody) by PCR
amplification using synthetic primers that hybridize to the 3' and
5' ends of the sequence or by cloning using an oligonucleotide
probe specific for the particular gene sequence to identify, e.g.,
a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be cloned into
replicable cloning vectors using any method well known in the
art.
[0192] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, Or example, the techniques described
in Current Protocols in Molecular Biology, F. M. Ausubel et al.,
ed., John Wiley & Sons (Chichester, England, 1998); Molecular
Cloning: A Laboratory Manual, 3nd Edition, J. Sambrook et al., ed.,
Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y.,
2001); Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed.,
Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y.,
1988); and Using Antibodies: A Laboratory Manual, E. Harlow and D.
Lane, ed., Cold Spring Harbor Laboratory (Cold Spring. Harbor,
N.Y., 1999) which are incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence by, for example, introducing deletions, and/or insertions
into desired regions of the antibodies.
[0193] In one embodiment, one or more substitutions are made within
the Fc region (e.g. supra) of an antibody able to
immunospecifically bind Integrin .alpha..sub.v.beta..sub.3. In
another embodiment, the amino acid substitutions modify binding to
one or more Fc ligand (e.g., Fc.gamma.Rs, C1q) and alter ADCC
and/or CDC activity.
[0194] In a specific embodiment, one or more of the CDRs is
inserted within framework regions using routine recombinant DNA
techniques. The framework regions may be naturally occurring or
consensus framework regions, specifically contemplated are human
framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol.
278: 457-479 for a listing of human framework regions). In one
embodiment, the polynucleotide generated by the combination of the
framework regions and CDRs encodes an antibody that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3. In
another embodiment, as discussed supra, one or more amino acid
substitutions may be made within the framework regions, it is
contemplated that the amino acid substitutions improve binding of
the antibody to its antigen. Additionally, such methods may be used
to make amino acid substitutions or deletions of one or more
variable region cysteine residues participating in an intrachain
disulfide bond to generate antibody molecules lacking one or more
intrachain disulfide bonds. Other alterations to the polynucleotide
are encompassed by the present invention and within the skill of
the art.
6.5 Polypeptides and Fusion Proteins That Bind to Integrin
.alpha..sub.v.beta..sub.3
[0195] The present invention encompasses polypeptides and fusion
proteins that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3.
[0196] In a specific embodiment, a polypeptide or a fusion protein
that immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3
inhibits or reduces the interaction between Integrin
.alpha..sub.v.beta..sub.3 and its ligands by about 25%, about 30%,
about 35%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or about 98% in an in vivo or in vitro assay described herein or
well-known to one of skill in the art. In this context "about"
means plus or minus 0.1% to 2.5%. In another specific embodiment, a
polypeptide or a fusion protein that immunospecifically binds to
Integrin .alpha..sub.v.beta..sub.3 inhibits or reduces the
interaction between Integrin .alpha..sub.v.beta..sub.3 and its
ligands by 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 98% in an in vivo or in vitro assay described
herein or well-known to one of skill in the art. In alternative
embodiment, a polypeptide or a fusion protein that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 does
not significantly inhibit the interaction between Integrin
.alpha..sub.v.beta..sub.3 and its ligands in an in vivo or in vitro
assay described herein or well-known to one of skill in the
art.
[0197] In a one embodiment, a polypeptide or a fusion protein that
immuno-specifically binds to Integrin .alpha..sub.v.beta..sub.3
comprises an Integrin .alpha..sub.v.beta..sub.3 ligand or a
fragment thereof which immunospecifically binds to an Integrin
.alpha..sub.v.beta..sub.3 fused to an Fc domain. It is specifically
contemplated that the Fc domain of said fusion protein comprises at
least one high effector function amino acid and/or substitution as
described supra. In another embodiment, said Fc domain is that of
an Fc variant of the present invention, the Fc domain of an Fc
variant is hereafter referred to as a variant Fc domain. Examples
of Integrin .alpha..sub.v.beta..sub.3 ligands include, but are not
limited to, vitronectin, osteopontin, bone sialoprotein,
echistatin, RGD-containing peptides, and RGD mimetics. (See e.g.,
Dresner-Pollak et al., J. Cell Biochem. 56(3): 323-30; Duong et
al., Front. Biosci. 1(3): d757-68).
[0198] In another embodiment, a polypeptide or a fusion protein
that immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3
comprises a bioactive molecule fused to a variant Fc domain of the
present invention. In accordance with these embodiments, the
bioactive molecule immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3. Bioactive molecules that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
include, but are not limited to, peptides, polypeptides, proteins,
small molecules, mimetic agents, synthetic drugs, inorganic
molecules, and organic molecules. In still another embodiment, a
bioactive molecule that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 is a polypeptide comprising at least 5,
at least 10, at least 20, at least 30, at least 40, at least 50, at
least 60, at least 70, at least 80, at least 90 or at least 100
contiguous amino acid residues, and is heterologous to the amino
acid sequence of the variant Fc domain of the invention.
[0199] In another embodiment, a peptide, a polypeptide or a fusion
protein that immunospecifically binds to Integrin
.alpha..sub.v.beta..sub.3 comprises a polypeptide having an amino
acid sequence that is at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, or at least 99% identical to the amino acid sequence of an
Integrin .alpha..sub.v.beta..sub.3 ligand or a fragment thereof
fused to a variant Fc domain of the present invention
[0200] The present invention provides polypeptides or fusion
proteins that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 comprising a variant Fc domain of the
present invention fused to a polypeptide encoded by a nucleic acid
molecule that hybridizes to the nucleotide sequence encoding an
Integrin .alpha..sub.v.beta..sub.3 ligand or a fragment
thereof.
[0201] In a specific embodiment, a polypeptide or a fusion protein
that immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3
comprises a variant Fc domain of the present invention fused to a
polypeptide encoded by a nucleic acid molecule that hybridizes to
the nucleotide sequence encoding an Integrin
.alpha..sub.v.beta..sub.3 ligand or a fragment thereof under
stringent conditions, e.g., hybridization to filter-bound DNA in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C. followed by one or more washes in 0.2.times.SSC/0.1% SDS at
about 50-65.degree. C., under highly stringent conditions, e.g.,
hybridization to filter-bound nucleic acid in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.1.times.SSC/0.2%
SDS at about 68.degree. C., or under other stringent hybridization
conditions which are known to those of skill in the art (see, for
example, Ausubel, F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and
John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and
2.10.3).
[0202] The present invention also encompasses polypeptides and
fusion proteins that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 comprising of a variant Fc domain, fused
to marker sequences, such as but not limited to, a peptide, to
facilitate purification. In other embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. Other peptide tags useful for purification include, but
are not limited to, the hemagglutinin "HA" tag, which corresponds
to an epitope derived from the influenza hemagglutinin protein
(Wilson et al., 1984, Cell 37:767) and the "flag" tag.
[0203] The present invention further encompasses polypeptides and
fusion proteins that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 fused to a variant Fc further conjugated
to a therapeutic moiety. A polypeptide or a fusion protein that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 may
be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a
cytostatic or cytocidal agent, an agent which has a potential
therapeutic benefit, or a radioactive metal ion, e.g.,
alpha-emitters. A cytotoxin or cytotoxic agent includes any agent
that is detrimental to cells. Examples of a therapeutic moieties
and cytotoxin or cytotoxic agents are listed supra (see section 6.3
entitled "Antibody Conjugates And Derivatives," infra).
[0204] Polypeptides, proteins and fusion proteins can be produced
by standard recombinant DNA techniques or by protein synthetic
techniques, e.g., by use of a peptide synthesizer. For example, a
nucleic acid molecule encoding a peptide, polypeptide, protein or a
fusion protein can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
reamplified to generate a chimeric gene sequence (see, e.g.,
Current Protocols in Molecular Biology, Ausubel et al., eds., John
Wiley & Sons, 1992). Moreover, a nucleic acid encoding a
bioactive molecule can be cloned into an expression vector
containing the variant Fc domain or a fragment thereof such that
the bioactive molecule is linked in-frame to the variant Fc domain
or variant Fc domain fragment.
[0205] Methods for fusing or conjugating polypeptides to the
constant regions of antibodies are known in the art. See, e.g.,
U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053,
5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and
5,112,946; EP 307,434; EP 367,166; EP 394,827; International
Publication Nos. WO 91/06570, WO 96/04388, WO 96/22024, WO
97/34631, and WO 99/04813; Ashkenazi et al., 1991, Proc. Natl.
Acad. Sci. USA 88: 10535-10539; Traunecker et al., 1988, Nature,
331:84-86; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil
et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341, which are
incorporated herein by reference in their entireties.
[0206] The nucleotide sequences encoding a bioactive molecule and
an Fc domain or fragment thereof may be obtained from any
information available to those of skill in the art (i.e., from
Genbank, the literature, or by routine cloning). The nucleotide
sequences encoding Integrin ligands may be obtained from any
available information, e.g., from Genbank, the literature or by
routine cloning. See, e.g., Xiong et al., Science, 12; 294(5541):
339-45 (2001). The nucleotide sequence coding for a polypeptide a
fusion protein can be inserted into an appropriate expression
vector, i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted protein-coding
sequence. A variety of host-vector systems may be utilized in the
present invention to express the protein-coding sequence. These
include but are not limited to mammalian cell systems infected with
virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems
infected with virus (e.g., baculovirus); microorganisms such as
yeast containing yeast vectors; or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used.
6.6 Recombinant Expression Of Antibodies and Fusion Proteins
[0207] Recombinant expression of an Fc variant or fusion protein
comprising a variant Fc domain (referred to herein as an "variant
Fc fusion protein", or "variant Fc fusion"), derivative, analog or
fragment thereof, (e.g., a heavy or light chain of an antibody of
the invention or a portion thereof or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody, or fusion
protein. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody, or fusion protein of the
invention has been obtained, the vector for the production of the
antibody or fusion protein molecule may be produced by recombinant
DNA technology using techniques well known in the art. Thus,
methods for preparing a protein by expressing a polynucleotide
containing an antibody or fusion protein encoding nucleotide
sequence are described herein. Methods that are well known to those
skilled in the art can be used to construct expression vectors
containing antibody or fusion protein coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an Fc variant or variant
Fc fusion of the invention, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., International
Publication No. WO 86/05807; International Publication No. WO
89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of
the antibody, or a polypeptide for generating an variant Fc fusion
may be cloned into such a vector for expression of the full length
antibody chain (e.g. heavy or light chain), or complete variant Fc
fusion protein.
[0208] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an Fc variant or variant Fc
fusion protein of the invention. Thus, the invention includes host
cells containing a polynucleotide encoding an Fc variant or variant
Fc fusion protein of the invention or fragments thereof, or a heavy
or light chain thereof, or portion thereof, or a single chain
antibody of the invention, operably linked to a heterologous
promoter. In other embodiments for the expression of double-chained
antibodies, vectors encoding both the heavy and light chains may be
co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
[0209] A variety of host-expression vector systems may be utilized
to express the antibody or fusion protein molecules of the
invention (see, e.g., U.S. Pat. No. 5,807,715). Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody or fusion protein molecule of the invention in situ. These
include but are not limited to microorganisms such as bacteria
(e.g., E. coli and B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody or fusion protein coding sequences; yeast
(e.g., Saccharomyces Pichia) transformed with recombinant yeast
expression vectors containing antibody or fusion protein coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody or
fusion protein 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 antibody or fusion protein coding sequences; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3
cells) harboring 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).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody or fusion protein molecules, are used
for the expression of a recombinant antibody or fusion protein
molecules. For example, mammalian cells such as Chinese hamster
ovary cells (CHO), in conjunction with a vector such as the major
intermediate early gene promoter element from human cytomegalovirus
is an effective expression system for antibodies (Foecking et al.,
1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2).
In a specific embodiment, the expression of nucleotide sequences
encoding antibodies or fusion protein that bind to Integrin
.alpha..sub.v.beta..sub.3 is regulated by a constitutive promoter,
inducible promoter or tissue specific promoter.
[0210] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody or fusion protein molecule being expressed. For example,
when a large quantity of such a protein is to be produced, for the
generation of pharmaceutical compositions of an antibody or fusion
protein molecule, vectors that direct the expression of high levels
of fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited to, the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791),
in which the antibody or fusion protein coding sequence may be
ligated individually into the vector in frame with the lac Z coding
region so that a lac Z-fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the
like. pGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione 5-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption and binding to matrix
glutathione agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0211] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
or fusion protein coding sequence may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter).
[0212] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody or fusion protein coding sequence
of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the antibody or fusion
protein molecule in infected hosts (e.g., see Logan & Shenk,
1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bittner et al., 1987, Methods in
Enzymol. 153:516-544).
[0213] The expression of an antibody or a fusion protein may be
controlled by any promoter or enhancer element known in the art.
Promoters which may be used to control the expression of the gene
encoding an antibody or fusion protein include, but are not limited
to, the SV40 early promoter region (Bernoist and Chambon, 1981,
Nature 290:304-310), the promoter contained in the 3' long terminal
repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell
22:787-797), the herpes thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory
sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-42), the tetracycline (Tet) promoter (Gossen et al.,
1995, Proc. Nat. Acad. Sci. USA 89:5547-5551); prokaryotic
expression vectors such as the .beta.-lactamase promoter
(Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. U.S.A.
75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc.
Natl. Acad. Sci. U.S.A. 80:21-25; see also "Useful proteins from
recombinant bacteria" in Scientific American, 1980, 242:74-94);
plant expression vectors comprising the nopaline synthetase
promoter region (Herrera-Estrella et al., Nature 303:209-213) or
the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,
1981, Nucl. Acids Res. 9:2871), and the promoter of the
photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286); neuronal-specific enolase (NSE) which is
active in neuronal cells (Morelli et al., 1999, Gen. Virol.
80:571-83); brain-derived neurotrophic factor (BDNF) gene control
region which is active in neuronal cells (Tabuchi et al., 1998,
Biochem. Biophysic. Res. Com. 253:818-823); glial fibrillary acidic
protein (GFAP) promoter which is active in astrocytes (Gomes et
al., 1999, Braz J Med Biol Res 32(5): 619-631; Morelli et al.,
1999, Gen. Virol. 80:571-83) and gonadotropic releasing hormone
gene control region which is active in the hypothalamus (Mason et
al., 1986, Science 234:1372-1378).
[0214] Expression vectors containing inserts of a gene encoding an
antibody or fusion protein can be identified by three general
approaches: (a) nucleic acid hybridization, (b) presence or absence
of "marker" gene functions, and (c) expression of inserted
sequences. In the first approach, the presence of a gene encoding a
peptide, polypeptide, protein or a fusion protein in an expression
vector can be detected by nucleic acid hybridization using probes
comprising sequences that are homologous to an inserted gene
encoding the peptide, polypeptide, protein or the fusion protein,
respectively. In the second approach, the recombinant vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype,
occlusion body formation in baculovirus, etc.) caused by the
insertion of a nucleotide sequence encoding an antibody or fusion
protein in the vector. For example, if the nucleotide sequence
encoding the antibody or fusion protein is inserted within the
marker gene sequence of the vector, recombinants containing the
gene encoding the antibody or fusion protein insert can be
identified by the absence of the marker gene function. In the third
approach, recombinant expression vectors can be identified by
assaying the gene product (e.g., antibody or fusion protein)
expressed by the recombinant. Such assays can be based, for
example, on the physical or functional properties of the fusion
protein in in vitro assay systems, e.g., binding with
anti-bioactive molecule antibody.
[0215] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence
of certain inducers; thus, expression of the genetically engineered
fusion protein may be controlled. Furthermore, different host cells
have characteristic and specific mechanisms for the translational
and post-translational processing and modification (e.g.,
glycosylation, phosphorylation of proteins). Appropriate cell lines
or host systems can be chosen to ensure the desired modification
and processing of the foreign protein expressed. For example,
expression in a bacterial system will produce an unglycosylated
product and expression in yeast will produce a glycosylated
product. Eukaryotic host cells that possess the cellular machinery
for proper processing of the primary transcript (e.g.,
glycosylation, and phosphorylation) of the gene product may be
used. Such mammalian host cells include, but are not limited to,
CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, NS0, and in
particular, neuronal cell lines such as, for example, SK-N-AS,
SK-N-FI, SK-N-DZ human neuroblastomas (Sugimoto et al., 1984, J.
Natl. Cancer Inst. 73: 51-57), SK-N-SH human neuroblastoma
(Biochim. Biophys. Acta, 1982, 704: 450-460), Daoy human cerebellar
medulloblastoma (He et al., 1992, Cancer Res. 52: 1144-1148)
DBTRG-05MG glioblastoma cells (Kruse et al., 1992, In Vitro Cell.
Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma (Cancer Res.,
1970, 30: 2110-2118), 1321N1 human astrocytoma (Proc. Natl. Acad.
Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br. J.
Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (Acta
Pathol. Microbiol. Scand., 1968, 74: 465-486), A172 human
glioblastoma (Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6
rat glioma cells (Benda et al., 1968, Science 161: 370-371),
Neuro-2a mouse neuroblastoma (Proc. Natl. Acad. Sci. USA, 1970, 65:
129-136), NB41A3 mouse neuroblastoma (Proc. Natl. Acad. Sci. USA,
1962, 48: 1184-1190), SCP sheep choroid plexus (Bolin et al., 1994,
J. Virol. Methods 48: 211-221), G355-5, PG-4 Cat normal astrocyte
(Haapala et al., 1985, J. Virol. 53: 827-833), Mpf ferret brain
(Trowbridge et al., 1982, In Vitro 18: 952-960), and normal cell
lines such as, for example, CTX TNA2 rat normal cortex brain
(Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6467-6471)
such as, for example, CRL7030 and Hs578Bst. Furthermore, different
vector/host expression systems may effect processing reactions to
different extents.
[0216] For long-term, high-yield production of recombinant
proteins, stable expression is often preferred. For example, cell
lines which stably express an antibody or fusion protein may be
engineered. Rather than using expression vectors that contain viral
origins of replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched medium, and then are
switched to a selective medium. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci that in turn can be cloned and expanded into cell
lines. This method may advantageously be used to engineer cell
lines that express an Fc variant or variant Fc fusion protein that
specifically binds to Integrin .alpha.v.beta..sub.3. Such
engineered cell lines may be particularly useful in screening and
evaluation of compounds that affect the activity of a polypeptide
or a fusion protein that immunospecifically binds to Integrin
.alpha.v.beta..sub.3.
[0217] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc.
Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1); and hygro, which confers resistance to hygromycin (Santerre
et al., 1984, Gene 30:147) genes.
[0218] Once a peptide, polypeptide, protein or a fusion protein of
the invention has been produced by recombinant expression, it may
be purified by any method known in the art for purification of a
protein, 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.
[0219] The expression levels of an antibody or fusion protein
molecule can be increased by vector amplification (for a review,
see Bebbington and Hentschel, The use of vectors based on gene
amplification for the expression of cloned genes in mammalian cells
in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a
marker in the vector system expressing an antibody or fusion
protein is amplifiable, increase in the level of inhibitor present
in culture of host cell will increase the number of copies of the
marker gene. Since the amplified region is associated with the
antibody gene, production of the antibody or fusion protein will
also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0220] The host cell may be co-transfected with two expression
vectors of the invention. For example, the first vector encoding a
heavy chain derived polypeptide and the second vector encoding a
light chain derived polypeptide. The two vectors may contain
identical selectable markers, which enable equal expression of
heavy and light chain polypeptides. Alternatively, a single vector
may be used which encodes, and is capable of expressing, a fusion
protein or both heavy and light chain polypeptides. The coding
sequences for the fusion protein or heavy and light chains may
comprise cDNA or genomic DNA.
6.7 Antagonists of Integrin .alpha..sub.v.beta..sub.3
[0221] The invention specifically encompasses Fc variants, or
variant Fc fusions, of the invention that are Integrin
.alpha..sub.v.beta..sub.3 antagonists. As used herein, the terms
"antagonist" and "antagonists" refer to any protein, polypeptide,
peptide, peptidomimetic, glycoprotein, antibody, antibody fragment,
carbohydrate, nucleic acid, organic molecule, inorganic molecule,
large molecule, or small molecule that blocks, inhibits, reduces or
neutralizes the function, activity and/or expression of another
molecule. In various embodiments, an antagonist reduces the
function, activity and/or expression of another molecule by at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95% or at least 99%
relative to a control such as phosphate buffered saline (PBS). More
specifically, an Integrin .alpha..sub.v.beta..sub.3 antagonist
inhibits, reduces or neutralizes the function, activity and/or
expression of Integrin .alpha..sub.v.beta..sub.3 or inhibits or
reduces Integrin .alpha..sub.v.beta..sub.3-mediated
pathologies.
[0222] In one embodiment, integrin .alpha..sub.v.beta..sub.3
antagonists inhibit or reduce angiogenesis. In particular
embodiments, integrin .alpha..sub.v.beta..sub.3 antagonists inhibit
or reduce angiogenesis in a subject by at least 5%, at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, or at least 99% relative
to a control such as PBS, as measured by, for example, changes in
regional blood volume using dynamic susceptibility
contrast-enhanced MRI.
[0223] The invention also provides methods for screening for
antagonists for Integrin .alpha..sub.v.beta..sub.3. Said screening
methods include but are not limited to assays that monitor Integrin
.alpha..sub.v.beta..sub.3 activity (e.g., cell adhesion,
angiogenesis, tumor cell growth and tumor progression) and/or
plasma concentration. These and additional methods are further
described infra (see section 6.8 entitled "Biological Assays,"
infra) and in PCT publications WO 02/12501, WO 03/075957, WO
04/066956 and U.S. patent applications 2003/0157098 among
others.
[0224] In addition, the invention provides methods for identifying
monoclonal antibodies that bind to the heterodimerized
.alpha..sub.v.beta..sub.3 but not the .alpha..sub.v or the
.beta..sub.3 chains when not included in a heterodimer. Further,
the invention provides for a method to manipulate both the ADCC
activity and the binding affinities for Fc.gamma.R of antibodies
identified using such screening methods.
[0225] Integrin .alpha..sub.v.beta..sub.3 and/or amino acid
substituted subunits of Integrin .alpha..sub.v.beta..sub.3 (see for
example PCT publication WO 03/075957) can be used for screening
antibodies with specific affinity for particular epitopes by
identifying monoclonal antibodies that bind to wild type Integrin
.alpha..sub.v.beta..sub.3 but not the altered form, or that bind
mouse .alpha..sub.v.beta..sub.3 integrins with a region substituted
with the corresponding region from the human
.alpha..sub.v.beta..sub.3 but do not bind to wild type mouse
Integrin .alpha..sub.v.beta..sub.3.
[0226] In addition, the invention provides methods for identifying
monoclonal antibodies and other molecules (e.g., Integrin
.alpha..sub.v.beta..sub.3 ligands and variants thereof) that bind
to the heterodimerized .alpha..sub.v.beta..sub.3 but not the
.alpha..sub.v or .beta..sub.3 chains when not included in a
heterodimer. Such screening can be accomplished by any routine
method for assaying antibody specificity and/or protein
interactions known in the art, for example, using cell lines that
do not express wild type Integrin .alpha..sub.v.beta..sub.3 to
recombinantly express the mutant Integrin .alpha..sub.v.beta..sub.3
or individual .alpha..sub.v or .beta..sub.3 chains. In one
embodiment, new identified antibodies that immunospecifically bind
Integrin .alpha..sub.v.beta..sub.3 are antagonists of Integrin
.alpha..sub.v.beta..sub.3. Assays to measure the antagonist
activity of a molecule include but are not limited to those
described infra.
[0227] The Fc of antibodies identified from such screening methods
can be substituted as described supra to alter ADCC and/or CDC
activity and to modify binding affinities for one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q). Other antagonistic binding molecules
(e.g., Integrin .alpha..sub.v.beta..sub.3 ligands and variants
thereof) identified from such screening methods can be fused to a
variant Fc domain of the invention. It is further contemplated that
the Fc variants of the newly identified Integrin
.alpha..sub.v.beta..sub.3 antagonistic antibodies and variant Fc
fusions of the newly identified Integrin .alpha..sub.v.beta..sub.3
antagonists are useful for the prevention, management and treatment
of Integrin .alpha..sub.v.beta..sub.3-mediated diseases and
disorders, including but not limited to inflammatory diseases,
autoimmune diseases, bone metabolism related disorders, angiogenic
related disorders, disorders related to aberrant expression and/or
activity of .alpha..sub.v.beta..sub.3, and cancer. Such Fc variants
and/or variant Fc fusions can be used in the methods and
formulations of the present invention.
6.8 Biological Assays
[0228] The antagonistic effect of one or more Fc variant, or
variant Fc fusion of the invention on Integrin
.alpha..sub.v.beta..sub.3 activity can be determined by any method
known in the art. Methods include but are not limited to those
described infra and in PCT publications WO 02/12501, WO 03/075957,
WO 03/075741, WO 04/066956 and U.S. patent applications
2003/0157098 among others, each of which is are incorporated herein
by reference in its entirety. For example, the blockage of Integrin
.alpha..sub.v.beta..sub.3 activity and/or the plasma concentration
of Integrin .alpha..sub.v.beta..sub.3 can be assayed by any
technique known in the art that measures the activity and/or
expression of Integrin .alpha..sub.v.beta..sub.3, including but not
limited to, Western blot, Northern blot, RNase protection assays,
enzymatic activity assays, in situ hybridization,
immunohistochemistry, and immunocytochemistry.
[0229] The binding specificity, affinity and functional activity of
an Fc variant, or variant Fc fusion protein of the invention can be
characterized in various in vitro binding and cell adhesion assays
known in the art, including but limited to, ELISA Western Blot
analysis, cell surface staining, inhibition of ligand-receptor
interactions, flow cytometric analysis and those disclosed in
International Publication Nos. WO 04/014292, WO 03/094859, WO
04/069264, WO 04/028551, WO 03/004057, WO 03/040304, WO 00/78815,
WO 02/070007 and WO 03/075957, U.S. Pat. Nos. 5,795,734, 6,248,326
and 6,472,403, Pecheur et al., 2002, FASEB J. 16(10): 1266-1268;
Almed et al., The Journal of Histochemistry & Cytochemistry
50:1371-1379 (2002), all of which are incorporated herein by
reference. For example, the binding affinity, specificity and the
off-rate of an Fc variant and/or variant Fc fusion protein can be
determined by a competitive binding assay with the parental
anti-Integrin .alpha..sub.v.beta..sub.3 antibody, by measuring the
inhibitory activity of an Fc variant, or variant Fc fusion protein
of the invention on binding to Integrin .alpha..sub.v.beta..sub.3.
One example of a competitive binding assay is a radioimmunoassay
comprising the incubation of labeled Integrin
.alpha..sub.v.beta..sub.3 (e.g., 3H or 125I) with the Fc variant of
interest in the presence of increasing amounts of unlabeled
Integrin .alpha..sub.v.beta..sub.3, and the detection of the
monoclonal antibody bound to the labeled Integrin
.alpha..sub.v.beta..sub.3. The affinity of an Fc variant for an
Integrin .alpha..sub.v.beta..sub.3 and the binding off-rates can be
determined from the data by scatchard plot analysis. Competition
with a second antibody can also be determined using
radioimmunoassays. In this case, an Integrin
.alpha..sub.v.beta..sub.3 is incubated with an Fc variant
conjugated to a labeled compound (e.g., 3H or 125I) in the presence
of increasing amounts of a second unlabeled monoclonal
antibody.
[0230] The kinetic parameters of an Fc variant, or variant Fc
fusion protein may also be determined using any surface plasmon
resonance (SPR) based assays known in the art. For a review of
SPR-based technology see Mullet et al., 2000, Methods 22: 77-91;
Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et
al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al.,
2000, Current Opinion in Biotechnology 11: 54-61; all of which are
incorporated herein by reference in their entirety. Additionally,
any of the SPR instruments and SPR based methods for measuring
protein-protein interactions described in U.S. Pat. Nos. 6,373,577;
6,289,286; 5,322,798; 5,341,215; 6,268,125 are contemplated in the
methods of the invention, all of which are incorporated herein by
reference in their entirety.
[0231] The binding specificity of an Fc variant, or variant Fc
fusion protein of the invention to Integrin
.alpha..sub.v.beta..sub.3 can be assessed by any method known in
the art including but not limited to, measuring binding to Integrin
.alpha..sub.v.beta..sub.3 and its crossreactivity to other
.alpha..sub.v- or .beta..sub.3-containing integrins, inhibition of
Integrin .alpha..sub.v.beta..sub.3 binding in cell adhesion assays.
In addition, binding affinity and specificity can be determined by
a competitive binding assay with the parental anti-Integrin
.alpha..sub.v.beta..sub.3 antibody against Integrin
.alpha..sub.v.beta..sub.3 or by measuring the inhibitory activity
of an Fc variant, or variant Fc fusion protein of the invention on
Integrin .alpha..sub.v.beta..sub.3 binding to fibrinogen.
[0232] The inhibitory and/or antagonistic activity of an Fc
variant, or variant Fc fusion of the invention can be tested in
cell proliferation assays, cell adhesion assays (Lawrenson et al.,
2002, J Cell Sci 115:1059 and Davy et al., 2000, EMBO 19:5396) and
in endothelial cell migration assays such as the transwell cell
migration assay (Choi et al., 1994, J Vascular Sur 19:125-134 and
Leavesly et al., 1993, J Cell Biol 121:163-170).
[0233] Additional examples of in vitro assays, e.g., Western
blotting analysis, flow cytometric analysis, cell adhesion assay to
cortical bone and extracellular matrix proteins, cell migration
assay, cell invasion assay, and cell proliferation assay, can be
found in Pecheur et al., 2002, FASEB J. 16(10): 1266-1268, of which
the entire text is incorporated herein by reference.
[0234] The anti-cancer activity of an Fc variant, or variant Fc
fusion of the invention can be determined by using various
experimental animal models for the study of cancer such as the
corneal micro pocket assay (see, e.g., Fournier et al., (1981)
Invest Opthalmol & Visual Sci. 21:351-54); scid mouse model or
transgenic mice where a mouse Integrin .alpha..sub.v.beta..sub.3 is
replaced with the human Integrin .alpha..sub.v.beta..sub.3, nude
mice with human xenografts, animal models wherein an antagonist of
Integrin .alpha..sub.v.beta..sub.3 recognizes the same target as
Vitaxin.RTM., such as hamsters, rabbits, etc. known in the art and
described in Relevance of Tumor Models for Anticancer Drug
Development (1999, eds. Fiebig and Burger); Contributions to
Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991,
eds. Boven and Winograd); and Anticancer Drug Development Guide
(1997 ed. Teicher), herein incorporated by reference in their
entireties.
[0235] Various animal models known in the art that are relevant to
a targeted disease or disorder, e.g., inflammatory diseases,
autoimmune diseases, diseases or disorders associated with aberrant
bone metabolism and/or aberrant angiogenesis, cancers, disorders
associated with aberrant integrin .alpha..sub.v.beta..sub.3
expression and/or activity can be used, including but not limited
to, those that are disclosed in International Publication No. WO
00/78815, U.S. Pat. No. 6,248,326, U.S. Pat. No. 6,472,403, Pecheur
et al., 2002, FASEB J. 16(10): 1266-1268; Almed et al., The Journal
of Histochemistry & Cytochemistry 50:1371-1379 (2002), all of
which are incorporated herein by reference. Models that can be used
include but are not limited to, growth factor or tumor-induced
angiogenesis in the chick chorioallantoic membrane (CAM) (see,
e.g., Ausprunk et al. (1980) Am. J. Pathol., 79:597-618; Ossonski
et al. (1975) Cancer Res., 40:2300-2309; Brooks et al. (1994)
Science, 264:569-571 and Brooks et al., (1994), Cell,
79:1157-1164), Vx2 carcinoma cells in rabbits (see, e.g., Voelkel
et al., (1975) Metabolism 24:973-86), tumors induced in BALB/c
nu/nu mice and SCID mice with subcutaneously implanted human bone
fragments (SCID-human-bone model). Additional examples of tumor
models can be found in Teicher et al., Tumor Models in Cancer
Research, (Humana Press, Totowa, N.J., 2001).
[0236] It is contemplated that the protocols and formulations of
the invention are tested in vitro, and then in vivo, for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, assays which can be used to determine whether
administration of a specific therapeutic protocol, formulation or
combination therapy of the invention is indicated, include in vitro
cell culture assays in which a patient tissue sample is grown in
culture, and exposed to or otherwise contacted with a formulation
of the invention, and the effect of such a formulation upon the
tissue sample is observed. The tissue sample can be obtained by
biopsy from the patient. This test allows the identification of the
therapeutically most effective prophylactic or therapeutic agent(s)
for each individual patient. In various specific embodiments, in
vitro assays can be carried out with representative cells of cell
types involved in an autoimmune disorder, an inflammatory disorder,
a disorder associated with aberrant expression and/or activity of
Integrin .alpha.V.beta.3, to determine if a formulation of the
invention has a desired effect upon such cell types. A lower level
of proliferation or survival of the contacted cells indicates that
the formulation is effective to treat the condition in the patient.
Alternatively, instead of culturing cells from a patient, a
formulation of the invention may be screened using cells of a tumor
or malignant cell line, osteoclasts, endothelial cells or an
endothelial cell line. Many assays standard in the art can be used
to assess such survival and/or growth; for example, cell
proliferation can be assayed by measuring .sup.3H-thymidine
incorporation, by direct cell count, by detecting changes in
transcriptional activity of known genes such as proto-oncogenes
(e.g., fos, myc) or cell cycle markers; cell viability can be
assessed by trypan blue staining, differentiation can be assessed
visually based on changes in morphology, etc.
[0237] Prophylactic or therapeutic agents can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to in rats, mice, chicken, cows, monkeys, rabbits,
hamsters, etc. The principle animal models for known in the art and
widely used are known and described in the art as described
above.
[0238] Further, any assays known to those skilled in the art can be
used to evaluate the prophylactic and/or therapeutic utility of the
combinatorial therapies disclosed herein for treatment or
prevention of cancer.
6.9 Prophylactic and Therapeutic Uses
[0239] As discussed above, agents that immunospecifically bind
Integrin .alpha..sub.v.beta..sub.3, can be utilized for the
inhibition of angiogenesis or the inhibition of other functions
mediated or influenced by Integrin .alpha..sub.v.beta..sub.3,
including but not limited to cell proliferation, cell attachment,
cell migration, granulation tissue development, and/or
inflammation. Accordingly, the present invention relates to the use
of agents that immunospecifically bind and in particular
embodiments, inhibit Integrin .alpha..sub.v.beta..sub.3 for the
prevention, management, treatment or amelioration of cancer or one
or more symptoms thereof and/or the inhibition of angiogenesis.
[0240] Angiogenesis, also called neovascularization, is the process
where new blood vessels form from pre-existing vessels within a
tissue. As described above, this process is mediated by endothelial
cells expressing Integrin .alpha..sub.v.beta..sub.3 and inhibition
of at least this integrin, inhibits new vessel growth. There are a
variety of pathological conditions that require new blood vessel
formation or tissue angiogenesis and inhibition of this process
inhibits the pathological condition. As such, pathological
conditions that require angiogenesis for growth or maintenance are
considered to be Integrin .alpha..sub.v.beta..sub.3-mediated
diseases. The extent of treatment, or reduction in severity, of
these diseases will therefore depend on the extent of inhibition of
angiogenesis. These Integrin .alpha..sub.v.beta..sub.3-mediated
diseases include, for example, inflammatory disorders such as
immune and non-immune inflammation, thrombosis, acute ischemic
stroke, chronic articular rheumatism, psoriasis, disorders
associated with inappropriate or inopportune invasion of vessels
such as diabetic retinopathy, neovascular glaucoma and capillary
proliferation in atherosclerotic plaques as well as cancer
disorders.
[0241] Such cancer disorders can include, for example, solid
tumors, tumor metastasis, angiofibromas, angiosarcomas,
retrolental, fibroplasia, hemangiomas, Kaposi's sarcoma,
carcinomas, carcinosarcomas, and other cancers which require
neovascularization to support tumor growth. Additional diseases
which are considered angiogenic include psoriasis and rheumatoid
arthritis as well as retinal diseases such as macular
degeneration.
[0242] Diseases other than those requiring new blood vessels which
are Integrin .alpha..sub.v.beta..sub.3-mediated diseases include,
for example, restenosis and osteoporosis.
[0243] Accordingly, the present invention relates to the use of
agents that immunospecifically bind and in particular embodiments,
inhibit Integrin .alpha..sub.v.beta..sub.3 for the prevention,
management, treatment or amelioration of cancer, solid tumor
metastasis, restenosis, thrombosis, acute ischemic stroke,
granulation tissue development in cutaneous wounds, osteoporosis,
age-related macular degeneration, diabetic retinopathy, as well as,
inflammatory diseases such as rheumatoid arthritis and psoriasis or
one or more symptoms thereof and/or the inhibition of angiogenesis
or conditions associated therewith.
[0244] In one embodiment, the methods and formulations of the
invention are used for inhibiting angiogenesis. In a specific
embodiment, the methods and formulations of the invention are used
for inhibiting angiogenesis in a solid tumor. In another
embodiment, the methods and formulations of the invention are used
for inhibiting angiogenesis in an inflamed, angiogenic tissue
including but not limited to retinal tissues and joint tissues.
[0245] Further, the present invention provides Fc variants that
immunospecifically bind and in particular embodiments, inhibit
Integrin .alpha..sub.v.beta..sub.3 which are useful for therapeutic
purposes, more specifically, for the treatment, prevention,
management or amelioration of cancer. Specific examples of cancers
that can be prevented, managed, treated or ameliorated in
accordance with the invention include, but are not limited to,
leukemias, such as but not limited to, acute leukemia, acute
lymphocytic leukemia, acute myelocytic leukemias, such as,
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia leukemias and myelodysplastic syndrome; chronic
leukemias, such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including but not limited to
adenocarcinoma, lobular (small cell) carcinoma, intraductal
carcinoma, medullary breast cancer, mucinous breast cancer, tubular
breast cancer, papillary breast cancer, Paget's disease, and
inflammatory breast cancer; adrenal cancer such as but not limited
to pheochromocytom and adrenocortical carcinoma; thyroid cancer
such as but not limited to papillary or follicular thyroid cancer,
medullary thyroid cancer and anaplastic thyroid cancer; pancreatic
cancer such as but not limited to, insulinoma, gastrinoma,
glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or
islet cell tumor; pituitary cancers such as but limited to
Cushing's disease, prolactin-secreting tumor, acromegaly, and
diabetes insipius; eye cancers such as but not limited to ocular
melanoma such as iris melanoma, choroidal melanoma, and cilliary
body melanoma, and retinoblastoma; vaginal cancers such as squamous
cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as
squamous cell carcinoma, melanoma, adenocarcinoma, basal cell
carcinoma, sarcoma, and Paget's disease; cervical cancers such as
but not limited to, squamous cell carcinoma, and adenocarcinoma;
uterine cancers such as but not limited to endometrial carcinoma
and uterine sarcoma; ovarian cancers such as but not limited to,
ovarian epithelial carcinoma, borderline tumor, germ cell tumor,
and stromal tumor; esophageal cancers such as but not limited to,
squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,
mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,
melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small
cell) carcinoma; stomach cancers such as but not limited to,
adenocarcinoma, fungating (polypoid), ulcerating, superficial
spreading, diffusely spreading, malignant lymphoma, liposarcoma,
fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers;
liver cancers such as but not limited to hepatocellular carcinoma
and hepatoblastoma; gallbladder cancers such as adenocarcinoma;
cholangiocarcinomas such as but not limited to pappillary, nodular,
and diffuse; lung cancers such as non-small cell lung cancer,
squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,
large-cell carcinoma and small-cell lung cancer; testicular cancers
such as but not limited to germinal tumor, seminoma, anaplastic,
classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,
teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate
cancers such as but not limited to, adenocarcinoma, leiomyosarcoma,
and rhabdomyosarcoma; penal cancers; oral cancers such as but not
limited to squamous cell carcinoma; basal cancers; salivary gland
cancers such as but not limited to adenocarcinoma, mucoepidermoid
carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but
not limited to squamous cell cancer, and verrucous; skin cancers
such as but not limited to, basal cell carcinoma, squamous cell
carcinoma and melanoma, superficial spreading melanoma, nodular
melanoma, lentigo malignant melanoma, acral lentiginous melanoma;
kidney cancers such as but not limited to renal cell carcinoma,
adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell
cancer (renal pelvis and/or uterer); Wilms' tumor; bladder cancers
such as but not limited to transitional cell carcinoma, squamous
cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers
include myxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lymphangioendotheliosarcoma, mesothelioma, synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J.B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
Inc., United States of America).
[0246] In a specific embodiment, the methods and formulations of
the invention are used for the prevention, management, treatment or
amelioration of a primary or secondary cancer that expresses
Integrin .alpha..sub.v.beta..sub.3. In another embodiment, the
methods and formulations of the invention are used for the
prevention, management, treatment or amelioration of a primary or
secondary cancer that does not express Integrin
.alpha..sub.v.beta..sub.3. In another embodiment, the methods and
formulations are used for the prevention, management, treatment or
amelioration of a cancer that has the potential to metastasize or
has metastasized to other tissues or organs (e.g., bone). In
another embodiment, the methods and formulations of the invention
are used for the prevention, management, treatment or amelioration
of lung cancer, prostate cancer, ovarian cancer, melanoma, bone
cancer or breast cancer. Methods using agents that
immunospecifically inhibit Integrin .alpha..sub.v.beta..sub.3
include but are not limited to those disclosed in PCT publications
WO 00/078815, WO 02/070007, WO 03/075957, WO 03/075741 and WO
04/066956, each of which is herein incorporated by reference in its
entirety.
[0247] The invention provides methods for screening for antibody
and other antagonists of Integrin .alpha..sub.v.beta..sub.3.
Further, the invention provides for a method to manipulate the ADCC
and/or CDC activity and the binding affinities for one or more Fc
ligand (e.g., Fc.gamma.R, C1q) of the antibodies and/or other
antagonists identified using such screening methods. The Integrin
.alpha..sub.v.beta..sub.3 antagonists identified and manipulated
utilizing such methods can be used for the prevention, treatment,
management or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated diseases and disorders or one or
more symptoms thereof, including but not limited to cancer,
inflammatory and autoimmune diseases either alone or in combination
with other therapies.
[0248] The invention also provides variant Fc fusion proteins that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3. Said
variant Fc fusion proteins can be used for the prevention,
treatment, management or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated diseases and disorders or one or
more symptoms thereof, including but not limited to cancer,
inflammatory and autoimmune diseases either alone or in combination
with other therapies.
[0249] In a specific embodiment, Fc variants and/or Fc variant
fusion proteins of the invention that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3 are used for the prevention,
management, treatment or amelioration of cancer or one or more
symptoms thereof. In another embodiment, Fc variant antibodies
and/or Fc variant fusion proteins of the invention used for the
prevention, management, treatment or amelioration of cancer or one
or more symptoms thereof are antagonists of Integrin
.alpha..sub.v.beta..sub.3.
[0250] The invention also encompasses the use of Fc variants and/or
variant Fc fusions with modified binding affinity to one or more Fc
ligand (e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC
activity that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 conjugated or fused to a moiety (e.g.,
therapeutic agent or drug) for prevention, treatment, management or
amelioration of Integrin .alpha..sub.v.beta..sub.3-mediated
diseases and disorders or one or more symptoms thereof, including
but not limited to cancer, inflammatory and autoimmune diseases.
The invention further encompasses treatment protocols that enhance
the prophylactic or therapeutic effect of said Fc variants and/or
variant Fc fusions.
[0251] The invention provides methods for preventing, managing,
treating or ameliorating cancer that has the potential to
metastasize or has metastasized to an organ or tissue (e.g., bone)
or one or more symptoms thereof, said methods comprising
administering to a subject in need thereof one or more doses of a
prophylactically or therapeutically amount of one or more Fc
variants and/or variant Fc fusion protein of the invention.
[0252] The invention provides methods for preventing, managing,
treating or ameliorating cancer or one or more symptoms thereof,
said methods comprising administering to a subject in need thereof
one or more doses of a prophylactically or therapeutically
effective amount of one or more Fc variants and/or variant Fc
fusion with modified binding affinity to one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity fused
or conjugated to a moiety (e.g., a therapeutic agent or drug).
Examples of a moiety that an Fc variant can be fused or conjugated
to include, but are not limited to those disclosed in PCT
publication WO 2003/075957 which is herein incorporated by
reference in its entirety. Examples of Fc variants and variant Fc
fusions with modified binding affinity to their one or more Fc
ligand (e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC
activity include, but are not limited to, those variants disclosed
supra.
[0253] The present invention encompasses protocols for the
prevention, management, treatment or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated diseases and disorders or one or
more symptoms thereof, including but not limited to cancer,
inflammatory and autoimmune diseases or one or more symptoms
thereof in which one or more Fc variants and/or variant Fc fusion
with modified binding affinity to one or more Fc ligand (e.g.,
Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically binds to Integrin .alpha..sub.v.beta..sub.3 is
used in combination with the administration of a dosage of a
prophylactically or therapeutically effective amount of one or more
other therapies other than an Fc variant and/or variant fusion
protein. The invention is based, in part, on the recognition that
the Fc variants and/or variant fusion proteins of the invention
potentiate and synergize with, enhance the effectiveness of,
improve the tolerance of, and/or reduce the side effects caused by,
other therapies, including current standard and experimental
chemotherapies. The combination therapies of the invention have
additive potency, an additive therapeutic effect or a synergistic
effect. The combination therapies of the invention enable lower
dosages of the therapy (e.g., prophylactic or therapeutic agents)
utilized in conjunction with Fc variants and/or variant Fc fusions
for the prevention, management, treatment or amelioration of
Integrin .alpha..sub.v.beta..sub.3-mediated diseases and disorders
or one or more symptoms thereof, including but not limited to,
cancer, inflammatory and autoimmune diseases and/or less frequent
administration of such prophylactic or therapeutic agents to a
subject with an Integrin .alpha..sub.v.beta..sub.3-mediated disease
(e.g., cancer) to improve the quality of life of said subject
and/or to achieve a prophylactic or therapeutic effect. Further,
the combination therapies of the invention reduce or avoid unwanted
or adverse side effects associated with the administration of
current single agent therapies and/or existing combination
therapies for diseases, such as cancer, which in turn improves
patient compliance with the treatment protocol.
[0254] In one embodiment, the invention provides methods for
preventing, managing, treating or ameliorating an Integrin
.alpha..sub.v.beta..sub.3-mediated disease (e.g., cancer) or one or
more symptoms thereof, said methods comprising administering to a
subject in need thereof a dose of a prophylactically or
therapeutically effective amount of an Fc variant and/or variant Fc
fusion in combination with the administration of an Integrin
antagonist, a standard or experimental chemotherapy, a hormonal
therapy, a biological therapy/immunotherapy and/or a radiation
therapy. In another embodiment, the invention provides methods for
preventing, managing, treating or ameliorating an Integrin
.alpha..sub.v.beta..sub.3-mediated disease (e.g., cancer) or one or
more symptoms thereof, said methods comprising administering to a
subject in need thereof a dose of a prophylactically or
therapeutically effective amount of an Fc variant and/or variant Fc
fusion in combination with surgery, alone or in further combination
with the administration of an Integrin antagonist, a standard or
experimental chemotherapy, a hormonal therapy, a biological
therapy/immunotherapy and/or a radiation therapy. In accordance
with these embodiments, the Fc variant and/or variant Fc fusion
utilized to prevent, manage, treat or ameliorate an Integrin
.alpha..sub.v.beta..sub.3-mediated disease (e.g., cancer) or one or
more symptoms thereof may or may not be conjugated or fused to a
moiety (e.g., therapeutic agent or drug) and said Fc variants
and/or variant Fc fusions are in particular embodiments,
antagonists that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3.
[0255] Therapeutic or prophylactic agents include, but are not
limited to, small molecules, synthetic drugs, peptides,
polypeptides, proteins, nucleic acids (e.g., DNA and RNA
nucleotides including, but not limited to, antisense nucleotide
sequences, triple helices and nucleotide sequences encoding
biologically active proteins, polypeptides or peptides),
antibodies, synthetic or natural inorganic molecules, mimetic
agents, and synthetic or natural organic molecules. Any agent which
is known to be useful, or which has been used or is currently being
used for the prevention, treatment or amelioration of Integrin
.alpha..sub.v.beta..sub.3-mediated disease or disorder including
but not limited to cancer, inflammatory and autoimmune diseases or
symptom associated therewith can be used in combination with an Fc
variant and/or variant Fc fusion in accordance with the invention
described herein.
[0256] Exemplary agents to be used in the combination therapies
described supra include but are not limited to Integrin antagonists
(e.g., RGD peptides and disintegrins), standard or experimental
chemotherapy agents (e.g., doxorubicin, epirubicin,
cyclophosphamide, 5-fluorouracil, taxanes such as docetaxel and
paclitaxel, leucovorin, levamisole, irinotecan, estramustine,
etoposide, vinblastine, dacarbazine, nitrosoureas such as
carmustine and lomustine, vinca alkaloids, platinum compounds,
cisplatin, mitomycin, vinorelbine, gemcitabine, carboplatin,
hexamethylmelamine and/or topotecan), immunomodulatory agents
(e.g., cytokines, antibodies, interleukins and hemapoietic
factors), biological therapies/immunotherapies (e.g., tamoxifen,
LHRH agonists, non-steroidal antiandrogens, steroidal
antiandrogens, estrogens, aminoglutethimide, hydrocortisone,
flutamide withdrawal, progesterone, ketoconazole, prednisone,
interferon-alpha, interferon-beta, interferon-gamma, interleukin-2,
tumor necrosis factor-alpha, and melphalan), anti-inflammatory
agents (e.g., non-steroidal anti-inflammatory drugs (NSAIDs),
steroidal anti-inflammatory drugs, beta-agonists, anticholingeric
agents, and methyl xanthines), analgesics (e.g., NSAIDs,
salicylates, acetominophen, narcotics, and non-narcotic and
anxiolytic combinations). Also contemplated is the use of the Fc
variants of the invention in combination with other anti-cancer
antibody agents including but not limited to, Avastin.TM.
(Genetech), Herceptin.TM. (Genentech), Rituxin.TM.
(Genentech/Biogen) and Zevalin (Biogen). Additional agents and
therapies and their dosages, routes of administration and
recommended usage are known in the art and have been described in
such literature as the Physician's Desk Reference (57.sup.th ed.,
2003). Other combination therapies are described in PCT
applications WO 02/070007; WO 03/075741; WO 03/075957 and WO
04/066956. Each of the above references and patent publications
each of which are incorporated herein in their entireties.
[0257] Examples of anti-cancer agents that can be used in
combination with the Fc variants and other embodiments of the
invention, including pharmaceutical compositions and dosage forms
and kits of the invention, include, but are not limited to:
acivicin, aclarubicin, acodazole hydrochloride, acronine,
adozelesin, aldesleukin, altretamine, ambomycin, ametantrone
acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin,
asparaginase, asperlin, azacitidine, azetepa, azotomycin,
batimastat, benzodepa, bicalutamide, bisantrene hydrochloride,
bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar
sodium, bropirimine, busulfan, cactinomycin, calusterone,
caracemide, carbetimer, carboplatin, carmustine, carubicin
hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin,
cisplatin, cladribine, crisnatol mesylate, cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride,
decarbazine, decitabine, dexormaplatin, dezaguanine, dezaguanine
mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin
hydrochloride, droloxifene, droloxifene citrate, dromostanolone
propionate, duazomycin, edatrexate, eflomithine hydrochloride,
elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin
hydrochloride, erbulozole, esorubicin hydrochloride, estramustine,
estramustine phosphate sodium, etanidazole, etoposide, etoposide
phosphate, etoprine, fadrozole hydrochloride, fazarabine,
fenretinide, floxuridine, fludarabine phosphate, fluorouracil,
flurocitabine, fosquidone, fostriecin sodium, gemcitabine,
gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride,
ifosfamide, ilmofosine, interleukin 2 (including recombinant
interleukin 2, or rIL2), interferon alpha 2a, interferon alpha 2b,
interferon alpha n1, interferon alpha n3, interferon beta I a,
interferon gamma I b, iproplatin, irinotecan hydrochloride,
lanreotide acetate, letrozole, leuprolide acetate, liarozole
hydrochloride, lometrexol sodium, lomustine, losoxantrone
hydrochloride, masoprocol, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, melphalan,
menogaril, mercaptopurine, methotrexate, methotrexate sodium,
metoprine, meturedepa, mitindomide, mitocarcin, mitocromin,
mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone
hydrochloride, mycophenolic acid, nitrosoureas, nocodazole,
nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase,
peliomycin, pentamustine, peplomycin sulfate, perfosfamide,
pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,
plomestane, porfimer sodium, porfiromycin, prednimustine,
procarbazine hydrochloride, puromycin, puromycin hydrochloride,
pyrazofurin, riboprine, rogletimide, safingol, safingol
hydrochloride, semustine, simtrazene, sparfosate sodium,
sparsomycin, spirogermanium hydrochloride, spiromustine,
spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,
tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin,
teniposide, teroxirone, testolactone, thiamiprine, thioguanine,
thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestolone
acetate, triciribine phosphate, trimetrexate, trimetrexate
glucuronate, triptorelin, tubulozole hydrochloride, uracil mustard,
uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine
sulfate, vindesine, vindesine sulfate, vinepidine sulfate,
vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate,
vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplatin,
zinostatin, zorubicin hydrochloride. Other anti cancer drugs
include, but are not limited to: 20 epi 1,25 dihydroxyvitamin D3, 5
ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol,
adozelesin, aldesleukin, ALL TK antagonists, altretamine,
ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin,
amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis
inhibitors, antagonist D, antagonist G, antarelix, anti dorsalizing
morphogenetic protein 1, antiandrogens, antiestrogens,
antineoplaston, aphidicolin glycinate, apoptosis gene modulators,
apoptosis regulators, apurinic acid, ara CDP DL PTBA, arginine
deaminase, asulacrine, atamestane, atrimustine, axinastatin 1,
axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine,
baccatin III derivatives, balanol, batimastat, BCR/ABL antagonists,
benzochlorins, benzoylstaurosporine, beta lactam derivatives, beta
alethine, betaclamycin B, betulinic acid, bFGF inhibitor,
bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,
bistratene A, bizelesin, breflate, bropirimine, budotitane,
buthionine sulfoximine, calcipotriol, calphostin C, camptothecin
derivatives, canarypox IL 2, capecitabine, carboxamide amino
triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage
derived inhibitor, carzelesin, casein kinase inhibitors (ICOS),
castanospermine, cecropin B, cetrorelix, chloroquinoxaline
sulfonamide, cicaprost, cis porphyrin, cladribine, clomifene
analogues, clotrimazole, collismycin A, collismycin B,
combretastatin A4, combretastatin analogue, conagenin, crambescidin
816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin
A, cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine
ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine,
dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide,
dexrazoxane, dexverapamil, diaziquone, didemnin B, didox,
diethylnorspermine, dihydro 5 azacytidine, dihydrotaxol,
dioxamycin, diphenyl spiromustine, docetaxel, docosanol,
dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA,
ebselen, ecomustine, edelfosine, edrecolomab, eflomithine, elemene,
emitefur, epirubicin, epristeride, estramustine analogue, estrogen
agonists, estrogen antagonists, etanidazole, etoposide phosphate,
exemestane, fadrozole, fazarabine, fenretinide, filgrastim,
finasteride, flavopiridol, flezelastine, fluasterone, fludarabine,
fluorodaunorunicin hydrochloride, forfenimex, formestane,
fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate,
galocitabine, ganirelix, gelatinase inhibitors, gemcitabine,
glutathione inhibitors, hepsulfam, heregulin, hexamethylene
bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene,
idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod,
immunostimulant peptides, insulin like growth factor 1 receptor
inhibitor, interferon agonists, interferons, interleukins,
iobenguane, iododoxorubicin, ipomeanol, iroplact, irsogladine,
isobengazole, isohomohalicondrin B, itasetron, jasplakinolide,
kahalalide F, lamellarin N triacetate, lanreotide, leinamycin,
lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia
inhibiting factor, leukocyte alpha interferon,
leuprolide+estrogen+progesterone, leuprorelin, levamisole,
liarozole, linear polyamine analogue, lipophilic disaccharide
peptide, lipophilic platinum compounds, lissoclinamide 7,
lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone,
lovastatin, loxoribine, lurtotecan, lutetium texaphyrin,
lysofylline, lytic peptides, maitansine, mannostatin A, marimastat,
masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase
inhibitors, menogaril, merbarone, meterelin, methioninase,
metoclopramide, MIF inhibitor, mifepristone, miltefosine,
mirimostim, mismatched double stranded RNA, mitoguazone,
mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast
growth factor saporin, mitoxantrone, mofarotene, molgramostim,
monoclonal antibody, human chorionic gonadotrophin, monophosphoryl
lipid A+myobacterium cell wall sk, mopidamol, multiple drug
resistance gene inhibitor, multiple tumor suppressor 1 based
therapy, mustard anticancer agent, mycaperoxide B, mycobacterial
cell wall extract, myriaporone, N acetyldinaline, N substituted
benzamides, nafarelin, nagrestip, naloxone+pentazocine, napavin,
naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid,
neutral endopeptidase, nilutamide, nisamycin, nitric oxide
modulators, nitroxide antioxidant, nitrullyn, O6 benzylguanine,
octreotide, okicenone, oligonucleotides, onapristone, ondansetron,
ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone,
oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogues,
paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronic
acid, panaxytriol, panomifene, parabactin, pazelliptine,
pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin,
pentrozole, perflubron, perfosfamide, perillyl alcohol,
phenazinomycin, phenylacetate, phosphatase inhibitors, picibanil,
pilocarpine hydrochloride, pirarubicin, piritrexim, placetin A,
placetin B, plasminogen activator inhibitor, platinum complex,
platinum compounds, platinum triamine complex, porfimer sodium,
porfiromycin, prednisone, propyl bis acridone, prostaglandin J2,
proteasome inhibitors, protein A based immune modulator, protein
kinase C inhibitor, protein kinase C inhibitors, microalgal,
protein tyrosine phosphatase inhibitors, purine nucleoside
phosphorylase inhibitors, purpurins, pyrazoloacridine,
pyridoxylated hemoglobin polyoxyethylene conjugate, raf
antagonists, raltitrexed, ramosetron, ras farnesyl protein
transferase inhibitors, ras inhibitors, ras GAP inhibitor,
retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin,
ribozymes, RII retinamide, rogletimide, rohitukine, romurtide,
roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU,
sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence
derived inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, single chain antigen
binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosic acid, spicamycin D, spiromustine, splenopentin,
spongistatin 1, squalamine, stem cell inhibitor, stem cell division
inhibitors, stipiamide, stromelysin inhibitors, sulfinosine,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, tallimustine,
tamoxifen methiodide, tauromustine, taxol, tazarotene, tecogalan
sodium, tegafur, tellurapyrylium, telomerase inhibitors,
temoporfin, temozolomide, teniposide, tetrachlorodecaoxide,
tetrazomine, thaliblastine, thalidomide, thiocoraline, thioguanine,
thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin
receptor agonist, thymotrinan, thyroid stimulating hormone, tin
ethyl etiopurpurin, tirapazamine, titanocene bichloride, topsentin,
toremifene, totipotent stem cell factor, translation inhibitors,
tretinoin, triacetyluridine, triciribine, trimetrexate,
triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors,
tyrphostins, UBC inhibitors, ubenimex, urogenital sinus derived
growth inhibitory factor, urokinase receptor antagonists,
vapreotide, variolin B, vector system, erythrocyte gene therapy,
velaresol, veramine, verdins, verteporfin, vinorelbine, vinxaltine,
vitaxin, vorozole, zanoterone, zeniplatin, zilascorb, and
zinostatin stimalamer. Additional anti-cancer drugs are
5-fluorouracil and leucovorin.
[0258] The methods and formulations of the invention are
particularly useful in preventing, managing, treating or
ameliorating cancers, including, but not limited to, cancer of the
head, neck, eye, mouth, throat, esophagus, chest, bone, lung,
colon, rectum, colorectal, or other gastrointestinal tract organs,
stomach, spleen, renal, skeletal muscle, subcutaneous tissue,
metastatic melanoma, endometrial, prostate, breast, ovaries,
testicles or other reproductive organs, skin, thyroid, blood, lymph
nodes, kidney, liver, pancreas, and brain or central nervous
system. Additional specific cancers are described supra. In a
specific embodiment, the methods and formulations of the invention
are used for the prevention, management, treatment or amelioration
of a primary or secondary cancer that expresses Integrin
.alpha..sub.v.beta..sub.3. In another embodiment, the methods and
formulations of the invention are used for the prevention,
management, treatment or amelioration of a primary or secondary
cancer that does not express Integrin
.alpha..sub.v.beta..sub.3.
[0259] The methods and formulations of the invention are useful not
only in untreated cancer patients but are also useful in the
management or treatment of cancer patients partially or completely
refractory to current standard and experimental cancer therapies,
including, but not limited to, chemotherapies, hormonal therapies,
biological therapies, radiation therapies, and/or surgery.
6.10 Formulations and Administration
[0260] As described above, the present invention relates to the use
of agents that immunospecifically bind and in particular
embodiments, inhibit Integrin .alpha..sub.v.beta..sub.3 for the
prevention, management, treatment or amelioration of an Integrin
.alpha..sub.v.beta..sub.3-mediated disease (e.g., cancer) or one or
more symptoms thereof and/or the inhibition of angiogenesis.
Accordingly, the present invention provides formulations (e.g., a
pharmaceutical composition) comprising one or more Fc variants
and/or Fc variant fusions with modified binding affinity to one or
more Fc ligand (e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC
activity that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 (also referred to herein as
"formulation(s) of the invention" or simply "formulation(s)"). In a
specific embodiment, said Fc variants and/or Fc variant fusions are
antagonists of Integrin .alpha..sub.v.beta..sub.3.
[0261] In one embodiment, formulations (e.g., a pharmaceutical
composition) comprising one or more Fc variants and/or Fc variant
fusions are liquid formulations (referred to herein as "liquid
formulation(s)" which are specifically encompassed by the more
generic terms "formulation(s) of the invention" and
"formulation(s)"). In a specific embodiment, the liquid
formulations are substantially free of surfactant and/or inorganic
salts. In another specific embodiment, the liquid formulations have
a pH ranging from about 5.0 to about 7.0, about 5.5 to about 6.5,
or about 5.8 to about 6.2, or about 6.0. In another specific
embodiment, the liquid formulations have a pH ranging from 5.0 to
7.0, 5.5 to 6.5, or 5.8 to 6.2, or 6.0. In yet another specific
embodiment, the liquid formulations comprise histidine at a
concentration ranging from about 1 mM to about 100 mM, or from
about 5 mM to about 50 mM, or about 10 mM to about 25 mM. In still
another specific embodiment, the liquid formulations comprise
histidine at a concentration ranging from 1 mM to 100 mM, or from 5
mM to 50 mM, or 10 mM to 25 mM
[0262] In another embodiment, the liquid formulations have a
concentration of one or more Fc variants and/or Fc variant fusions
is about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125
mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225
mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml. In
another embodiment, the liquid formulations have a concentration of
one or more Fc variants and/or Fc variant fusions is 50 mg/ml, 75
mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, 225
mg/ml, 250 mg/ml, 275 mg/ml, or 300 mg/ml. In still another
embodiment, the liquid formulations should exhibit one, or more of
the following characteristics, stability, low to undetectable
levels of antibody fragmentation and/or aggregation, very little to
no loss of the biological activities of the antibodies or antibody
fragments during manufacture, preparation, transportation, and
storage. In certain embodiments the liquid formulations lose less
than 50%, or less than 30%, or less than 20%, or less than 10% or
even less than 5% or 1% of the antibody activity within 1 year
storage under suitable conditions at about 4.degree. C. The
activity of an antibody can be determined by a suitable
antigen-binding or effector function assay for the respective
antibody. In yet another embodiment, the liquid formulations are of
low visocisty and turbidity. In a particular embodiment, the liquid
formulations have a viscosity of less than 10.00 cP at any
temperature in the range of 1 to 26.degree. C. Viscosity can be
determined by numerous method well known in the art. For example,
the viscosity of a polypeptide solution can be measured using a
ViscoLab 4000 Viscometer System (Cambridge Applied Systems)
equipped with a ViscoLab Piston (SN:7497, 0.3055'', 1-20 cP) and
S6S Reference Standard (Koehler Instrument Company, Inc.) and
connected to a water bath to regulate the temperature of the
samples being analyzed. The sample is loaded into the chamber at a
desired starting temperature (e.g., 2.degree. C.) and the piston
lowered into the sample. After sample was equilibrated to the
temperature of the chamber, measurement is initiated. The
temperature is increased at a desired rate to the desired final
temperature (e.g., >25.degree. C.). And the viscosity over time
is recorded.
[0263] It is contemplated that the liquid formulations may further
comprise one or more excipients such as a saccharide, an amino acid
(e.g. arginine, lysine, and methionine) and a polyol. Additional
descriptions and methods of preparing and analyzed liquid
formulations can be found, for example, in PCT publications WO
03/106644; WO 04/066957; WO 04/091658 each of which is herein
incorporated by reference in its entirety.
[0264] In one embodiment the formulations (e.g., liquid
formulations) of the invention are pyrogen-free formulations which
are substantially free of endotoxins and/or related pyrogenic
substances. Endotoxins include toxins that are confined inside a
microorganism and are released when the microorganisms are broken
down or die. Pyrogenic substances also include fever-inducing,
thermostable substances (glycoproteins) from the outer membrane of
bacteria and other microorganisms. Both of these substances can
cause fever, hypotension and shock if administered to humans. Due
to the potential harmful effects, it is advantageous to remove even
low amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight, as can be the case
with monoclonal antibodies, it is advantageous to remove even trace
amounts of endotoxin. In one embodiment, endotoxin and pyrogen
levels in the composition are less then 10 EU/mg, or less then 5
EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then
0.01 EU/mg, or less then 0.001 EU/mg.
[0265] It will be apparent to one skilled in the art that a
formulation comprising one or more Fc variants and/or Fc variant
fusions to be administered to a subject (e.g., a human) in need
thereof should be formulated in a pharmaceutically-acceptable
excipient. Examples of formulations, pharmaceutical compositions in
particular, of the invention include but are not limited to those
disclosed in PCT publications WO 02/070007, WO 03/075957 and WO
04/066957 each of which is herein incorporated by reference in its
entirety. Briefly, the excipient that is included with the Fc
variants and/or variant Fc fusion of the present invention in these
formulations (e.g., liquid formulations) can be selected based on
the expected route of administration of the formulations in
therapeutic applications. The route of administration of the
formulations depends on the condition to be treated. For example,
intravenous injection may be preferred for treatment of a systemic
disorder such as a lymphatic cancer or a tumor which has
metastasized. The dosage of the formulations to be administered can
be determined by the skilled artisan without undue experimentation
in conjunction with standard dose-response studies. Relevant
circumstances to be considered in making those determinations
include the condition or conditions to be treated, the choice of
formulations to be administered, the age, weight, and response of
the individual patient, and the severity of the patient's symptoms.
For example, the actual patient body weight may be used to
calculate the dose of the Fc variants and/or variant Fc fusion of
the present invention in these formulations in milliliters (mL) to
be administered. There may be no downward adjustment to "ideal"
weight. In such a situation, an appropriate dose may be calculated
by the following formula: Dose (mL)=[patient weight (kg).times.dose
level (mg/kg)/drug concentration (mg/mL)]
[0266] Depending on the condition, the formulations can be
administered orally, parenterally, intramuscularly, intranasally,
vaginally, rectally, lingually, sublingually, buccally,
intrabuccally, intravenously, cutaneously, subcutaneously and/or
transdermally to the patient.
[0267] Accordingly, formulations designed for oral, parenteral,
intramuscular, intranasal, vaginal, rectal, lingual, sublingual,
buccal, intrabuccal, intravenous, cutaneous, subcutaneous and/or
transdermal administration can be made without undue
experimentation by means well known in the art, for example, with
an inert diluent or with an edible carrier. The formulations may be
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the formulations of the
present invention may be incorporated with excipients and used in
the form of tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, chewing gums, and the like.
[0268] Tablets, pills, capsules, troches and the like may also
contain binders, recipients, disintegrating agent, lubricants,
sweetening agents, and/or flavoring agents. Some examples of
binders include microcrystalline cellulose, gum tragacanth and
gelatin. Examples of excipients include starch and lactose. Some
examples of disintegrating agents include alginic acid, cornstarch,
and the like. Examples of lubricants include magnesium stearate and
potassium stearate. An example of a glidant is colloidal silicon
dioxide. Some examples of sweetening agents include sucrose,
saccharin, and the like. Examples of flavoring agents include
peppermint, methyl salicylate, orange flavoring, and the like.
Materials used in preparing these various formulations should be
pharmaceutically pure and non-toxic in the amounts used.
[0269] The formulations of the present invention can be
administered parenterally, such as, for example, by intravenous,
intramuscular, intrathecal and/or subcutaneous injection.
Parenteral administration can be accomplished by incorporating the
formulations of the present invention into a solution or
suspension. Such solutions or suspensions may also include sterile
diluents, such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol and/or other
synthetic solvents. Parenteral formulations may also include
antibacterial agents, such as, for example, benzyl alcohol and/or
methyl parabens, antioxidants, such as, for example, ascorbic acid
and/or sodium bisulfite, and chelating agents, such as EDTA.
Buffers, such as acetates, citrates and phosphates, and agents for
the adjustment of tonicity, such as sodium chloride and dextrose,
may also be added. The parenteral preparation can be enclosed in
ampules, disposable syringes and/or multiple dose vials made of
glass or plastic. Rectal administration includes administering the
formulation into the rectum and/or large intestine. This can be
accomplished using suppositories and/or enemas. Suppository
formulations can be made by methods known in the art. Transdermal
administration includes percutaneous absorption of the formulation
through the skin. Transdermal formulations include patches,
ointments, creams, gels, salves, and the like. The formulations of
the present invention can be administered nasally to a patient. As
used herein, nasally administering or nasal administration includes
administering the formulations to the mucous membranes of the nasal
passage and/or nasal cavity of the patient.
[0270] In certain embodiments, the formulations (e.g., liquid
formulations) are administered to the mammal by subcutaneous (i.e.,
beneath the skin) administration. For such purposes, the
formulations may be injected using a syringe. However, other
devices for administration of the formulations are available such
as injection devices (e.g. the Inject-ease_and Genject_devices),
injector pens (such as the GenPen.TM.); auto-injector devices,
needleless devices (e.g., MediJector and BioJector); and
subcutaneous patch delivery systems.
[0271] In another aspect of the invention there is provided a slow
release formulations. In a specific embodiment, a slow release
formulation comprises a liquid formulation. Slow release
formulations may be formulated from a number of agents including,
but not limited to, polymeric nano or microparticles and gels
(e.g., a hyaluronic acid gel). Besides convenience, slow release
formulations offer other advantages for delivery of protein drugs
including protecting the protein (e.g., Fc variant and/or variant
Fc fusion) over an extended period from degradation or elimination,
and the ability to deliver the protein locally to a particular site
or body compartment thereby lowering overall systemic exposure.
[0272] The present invention, for example, also contemplates
injectable depot formulations in which the protein (e.g., Fc
variant and/or variant Fc fusion) is embedded in a biodegradable
polymeric matrix. Polymers that may be used include, but are not
limited to, the homo- and co-polymers of lactic and glycolic acid
(PLGA). PLGA degrades by hydrolysis to ultimately give the acid
monomers and is chemically unreactive under the conditions used to
prepare, for example, microspheres and thus does not modify the
protein. After subcutaneous or intramuscular injection, the protein
is released by a combination of diffusion and polymer degradation.
By using polymers of different composition and molecular weight,
the hydrolysis rate can be varied thereby allowing release to last
from days to months. In a further aspect the present invention
provides a nasal spray formulation. In a specific embodiment, a
nasal spray formulation comprises the liquid formulation of the
present invention.
[0273] The formulations of the invention may be used in accordance
with the methods of the invention for the prevention, management,
treatment or amelioration of cancer, inflammatory and autoimmune
diseases (in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease) or one or more symptoms
thereof. In one embodiment, the formulations of the invention are
sterile and in suitable form for a particular method of
administration to a subject with cancer, inflammatory and
autoimmune diseases, in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease.
[0274] The invention provides methods for preventing, managing,
treating or ameliorating cancer, inflammatory and autoimmune
diseases (in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease) or one or more symptoms
thereof, said method comprising: (a) administering to a subject in
need thereof a dose of a prophylactically or therapeutically
effective amount of a formulation comprising one or more Fc
variants and/or variant Fc fusions, that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3 and (b) administering one or
more subsequent doses of said formulation, to maintain a plasma
concentration of the antagonist at a desirable level (e.g., about
0.1 to about 100 .mu.g/ml), which continuously blocks the Integrin
.alpha..sub.v.beta..sub.3 activity. In a specific embodiment, the
plasma concentration of the Fc variants and/or variant Fc fusions
is maintained at 10 .mu.g/ml, 15 .mu.g/ml, 20 .mu.g/ml, 25
.mu.g/ml, 30 .mu.g/ml, 35 .mu.g/ml, 40 .mu.g/ml, 45 .mu.g/ml or 50
.mu.g/ml. In a specific embodiment, said effective amount of Fc
variant and/or variant Fc fusion to be administered is between at
least 1 mg/kg and 100 mg/kg per dose. In another specific
embodiment, said effective amount of Fc variant and/or variant Fc
fusion to be administered is between at least 1 mg/kg and 20 mg/kg
per dose. In another specific embodiment, said effective amount of
Fc variant and/or variant Fc fusion to be administered is between
at least 4 mg/kg and 10 mg/kg per dose. In yet another specific
embodiment, said effective amount of Fc variant and/or variant Fc
fusion to be administered is between 50 mg and 250 mg per dose. In
still another specific embodiment, said effective amount of Fc
variant and/or variant Fc fusion to be administered is between 100
mg and 200 mg per dose.
[0275] The present invention provides kits comprising one or more
Fc variants and/or variant Fc fusions with modified binding
affinity to one or more Fc ligand (e.g., Fc.gamma.Rs, C1q) and
altered ADCC and/or CDC activity that immunospecifically bind to
Integrin .alpha..sub.v.beta..sub.3 conjugated or fused to a
detectable agent, therapeutic agent or drug, in one or more
containers, for use in the prevention, treatment, management,
amelioration, detection, monitoring or diagnosis of cancer,
inflammatory and autoimmune diseases, in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease.
[0276] The invention also provides kits comprising one or more Fc
variants and/or variant Fc fusions with modified binding affinity
to one or more Fc ligand (e.g., Fc.gamma.Rs, C1q and altered ADCC
and/or CDC activity that immunospecifically bind to Integrin
.alpha..sub.v.beta..sub.3 in a first vial and one or more
prophylactic or therapeutic agents, other than Fc variants that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3, in a
second vial for use in the prevention, treatment, management,
amelioration, detection, monitoring or diagnosis of cancer,
inflammatory and autoimmune diseases, in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease. The invention also
provides kits comprising one or more Fc variants and/or variant Fc
fusions with modified binding affinity to one or more Fc ligand
(e.g., Fc.gamma.Rs, C1q) and altered ADCC and/or CDC activity that
immunospecifically bind to Integrin .alpha..sub.v.beta..sub.3
conjugated or fused to a therapeutic agent or drug in a first vial
and one or more prophylactic or therapeutic agents, other than
antagonists of Integrin .alpha..sub.v.beta..sub.3, in a second vial
for use in the prevention, treatment, management, amelioration,
detection, monitoring or diagnosis of cancer, inflammatory and
autoimmune diseases, in particular an Integrin
.alpha..sub.v.beta..sub.3-mediated disease. The kits may further
comprise packaging materials and/or instructions.
7. EXAMPLES
[0277] The invention is now described with reference to the
following examples. These examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these examples but rather should be construed
to encompass any and all variations which become evident as a
result of the teachings provided herein.
7.1 Example 1
Construction and Expression of Novel Fc Variants of Antibodies
[0278] Based on the structural information available for the
Fc-Fc.gamma.RIIIB complex, each of the putative Fc.gamma.R contact
residues of the IgG1 Fc portion was randomly mutated by using
degenerated oligonucleotides incorporating all possible single
mutations. The contact residues were divided into four regions (RI:
Leu.sup.234, Leu.sup.235, Gly.sup.236, Gly.sup.237, Pro.sup.238,
Ser239; RII: Asp.sup.265, Ser.sup.267, Glu.sup.269; RIII:
Ser.sup.298; and RIV: Ala.sup.327, Leu.sup.328, Pro.sup.329,
Ala.sup.330, and Ile.sup.332). Primers used for the amplification
and library construction are listed in table 4. The IgG1 of
antibody Vitaxin.TM., converted into scFv-Fc format, was used as
the model for this study. The DNA and corresponding amino acid
sequences of the variable regions of the Vitaxin.RTM. heavy and
light chains used to generate the scFv-Fc are shown in FIG. 1
(panels A and B, respectively). The scFv-Fc was then harnessed as
the template to build three Fc mutant libraries containing single
mutations in the Fc region. Library I contains all single mutations
in the RI region; library II covers the RII and RIII regions; and
library III covers the RIV region. Overlapping PCR approach was
used to synthesize entire Fc region containing mutations.
TABLE-US-00004 TABLE 4 Primers SEQ Primer Sequence Notes ID MDAD-1
CCG TGC CCA GCA CCT GAA NNK CTG GGG GGA CCG contact Region I 11 TCA
GTC MDAD-2 CCG TGC CCA GCA CCT GAA CTC NNK GGG GGA CCG contact
Region I 12 TCA GTC TTC MDAD-3 CCG TGC CCA GCA CCT GAA CTC CTG NNK
GGA CCG contact Region I 13 TCA GTC TTC CTC MDAD-4 CCG TGC CCA GCA
CCT GAA CTC CTG GGG NNK CCG contact Region I 14 TCA GTC TTC CTC TTC
MDAD-5 CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA NNK contact Region I
15 TCA GTC TTC CTC TTC CCC MDAD-6 CCG TGC CCA GCA CCT GAA CTC CTG
GGG GGA CCG contact Region I 16 NNK CT TT CT T CC CC NNK GTC TTC
CTC TTC CCC CCA MDAD-7 GTC ACA TGC GTG GTG GTG NNK GTG AGC CAC GAA
contact Region II 17 GAC CCT MDAD-8 GTC ACA TGC GTG GTG GTG GAG GTC
NNK CAC GAA contact Region II 18 GAC CCT GAG GTC MDAD-9 GTC ACA TGC
GTG GTG GTG GAC GTG AGC CAC NNK contact Region II 19 GAC CCT GAG
GTC AAG TTC MDAD-10 CGG GAG GAG CAG TAC AAC NNK ACG TAC CGT GTG
contact Region III 20 GTC AGC MDAD-11 TGC AAG GTC TCC AAC AAA NNK
CTC CCA GCC CCC contact Region IV 21 ATC GAG MDAD-12 TGC AAG GTC
TCC AAC AAA GCC NNK CCA GCC CCC contact Region IV 22 ATC GAG AAA
MDAD-13 TGC AAG GTC TCC AAC AAA GCC CTC NNK GCC CCC contact Region
IV 23 ATC GAG AAA ACC MDAD-14 TGC AAG GTC TCC AAC AAA GC CTC CCA
NNK CCC contact Region IV 24 ATC GAG AAA ACC ATC MDAD-15 TGC AAG
GTC TCC AAC AAA GCC CTC CCA GCC CCC contact Region IV 25 NNK GAG
AAA ACC ATC TCC AAA MDAD-16 ACT CAC ACA TGT CCA CCG TGC CCA GCA CCT
GAA Fc N-terminus 26 MDAD-17 CAC CAC CAC GCA TGT GAC RII primer 27
MDAD-18 GTT GTA CTG CTC CTC CCG RIII primer 28 MDAD-19 TTT GTT GGA
GAC CTT GCA RIV primer 29 MDAD-20 AAC CTC TAC AAA TGT GGT ATG GCT
Fc C- terminus 30 A1 AAG CTT CGG TCC GCC ACC ATG GCA ACT GAA GAT
Fc.gamma.RIIIA primer 31 CTC CCA AAG A2 GTC TGC CGA ACC GCT GCC TGC
CAA ACC TTG AGT Fc.gamma.RIIIA primer 32 GAT GGT B1 AGC TTC GGT CCG
CCA CCA TGG CTG TGC TAT TCC Fc.gamma.RIIB primer 33 TGG CAG CTC CCC
CAA B2 GTC TGC CGA ACC GCT GCC CCC CAT CGG TGA AGA Fc.gamma.RIIB
primer 34 GCT GGG AGC SA1 GGC AGC GGT TCG GCA GAC CCC TCC AAG GAC
Streptavidin primer 35 SA2 CAG GGG CTA GCT TAC TGC TGA ACG GCG TCG
AGC Streptavidin primer 36 GG EA1 TCC ACA GGT GTC CAC TCC CGG ACT
GAA GAT CTC Fc.gamma.RIIIA primer 37 CCA AAG EA2 GGG AGA ATT CCG
CGG CCG CTT ATT TGT CAT CGT Fc.gamma.RIIIA primer 38 CAT CTT TGT
AGT CAT GGT GAT GGT GAT GGT GTG CGC CTG CCA AAC CTT GAG TGA TGG T
EB1 TCC ACA GGT GTG CAC TCC GCT GTG CTA TTC CTG Fc.gamma.RIIB
primer 39 GCA GCT CCC CCA AAG EB2 GGG AGA ATT CCG CGG CCG CTT ATT
TGT CAT CGT Fc.gamma.RLIB primer 40 CAT CTT TGT AGT CAT GGT GAT GGT
GAT GGT GTG CGC CCC CCA TCG GTG AAG AGC TGG GAG C Oligo 1 GCC CTC
CCA GCC CCC gag GAG AAA ACC ATC TCC I332E 41 Oligo 2 GCC CTC CCA
GCC CCC cag GAG AAA ACC ATC TCC I332Q 42 Oligo 3 GCC CTC CCA GCC
CCC ggc GAG AAA ACC ATC TCC I332G 43 Oligo 4 GCC CTC CCA GCC CCC
gcc GAG AAA ACC ATC TCC I332A 44 Oligo 5 GCC CTC CCA GCC CCC tac
GAG AAA ACC ATC TCC I332Y 45 Oligo 6 GCC CTC CCA GCC CCC gac GAG
AAA ACC ATC TCC I332D 46 Oligo 7 GCC CTC CCA GCC CCC aac GAG AAA
ACC ATC TCC I332N 47 Oligo 8 GCC CTC CCA GCC CCC gtg GAG AAA ACC
ATC TCC I332V 48 Oligo 9 GCC CTG CCA GCC CCC tgg GAG AAA ACC ATC
TCC I332W 49 Oligo 10 GCC CTC CCA GCC CCC cgc GAG AAA ACC ATC TCC
I332R 50 Oligo 11 GCC CTC CCA GCC CCC agc GAG AAA ACC ATC TCC I332S
51 Oligo 12 GCC CTC CCA GCC CCC aag GAG AAA ACC ATC TCC I332K 52
Oligo 13 GCC CTC CCA GCC CCC atg GAG AAA ACC ATC TCC I332M 53 Oligo
14 GCC CTC CCA GCC CCC acc GAG AAA ACC ATC TCC I332T 54 Oligo 15
GCC CTC CCA GCC CCC tgc GAG AAA ACC ATC TCC I332C 55 Oligo 16 GCC
CTC CCA GCC CCC ctg GAG AAA ACC ATC TCC I332L 56 Oligo 17 GCC CTC
CCA GCC CCC ttc GAG AAA ACC ATC TCC I332F 57 Oligo 18 GCC CTC CCA
GCC CCC cac GAG AAA ACC ATC TCC I332H 58 Oligo 19 GCC CTC CCA GCC
CCC cct GAG AAA ACC ATC TCC I332P 59 Oligo 20 CTGGGGGGACCG gac
GTCTTCCTCTTC S239D 60 Oligo 21 AAAGCCCTCCCA ctg CCCgagGAGAAA
A330L/I332E 61
7.1.1 Materials and Methods
[0279] Construction of Fc Libraries: For constructing Fc library I,
primers MDAD-16, equimolar mixture of MAD-2 to -6, and MDAD-20 were
used in the PCR reaction. The PCR products were gel purified and
digested by restriction enzymes Not I/Pci I, and ligated into the
expression vector pMI under the control of the CMV promoter. For
constructing Fc library II, two PCR products incorporating RII and
RIII mutations were mixed at 3:1 molar ratio for cloning into pMI
vector. Primers MDAD-16, MDAD-17, equimolar mixture of MDAD-7 to
-9, and MDAD-20 were used to amplify Fc region to incorporate RII
mutations, and primers MDAD-16, -18, -10, and -20 were used to
amplify Fc region to incorporate RIII mutations. For Fc library
III, primers MDAD-16, MDAD-19, equimolar mixture of MDAD-11 to -15,
and MDAD-20 were used in the PCR reaction.
[0280] Transfection: The plasmids of three Fc libraries (I, II, and
III) were linearized by Sal I, ethanol precipitated and resuspended
in H.sub.2O. 50 .mu.g of each linearized library DNA was
individually transfected into 10.sup.7 NS0 cells by
electroporation. After electroporation, the cells were transferred
to a tube containing 30 ml of growth medium (Glutamine-free IMDM,
1.times.GS supplement and 2 mM L-glutamine) and seeded in 96-well
plates (50 .mu.l/well) at variable dilutions. The cells were
cultured at 37.degree. C. in humid air containing 5% CO.sub.2.
[0281] Selection of Stable Transfectants: The selection of stably
transfected NS0 cells expressing scFv-Fc mutants was started 18-24
hours after transfection by converting to selection medium (same as
growth medium but without glutamine). The medium was changed twice
a week at one half of the total volume. After 2-3 weeks of
incubation, the culture supernatants were collected for screening
of antibody expression.
[0282] Purification of scFv-Fc Variants: The culture supernatants
containing scFv-Fc mutants were purified by using a Protein A spin
chromatography kit following manufacturer's protocol (Pierce). The
bound scFv-Fc mutants were eluted with 0.1 M citrate buffer and
then dialyzed in PBS. All proteins were analyzed by
SDS-polyacrylamide gel electrophoresis and were applied to
quantitative ELISA using anti-human IgG assay plates (Becton
Dickson) or BCA kits (PIERCE) to determine scFv-Fc
concentrations.
[0283] Antibody Ouantitation by ELISA: To determine the expression
level of the Fc variants, anti-human IgG-coated microtiter plates
(Becton Dickson) were used. The culture supernatants were added to
the wells at dilutions of 1:10 and 1:100. After a 1 hour incubation
at room temperature, the plates were washed with PBST (PBS+0.1%
Tween 20) and incubated at room temperature for an additional hour
with anti-human IgG (Pierce) at a 1:60000 dilution. The signals
were detected by TME substrate (Pierce) and read by an ELISA reader
at 450 nm. Purified parental Vitaxin.TM. scFv-Fc expressed in a pMI
vector was employed as a standard (at serial dilutions of 0.003
.mu.g-10 .mu.g/ml).
7.2 Example 2
Construction and Expression of the Extracellular Domains of
Fc.gamma.RIIIA and Fc.gamma.RIIB
[0284] To facilitate the binding studies of the Fc variants to
Fc.gamma.Rs the extracellular domains of Fc.gamma.RIIIA and
Fc.gamma.RIIB were subcloned for expression as strepavidin fusion
proteins in E. coli and for expression in mammalian cells. The
Fc.gamma.RIIIA prepared for analysis is the low affinity (F158)
allotype. Two forms of Fc.gamma.RIIIA and Fc.gamma.RIIB were
prepared, a "tetramer" form, generated as as Strepavidin fusion,
and a "monomer" form generated as a Flag-tagged.
7.2.1 Materials and Methods
[0285] Construction and Bacterial Expression of the Extracellular
Domains of Fc.gamma.RIIIA- and Fc.gamma.RIIB-Strepavidin Fusion
Proteins (Tetramer): Primer pairs SA1/SA2, A1/A2, and B1/B2 (see
primer list, Table 4) were used to PCR amplify streptavidin and the
extracellular domains of Fc.gamma.R IIIA and Fc.gamma.R IIB,
respectively. The cDNA library of human bone marrow (Clontech) was
used as a template for Fc.gamma.R IIIA and Fc.gamma.R IIB
amplification, and the genomic DNA of Streptomyces avidinii was
used as the template for the amplification of Streptavidin.
Overlapping PCR was used to assemble fusion genes of Fc.gamma.R
IIIA-streptavidin and Fc.gamma.R IIB-strepavidin. The fusion genes
were digested by the restriction enzymes Nco I/Nhe I and cloned
into the expression vector pET-28a. The fusion proteins were
expressed as inclusion bodies and refolded by dialysis to slowly
remove urea as described by C. Gao, et al. (1997, PNAS USA
94:11777-82). The refolded fusion proteins were then purified by an
immunobiotin column (PIERCE) according to manufacturer's
instructions.
[0286] Construction and Mammalian Expression of the Extracellular
Domains of Fc.gamma.RIIIA and Fc.gamma.RIIB (Monomer): The
extracellular domains of Fc.gamma.R IIIA and Fc.gamma.R IIB were
PCR amplified from the cDNA library of human bone marrow (Clontech)
with primers EA1/EA2 and EB1/EB2, respectively (see primer list,
Table 4). The PCR products were digested by Xba I/Not I and cloned
into the mammalian cell expression vector pMI226 under the control
of the CMV promoter to generate proteins in which the extracelluar
domains of Fc.gamma.R IIIA and Fc.gamma.R IIB are tagged with
His6-tag followed by FLAG tag at the C-terminal end. The plasmid
DNA was transiently transfected into 293H cells by Lipofectamine
2000 Transfection Reagent (Invitrogen). After three collections
within 9 days, the proteins were purified by passing the culture
supernatant through anti-FLAG M2 agarose columns (Sigma). The
FLAG-tagged Fc.gamma.RIIIA/IIB proteins were eluted from the column
and dialyzed against PBS.
7.3 Example 3
Characterization of the Fc Variants
[0287] After mutagenesis of the Fc domain (see example 1 supra) Fc
variants, in the scFV-Fc fusion format, were screened for enhanced
binding to Fc.gamma.RIIIA tetramer by ELISA as detailed below. The
results for several clones are shown in FIG. 5. In addition, the
ADCC activity of these clones was determined against M21 cells. The
results for several clones are shown in FIG. 6. Based on these
studies three substitutions were chosen for further study, S239D,
A330L and I332E. These substitutions were introduced into the Fc
region of the intact Vitaxin.RTM. IgG1 heavy chain and coexpressed
with Vitaxin.RTM. light chain to produce full length Vitaxin.RTM.
Fc variant IgG1 molecules. The Vitaxin.RTM. Fc variant having the
I332E substitution was designated Vitaxin.RTM.-1M, the Vitaxin.RTM.
Fc variant having the S239D, A330L, I332L triple substitution was
designated Vitaxin.RTM.-3M.
[0288] A panel of Vitaxin.RTM. Fc variants, in IgG format, was
generated in which each of the standard 20 amino acids was
substituted at position 332. These variants were characterized.
FIG. 7A shows the relative binding to Fc.gamma.RIIIA of these Fc
variants, as determined by ELISA. It can be seen that under these
conditions several substitutions showed enhanced binding including
I332T, I332L, I332F and most dramatically, I332E. However, as shown
in FIG. 7B, only the I332E substitution showed a similar increase
in ADCC activity.
[0289] Representative binding curves for Vitaxin.RTM. and one Fc
variant of Vitaxin.RTM. (I332E; Vitaxin-IM) to Fc.gamma.RIIIA and
Fc.gamma.RIIB are shown in FIGS. 8A and 8B respectively.
Vitaxin.RTM. was prepared from two cell sources, NSO and HEK293
cells, no difference in binding was observed between these two
sources of Vitaxin. The Vitaxin.RTM. Fc variant was then prepared
from HEK293 cells. The Vitaxin.RTM. Fc variant showed approximately
a 2.5 fold increase in binding affinity to Fc.gamma.RIIIA (FIG. 8A)
with no corresponding change in binding to Fc.gamma.RIIB as
determined by ELISA (FIG. 8B).
[0290] The binding of Vitaxin.RTM. and the Vitaxin.RTM. Fc variants
to Fc.gamma.RIIIA was further analyzed by BIAcore analysis. The
binding of Vitaxin.RTM. and the Vitaxin.RTM. Fc variants were
analyzed with the receptor soluble and the antibody immobile (see
methods below). The Vitaxin-1M Fc variant was shown to have a
roughly 7 fold increase in binding affinity to Fc.gamma.IIIA as
compared to that of the parental wild type Vitaxin antibody. The
interaction of the Vitaxin-3M Fc variant to Fc.gamma.RIIIA was also
analyzed by BIAcore and found to have a binding affinity of
.about.114 nM, nearly 80 time better than that of the parental wild
type Vitaxin antibody. The results are summarized in Table 5.
TABLE-US-00005 TABLE 5 Binding Constants (K.sub.D) of wild type
antibodies and Fc variants to Fc.gamma.RIIIA Run RUs K.sub.D
K.sub.D Fold increase Antibody # Immobilized Isotherm Scatchard
over WT.sup.a Vitaxin .RTM. 1 9608 3.47 .mu.M 3.26 .mu.M Vitaxin-1M
1 9331 458 nM 458 nM 6.5 Vitaxin .RTM. 2 9434 8.9 .mu.M 7.6 .mu.M
Vitaxin-1M 2 9383 1.28 .mu.M 1.22 .mu.M 7.0 Vitaxin-3M 2 8284 114
nM 113 nM 78.0 3F2 3 8568 15.6 .mu.M 14.2 .mu.M 3F2-1M 3 7718 1.77
.mu.M 1.68 .mu.M 8.8 3F2-3M 3 7809 158 nM 162 nM 99
.sup.acalculated using Isoltherm values
[0291] The Vitaxin-IM Fc variant was further characterized in ADCC
assays against M21 cells. First, the ratio of target to effector
cells was kept constant at 50:1 and the concentration of the two
antibodies was varied from 0.4 to 1000 ng/ml (FIG. 9). Next, the
concentration of antibody was varied for several different ratios
of target to effector cell (6.25:1, 12.5:1, 25:1 and 50:1) (FIG.
10). In both assays the ADCC activity of the Vitaxin-IM Fc (I332E)
variant was approximately 3 fold higher than that of the parent
Vitaxin.RTM. antibody.
[0292] The Vitaxin-3M Fc variant was also characterized in ADCC
assays against a target cells expressing differing levels of
Integrin .alpha.V.beta.3 (FIG. 11). The target cell lines used were
M21 (a high expresser), DU145 (a low expressor), A498 and ACHN
(moderate expressors). The assays were performed using two
different ratios of target to effector cell (50:1 and 25:1) and
antibody concentrations ranging from 4 to 400 ng per well. In all
cases the ADCC activity of the Vitaxin-3M Fc variant was seen to be
higher than wild type Vitaxin.RTM.. Vitaxin-3M Fc variant was also
shown to have higher ADCC activity compared to the wild type
Vitaxin.RTM. antibody against SKMEL28 target cells which express
Integrin .alpha..sub.v.beta..sub.3 (FIG. 18).
7.3.1 Materials and Methods
[0293] ELISA Receptor Binding Assay: Microtiter plates were coated
with protein A/G (PIERCE) solution (0.25 .mu.g/ml) and incubated at
4.degree. C. overnight. Any remaining binding sites were blocked
with 4% skim milk. Approximately 25 .mu.l per well of mutant
antibody solution was added to each well and incubated for 1 h at
37.degree. C. After washing, Fc.gamma.RIIIA-streptavidin or
Fc.gamma.RIIB-streptavidin fusion protein (in 1% BSA) was added for
1 hour at 37.degree. C., followed by washing and biotin-conjugated
HRP for 30 min. Detection was carried out by adding 30 .mu.l of
tetramethylbenzidine substrate (Pierce) followed by neutralization
with 30 .mu.l of 0.2 M H.sub.2SO.sub.4. The absorbance was read at
450 nm
[0294] Generation of 332 Amino Acid Substitutions: QuikChange.RTM.
II XL site-directed mutagenesis kit (Stratagene, San Diego) was
used to generate all the amino acid substitutions at position 332
of the gene encoding the heavy chain of wild type Vitaxin.RTM. in
the plasmid pMI331 (see FIG. 4). Oligos 1 to 19 (see Table 4) were
applied to change the Isoleucine to all other 19 different amino
acids at the position 332, using Vitaxin as the template. The
mutation was further confirmed by DNA sequencing.
[0295] The plasmid DNA containing antibody genes was transiently
transfected into 293H cells by Lipofectamine 2000 Transfection
Reagent (Invitrogen). After three collections within 9 days, the
culture supernatants containing antibody were affinity purified by
using a pre-packed Protein A column (Amersham Biosciences, now
belongs to GE healthcare). The bound antibody were eluted with
elution buffer (100 mM Glycine, pH3.2), neutralized by 1 M Tris
buffer (pH 8.0) and then dialyzed in PBS. All purified antibodies
were analyzed by SDS-polyacrylamide gel electrophoresis and were
applied to quantitative ELISA using anti-human IgG assay plates
(Becton Dickson) or BCA kits (PIERCE) to determine IgG
concentrations.
[0296] Generation of Vitaxin.RTM.--1M and 3M Fc variants: The I332E
substitution was generated by site directed mutagenesis (as
described above) of the gene encoding the heavy chain of wild type
Vitaxin.RTM. in the plasmid pMI331 (see FIG. 4). The mutant I332E
was designated as Vitaxin 1M. The Vitaxin 3M was further generated
by two sequential site directed mutagenesis (as described above),
using oligo 20 and 21 (see Table 4) as primers and Vitaxin 1M as
template. Expression and purification of the 1M and 3M Vitaxin.RTM.
Fc variants was the same as described above.
[0297] Kinetic Analysis via BIAcore: for Run 1 the interaction of
Fc.gamma.RIIIA with immobilized Vitaxin.RTM. and Vitaxin.RTM. Fc
variant IgGs were monitored by surface plasmon resonance detection
using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala,
Sweden). Vitaxin.RTM. and Vitaxin.RTM. Fc variant IgGs were coupled
to the dextran matrix of a CM5 sensor chip (Pharmacia Biosensor)
using an Amine Coupling Kit, as described (Johnsson et al., 1992,
Anal Biochem 198:268-277), at a surface density of approximately
9400 RUs (see Table 5). Fc.gamma.RIIIA was serially diluted in 0.01
M HEPES pH 7.4 containing 0.15 M NaCl, 3 mM EDTA and 0.005% P20, at
concentrations ranging from 2 .mu.M down to 7.8 nM. Duplicate
injections of each concentration were made. All binding experiments
were performed at 25.degree. C., and at a flow rate of 10
.mu.L/min. Binding was monitored for 25 min. Following each
injection of Fc.gamma.RIIIA, the IgG surfaces were regenerated with
a 30 sec. pulse of 5 mM HCl. Fc.gamma.RIIIA was also passed over a
blank reference cell which is connected, in series, to the
IgG-containing flow cells. The steady-state binding curves were
also corrected for injection artifacts by subtraction of buffer
injections. This doubly-corrected data was then fit to a
steady-state isotherm provided by the instrument manufacturer
(Pharmacia Biosensor, Uppsala, Sweden) to derive the respective
equilibrium binding constants (K.sub.D). Separately, a Scatchard
plot of the Req data from each IgG surface was constructed to
confirm the results of the binding isotherms.
[0298] For Run 2 the interaction of The interaction of
Fc.gamma.RIIIA with immobilized Vitaxin.RTM. and Vitaxin.RTM. Fc
variant IgGs were monitored by surface plasmon resonance detection
using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala,
Sweden). Vitaxin.RTM. and Vitaxin.RTM. Fc variant IgGs were coupled
to the dextran matrix of a CM5 sensor chip (Pharmacia Biosensor)
using an Amine Coupling Kit, as described (Johnsson et al., 1992,
Anal Biochem 198:268-277), at a surface density of between
approximately 8200 and 9400 RUs. Fc.gamma.RIIIA was serially
diluted in 0.01 M HEPES pH 7.4 containing 0.15 M NaCl, 3 mM EDTA
and 0.005% P20, at concentrations ranging from 16 .mu.M down to 7.8
nM. Duplicate injections of each concentration were made. All
binding experiments were performed at 25.degree. C., and at a flow
rate of 10 .mu.L/min. Binding was monitored for 25 min. Following
each injection of Fc.gamma.RIIIA, the IgG surfaces were regenerated
with a 30 sec. pulse of 5 mM HCl. Fc.gamma.RIIIA was also passed
over a blank reference cell which is connected, in series, to the
IgG-containing flow cells. The steady-state binding curves were
also corrected for injection artifacts by subtraction of buffer
injections. This doubly-corrected data was then fit to a
steady-state isotherm provided by the instrument manufacturer
(Pharmacia Biosensor, Uppsala, Sweden) to derive the respective
equilibrium binding constants (K.sub.D). Separately, a Scatchard
plot of the Req data from each IgG surface was constructed to
confirm the results of the binding isotherms.
[0299] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay:
Antibody-dependent cell cytotoxicity (ADCC) was assayed in a
four-hour non-radioactive lactate dehydrogenase (LDH) release assay
(Promega Corporation, Madison, Wis.). Briefly, M21, A549, or H358
target cells were distributed into 96-well U-bottomed plates
(1.times.10.sup.4/50 .mu.l) and pre-incubated with serial dilution
of antibodies (50 .mu.l) for 20 min at 37.degree. C. Human effector
cells (100 .mu.l) were then added at effector to target ratios of
50:1 and 25:1. Human effector cells were peripheral blood
mononuclear cells (PBMC) purified from healthy donors using
Lymphocyte Separation Medium (MP Biomedicals, Irvine, Calif.).
After a 4-h incubation at 37.degree. C., plates were centrifuged,
and cell death was analyzed by measuring the release of LDH into
the cell supernatant with a 30-minute coupled enzymatic assay. The
percentage of specific lysis was calculated according to the
formula: % specific
lysis=100.times.(E.sub.x-E.sub.spon-T.sub.spon)/(T.sub.max-T.sub-
.spon) where E.sub.x represents the release from experimental
wells, E.sub.spon is the spontaneous release of effector cells
alone, T.sub.spon is spontaneous release of target cells alone, and
T.sub.max is the maximum release from lysed target cells.
[0300] The cell lines used for the ADCC studies included the
following: A498 and ACHN renal cell carcinomas with moderate
expression of Integrin .alpha.V.beta.3, M21 a melanoma cell line
with high Integrin .alpha..sub.v.beta..sub.3 expression, DU145 a
prostate cancer cell line with low levels of Integrin
.alpha.V.beta.3, SKMEL28 a human melanoma expressing Integrin
.alpha..sub.v.beta..sub.3 but little or no human EphA2.
7.4 Example 4
Fe Variants of Antibodies Recognizing Other Epitopes
[0301] Given the remarkable improvement in ADCC activity of the
Vitaxin.RTM. Fc (I332E) variant the (I332E) substitution was made
in two other antibodies designated 12G3H11 (abbreviated 12G3) and
3F2, both of which bind the EphA2 tyrosine receptor kinase. The
variable regions of 12G3 (FIG. 2A) and 3F2 (FIG. 3A) heavy chain
were fused to the wt and variant Fc domains generated above (see
sections 7.1 and 7.3). The variable region of the light chain of
Vitaxin.RTM. was replaced with the corresponding light chain
variable region (i.e., 12G3 or 3F2, see FIGS. 2B and 3B,
resectively) such that an intact 12G3 or 3F2 antibody was encoded
by the plasmid (see FIG. 4 for a map of the plasmid encoding
Vitaxin.RTM.). The antibodies containing the single substitutions
were designated 12G3-1M and 3F2-1M, respectively. In addition, the
S239D, A330L, I332L triple substitution was generated in 3F2,
designated 3F2-3M.
[0302] The binding characteristics of the 3F2-wt, 3F2-1M and 3F2-3M
Fc variants to several Fc ligands were examined in vitro by ELISA
(FIG. 12). Representative binding curves for 3F2 and the Fc
variants of 3F2 (3F2-1 M and 3F2-3M) to Fc.gamma.RIIIA tetramers
(FIG. 12, top panel), Fc.gamma.RIIIA monomers (FIG. 12, middle
panel) and C1q (FIG. 12, bottom panel). From these data it can be
seen that both the 3F2 Fc variants have improved binding to the
monomeric and tetrameric forms of Fc.gamma.RIIIA. In contrast both
the 3F2 Fc variants have reduced C1q binding with 3F2-3M having the
largest reduction in C1q binding (FIG. 12, bottom panel).
[0303] The binding of the 3F2 and the 3F2 Fc variants to
Fc.gamma.RIIIA was further analyzed by BIAcore analysis. The
binding of 3F2 and the 3F2 Fc variant was analyzed with the
receptor soluble and the antibody immobile (see methods below). The
data obtained for 3F2 and the 3F2 Fc variants (Run 3) is similar to
that obtained for Vitaxin.RTM. and the Vitaxin.RTM. Fc variants
(Runs 1 & 2) with improvements in binding of about 7 fold and
about 80 fold for the Vitaxin.RTM. 1M and 3M Fc variants,
respectively, and about 9 fold and about 100 fold for the 3F2-1M
and 3M Fc variants, respectively. The small differences between
these numbers may reflect subtle differences in glycosylation
between antibody produced in 293H cells vs NSO cells (Vitaxin
antibodies and 3F2 antibodies, respectively) as the variable domain
is generally not thought to affect Fc.gamma.RIIIA binding. The
results are summarized in Table 5.
[0304] The binding of 3F2-wt, 3F2-1M and 3F2-3M Fc variants to the
surface of cells via Fc ligand interactions was examined. Two cell
types were utilized, THP-1 cells and NK cells. To determine which
Fc ligands were present on the surface both cell types were stained
with antibodies recognizing CD32 (Fc.gamma.RII); CD64 (Fc.gamma.RI)
or CD16 (Fc.gamma.III) and analyzed by FACS. The percent of cell
staining positive for each Fc ligand are plotted in FIG. 13. As can
be seen in FIG. 14 panel A, THP-1 cells predominantly express CD32
with a small amount of CD64 present on the cell surface. In
contrast NK cells express CD16 almost exclusively (FIG. 13, panel
B). All three versions of 3F2 (wt, 1M and 3M) bound to a similar
degree to THP-1 cells (FIG. 13, panel C). However, the two Fc
variants (3F2-IM and 3F2-3M) were seen to bind to a greater extent
to NK cells, with the 3F2-3M Fc variant showing the largest
increase in binding (FIG. 13, panel D).
[0305] The ADCC activity of all the variants was examined. Shown in
FIGS. 14A and 14B are ADCC assays performed using the 12G3H11-Fc
(I332E) variant and the parental 12G3H11 antibody against A549
target cells using effector cells from two donors. The assays were
performed using two different ratios of target to effector cell
(50:1 and 25: 1) and antibody concentrations ranging from 4 to 400
ng per well. Remarkably, a 10 fold increase in ADCC activity is
seen for the 12G3H11-Fc (I332E) variant compared to the parent
antibody.
[0306] FIGS. 15, 16 and 17 are ADCC assays comparing the activity
of 3F2-wt and the 3F2 Fc variants against target cells expressing
different levels of EphA2. The target cell lines used were T23,1
A549 and Hey8 (high expressors), SKOV3 (a moderate expressor), A498
and SKMEL28 (low expressors). The assays were performed using three
different ratios of target to effector cell (between 12.5:1 and
100:1) and antibody concentrations ranging from 0.02 to 2 .mu.g/ml.
In all cases the ADCC activity of the 3F2-3M Fc variant was seen to
be higher than wild type 3F2. The activity of the 3F2-1M Fc variant
was also higher than the 3F2-wt.
7.4.1 Materials and Methods
[0307] Generation of 12G3 and 3F2 Fc variants: To generate the 12G3
and 3F2 Fc variants, the DNA sequences encoding the variable region
of Vitaxin.RTM. 1M or 3M heavy chain (VH) was replaced with the
variable region of 12G3 or 3F2 heavy chain to create 12G3-1M,
3F2-1M and 3F2-3M Fc variants using Xba I/Apa I restriction sites
(see plasmid map, FIG. 4). The DNA sequences encoding the variable
region of Vitaxin.RTM. light chain were also replaced with the
variable region of 12G3 or 3F2 light chain using SmaII/BsiWI
restriction sites (see plasmid map, FIG. 4). The nucleotide
sequence of the 12G3 heavy and light chain variable regions are
listed as SEQ ID NO.: 62 and 63 respectively. The amino acid
sequence of the 12G3 heavy and light chain variable regions are
listed as SEQ ID NO.: 64 and 65 respectively. The nucleotide
sequence of the 3F2 heavy and light chain variable regions are
listed as SEQ ID NO.: 66 and 67 respectively. The amino acid
sequence of the 3F2 heavy and light chain variable regions are
listed as SEQ ID NO.: 68 and 69 respectively.
[0308] The plasmid DNA containing the 12G3 antibody genes was
stably transfected into 293H cells by Lipofectamine 2000
Transfection Reagent (Invitrogen). The plasmid DNA containing the
3F2 antibody genes was stably transfected into NSO by
electroporation. Antibodies were purified from cell culture
supernatants by using a pre-packed Protein A column (Amersham
Biosciences, now belongs to GE healthcare). The bound antibody were
eluted with elution buffer (100 mM Glycine, pH3.2), neutralized by
1M Tris buffer (pH 8.0) and then dialyzed in PBS. All purified
antibodies were analyzed by SDS-polyacrylamide gel electrophoresis
and were applied to quantitative ELISA using anti-human IgG assay
plates (Becton Dickson) or BCA kits (PIERCE) to determine IgG
concentrations.
[0309] Kinetic Analysis via BIAcore: for Run 3 the interaction of
Fc.gamma.RIIIA with immobilized Vitaxin.RTM. and Vitaxin.RTM. Fc
variant IgGs were monitored by surface plasmon resonance detection
using a BIAcore 3000 instrument (Pharmacia Biosensor, Uppsala,
Sweden). Vitaxin.RTM. and Vitaxin.RTM. Fc variant IgGs were coupled
to the dextran matrix of a CM5 sensor chip (Pharmacia Biosensor)
using an Amine Coupling Kit, as described (Johnsson et al., 1992,
Anal Biochem 198:268-277), at a surface density of between
approximately 7700 and 9400 RUs (see Table 5). Fc.gamma.RIIIA was
serially diluted in 0.01 M HEPES pH 7.4 containing 0.15 M NaCl, 3
mM EDTA and 0.005% P20, at concentrations ranging from 16 uM down
to 7.8 nM. Duplicate injections of each concentration were made.
All binding experiments were performed at 25.degree. C., and at a
flow rate of 10 uL/min. Binding was monitored for 25 min. Following
each injection of Fc.gamma.RIIIA, the IgG surfaces were regenerated
with a 30 sec. pulse of 5 mM HCl. Fc.gamma.RIIIA was also passed
over a blank reference cell which is connected, in series, to the
IgG-containing flow cells. The steady-state binding curves were
also corrected for injection artifacts by subtraction of buffer
injections. This doubly-corrected data was then fit to a
steady-state isotherm provided by the instrument manufacturer
(Pharmacia Biosensor, Uppsala, Sweden) to derive the respective
equilibrium binding constants (K.sub.D). Separately, a Scatchard
plot of the Req data from each IgG surface was constructed to
confirm the results of the binding isotherms.
[0310] Cell Surface Binding: NK cells were isolated from healthy
donor by using NK cell isolation kit from Miltenybiotec (Cat#
130-091-152) THP-1: early passage of THP-1 cells were used. For
FACS staining of Fc.gamma.Rs, either THP-1 or human NK cells were
resuspended in FACS buffer (1% BSA in PBS, pH 7.2) at
1.times.10.sup.6 cells/ml and 0.5 ml of the cells were transfered
into 96 deep well plate, 10 .mu.l of the anti-CD32-PE (Immunotech),
anti-CD16-FITC (Pharmingen) or anti-CD64-FITC (PharMingen) was
added to the tubes. The samples were incubated at 40C for 30 min.
After incubation, the cells were washed with FACS buffer and the
samples were analyzed by using Guava EasyCyte
[0311] For binding of antibody 3F2 to Human NK cell surface
(Fc.gamma.RIIIA), 10 .mu.l of the antibody dilution (10 .mu.g/ml or
1 .mu.g/ml) was added to the cells and incubated at 4.degree. C.
for 30 min. The cells were washed with FACS buffer, then stained
with goat ant-human IgG (H+L)-FITC (Pierce) for 30 min at 4.degree.
C. The cells were washed and analyzed by Guava EasyCyte.
[0312] For binding of antibody 3F2 to THP-1 cell surface
(Fc.gamma.RI and Fc.gamma.RII), 10 .mu.l of the antibody dilution
(10 .mu.g/ml or 1 .mu.g/ml) were added to the cells, incubatee at
4.degree. C. for 30 min. The cells were washed with FACS buffer,
then stained with goat ant-human IgG (H+L)-FITC (Pierce) for 30 min
at 4.degree. C. The cells were washed and analyzed by Guava
EasyCyte.
[0313] ELISA for Fc.gamma.RIIIA Tetramer Binding: Microtiter plates
were coated with protein A/G (PIERCE) solution (0.25 .mu.g/ml) and
incubated at 4.degree. C. overnight. The plates were then washed
with PB S/0.1% Tween and any remaining binding sites were blocked
with 1% BSA. 50 .mu.l of test antibody at 1:1 dilution (from 5000
ng/ml to 4.9 ng/ml), was added to each well and inclubated for 60
min at 37.degree. C. 50 .mu.l of 1:500 dilution of the
Fc.gamma.tetramer was added to each well and incubated for 60 min
at 37.degree. C. followed by washing. 50 .mu.l of 1:1000 dilution
of biotin-conjugated HRP (PIERCE) was added to each well and
incubated for 30 min at 37.degree. C. Detection was carried out by
adding 30 .mu.l of tetramethylbenzidine (TMB) substrate (Pierce)
followed by neutralization with 30 .mu.l of 0.2 M H.sub.2SO.sub.4.
The absorbance was read at 450 nm.
[0314] ELISA for Fc.gamma.RIIIA Monomer Binding: Microtiter plates
were coated with 50 .mu.l to test antibody at concentration range
from 20 .mu.g/ml to 0.0019 .mu.g/ml and incubated at 4.degree. C.
overnight. 50 .mu.l of 10 .mu.g/ml Fc.gamma.RIIIA-flag protein was
added to each well and incubated for 60 min at 37.degree. C. 50
.mu.l of 2.5 .mu.g/ml anti-flag-ME-biotin (Sigma) was added to each
well and incubated for 30 min at 37.degree. C. 50 .mu.l of 1:1000
diulation of avidin-conjugated HRP (PIERCE) was added to each well
and incubated for 30 min at 37.degree. C. Detection was carried out
by adding 30 .mu.l of tetramethylbenzidine (TMB) substrate (Pierce)
followed by neutralization with 30 .mu.l of 0.2 M H.sub.2SO.sub.4.
The absorbance was read at 450 nm.
[0315] ELISA for C1q Binding: Microtiter plates were coated with 50
.mu.l of test antibody at concentration range from 20 .mu.g/ml to
0.0019 g/ml and incubated at 4.degree. C. overnight. The plate was
then blocked with 5% nonfat powdered milk for 60 min at 37.degree.
C. 50 .mu.l of 5 .mu.g/ml human C1q complement protein (Quidal,
SanDiego) was added to each well and inclubated for 60 min at
37.degree. C. 50 .mu.l of 1:1000 dilution of anti-complement C1q
antibody (Biodesign) was added to each well and incubated for 60
min at 37.degree. C. 50 .mu.l of 1:1000 dilution of donkey
anti-sheep/goat antibody-conjugated HRP (PIERCE) was added to each
well and incubated for 60 min at 37.degree. C. Detection was
carried out by adding 30 .mu.l of tetramethylbenzidine (TMB)
substrate (Pierce) followed by neutralization with 30 .mu.l of 0.2
M H.sub.2SO.sub.4. The absorbance was read at 450 nm.
[0316] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay:
Antibody-dependent cell cytotoxicity (ADCC) was assayed as
described above in section 7.3.1 using different target cells. The
target cell lines used for these assays are A549 a human non-small
cell lung adenocarcinoma cell line expressing high levels of human
EphA2, T231 a more metastatic variant of MDA-MB-231 human breast
adenocarcinoma cell line obtained from collaborator Kathy Miller at
Indiana University Medical Center expressing high levels of human
EphA2, HeyA8 a human ovarian carcinoma expressing high levels of
human EphA2, SKOV3 a human ovarian adenocarcinoma derived from
ascites expressing moderate levels of human EphA2, A498 a human
renal cell carcinoma expressing low levels of human EphA2, SKMEL28
a human melanoma expressing Integrin .alpha..sub.v.beta..sub.3 but
little or no human EphA2.
[0317] Whereas, particular embodiments of the invention have been
described above for purposes of description, it will be appreciated
by those skilled in the art that numerous variations of the details
may be made without departing from the invention as described in
the appended claims.
[0318] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference. In addition, U.S.
Provisional Patent Application Nos.: 60/601,634, filed, Aug. 16,
2004 and 60/608,852, filed, Sep. 13, 2004, and U.S. patent
application entitled "Eph Receptor Fc Variants With Enhanced
Antibody Dependent Cell-Mediated Cytotoxicity Activity," Attorney
Docket No.: AE702US, filed Aug. 15, 2005, are incorporated by
reference in their entirety
Sequence CWU 1
1
75 1 351 DNA Artificial recombinant antibody variable region 1
caggtgcagc tggtggagtc tgggggaggc gttgtgcagc ctggaaggtc cctgagactc
60 tcctgtgcag cctctggatt caccttcagt agctatgaca tgtcttgggt
tcgccaggct 120 ccgggcaagg gtctggagtg ggtcgcaaaa gttagtagtg
gtggtggtag cacctactat 180 ttagacactg tgcagggccg attcaccatc
tccagagaca atagtaagaa caccctatac 240 ctgcaaatga actctctgag
agccgaggac acagccgtgt attactgtgc aagacatctg 300 catggcagtt
ttgcttcttg gggccaaggg actacagtga ctgtttctag t 351 2 321 DNA
Artificial recombinant antibody variable region 2 gagattgtgc
taactcagtc tccagccacc ctgtctctca gcccaggaga aagggcgact 60
ctttcctgcc aggccagcca aagtattagc aacttcctac actggtatca acaaaggcct
120 ggtcaagccc caaggcttct catccgctat cgttcccagt ccatctctgg
gatccccgcc 180 aggttcagtg gcagtggatc agggacagat ttcaccctca
ctatctccag tctggagcct 240 gaagattttg cagtctatta ctgtcaacag
agtggcagct ggcctctgac gttcggaggg 300 gggaccaagg tggaaattaa g 321 3
117 PRT Artificial recombinant antibody variable region 3 Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Lys Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Leu
Asp Thr Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr 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 Arg His Leu His Gly Ser
Phe Ala Ser Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser
115 4 107 PRT Artificial recombinant antibody variable region 4 Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Gln Ala Ser Gln Ser Ile Ser Asn Phe
20 25 30 Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Arg Tyr Arg Ser Gln Ser Ile Ser Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Ser Gly Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 5 5 PRT Mus musculus 5 Ser Tyr Asp Met
Ser 1 5 6 17 PRT Mus musculus 6 Lys Val Ser Ser Gly Gly Gly Ser Thr
Tyr Tyr Leu Asp Thr Val Gln 1 5 10 15 Gly 7 8 PRT Mus musculus 7
His Asn Tyr Gly Ser Phe Ala Tyr 1 5 8 11 PRT Mus musculus 8 Gln Ala
Ser Gln Ser Ile Ser Asn His Leu His 1 5 10 9 7 PRT Mus musculus 9
Tyr Arg Ser Gln Ser Ile Ser 1 5 10 9 PRT Mus musculus 10 Gln Gln
Ser Gly Ser Trp Pro His Thr 1 5 11 39 DNA Artificial synthetic
primer 11 ccgtgcccag cacctgaann kctgggggga ccgtcagtc 39 12 42 DNA
Artificial synthetic primer 12 ccgtgcccag cacctgaact cnnkggggga
ccgtcagtct tc 42 13 45 DNA Artificial synthetic primer 13
ccgtgcccag cacctgaact cctgnnkgga ccgtcagtct tcctc 45 14 48 DNA
Artificial synthetic primer 14 ccgtgcccag cacctgaact cctggggnnk
ccgtcagtct tcctcttc 48 15 51 DNA Artificial synthetic primer 15
ccgtgcccag cacctgaact cctgggggga nnktcagtct tcctcttccc c 51 16 54
DNA Artificial synthetic primer 16 ccgtgcccag cacctgaact cctgggggga
ccgnnkgtct tcctcttccc ccca 54 17 39 DNA Artificial synthetic primer
17 gtcacatgcg tggtggtgnn kgtgagccac gaagaccct 39 18 45 DNA
Artificial synthetic primer 18 gtcacatgcg tggtggtgga cgtgnnkcac
gaagaccctg aggtc 45 19 51 DNA Artificial synthetic primer 19
gtcacatgcg tggtggtgga cgtgagccac nnkgaccctg aggtcaagtt c 51 20 39
DNA Artificial synthetic primer 20 cgggaggagc agtacaacnn kacgtaccgt
gtggtcagc 39 21 39 DNA Artificial synthetic primer 21 tgcaaggtct
ccaacaaann kctcccagcc cccatcgag 39 22 42 DNA Artificial synthetic
primer 22 tgcaaggtct ccaacaaagc cnnkccagcc cccatcgaga aa 42 23 45
DNA Artificial synthetic primer 23 tgcaaggtct ccaacaaagc cctcnnkgcc
cccatcgaga aaacc 45 24 48 DNA Artificial synthetic primer 24
tgcaaggtct ccaacaaagc cctcccannk cccatcgaga aaaccatc 48 25 54 DNA
Artificial synthetic primer 25 tgcaaggtct ccaacaaagc cctcccagcc
cccnnkgaga aaaccatctc caaa 54 26 33 DNA Artificial synthetic primer
26 actcacacat gtccaccgtg cccagcacct gaa 33 27 18 DNA Artificial
synthetic primer 27 caccaccacg catgtgac 18 28 18 DNA Artificial
synthetic primer 28 gttgtactgc tcctcccg 18 29 18 DNA Artificial
synthetic primer 29 tttgttggag accttgca 18 30 24 DNA Artificial
synthetic primer 30 aacctctaca aatgtggtat ggct 24 31 42 DNA
Artificial synthetic primer 31 aagcttcggt ccgccaccat ggcaactgaa
gatctcccaa ag 42 32 39 DNA Artificial synthetic primer 32
gtctgccgaa ccgctgcctg ccaaaccttg agtgatggt 39 33 48 DNA Artificial
synthetic primer 33 agcttcggtc cgccaccatg gctgtgctat tcctggcagc
tcccccaa 48 34 42 DNA Artificial synthetic primer 34 gtctgccgaa
ccgctgcccc ccatcggtga agagctggga gc 42 35 30 DNA Artificial
synthetic primer 35 ggcagcggtt cggcagaccc ctccaaggac 30 36 35 DNA
Artificial synthetic primer 36 caggggctag cttactgctg aacggcgtcg
agcgg 35 37 39 DNA Artificial synthetic primer 37 tccacaggtg
tccactcccg gactgaagat ctcccaaag 39 38 91 DNA Artificial synthetic
primer 38 gggagaattc cgcggccgct tatttgtcat cgtcatcttt gtagtcatgg
tgatggtgat 60 ggtgtgcgcc tgccaaacct tgagtgatgg t 91 39 48 DNA
Artificial synthetic primer 39 tccacaggtg tccactccgc tgtgctattc
ctggcagctc ccccaaag 48 40 94 DNA Artificial synthetic primer 40
gggagaattc cgcggccgct tatttgtcat cgtcatcttt gtagtcatgg tgatggtgat
60 ggtgtgcgcc ccccatcggt gaagagctgg gagc 94 41 33 DNA Artificial
synthetic primer 41 gccctcccag cccccgagga gaaaaccatc tcc 33 42 33
DNA Artificial synthetic primer 42 gccctcccag ccccccagga gaaaaccatc
tcc 33 43 33 DNA Artificial synthetic primer 43 gccctcccag
cccccggcga gaaaaccatc tcc 33 44 33 DNA Artificial synthetic primer
44 gccctcccag cccccgccga gaaaaccatc tcc 33 45 33 DNA Artificial
synthetic primer 45 gccctcccag ccccctacga gaaaaccatc tcc 33 46 33
DNA Artificial synthetic primer 46 gccctcccag cccccgacga gaaaaccatc
tcc 33 47 33 DNA Artificial synthetic primer 47 gccctcccag
cccccaacga gaaaaccatc tcc 33 48 33 DNA Artificial synthetic primer
48 gccctcccag cccccgtgga gaaaaccatc tcc 33 49 33 DNA Artificial
synthetic primer 49 gccctcccag ccccctggga gaaaaccatc tcc 33 50 33
DNA Artificial synthetic primer 50 gccctcccag ccccccgcga gaaaaccatc
tcc 33 51 33 DNA Artificial synthetic primer 51 gccctcccag
cccccagcga gaaaaccatc tcc 33 52 33 DNA Artificial synthetic primer
52 gccctcccag cccccaagga gaaaaccatc tcc 33 53 33 DNA Artificial
synthetic primer 53 gccctcccag cccccatgga gaaaaccatc tcc 33 54 33
DNA Artificial synthetic primer 54 gccctcccag cccccaccga gaaaaccatc
tcc 33 55 33 DNA Artificial synthetic primer 55 gccctcccag
ccccctgcga gaaaaccatc tcc 33 56 33 DNA Artificial synthetic primer
56 gccctcccag cccccctgga gaaaaccatc tcc 33 57 33 DNA Artificial
synthetic primer 57 gccctcccag cccccttcga gaaaaccatc tcc 33 58 33
DNA Artificial synthetic primer 58 gccctcccag ccccccacga gaaaaccatc
tcc 33 59 33 DNA Artificial synthetic primer 59 gccctcccag
ccccccctga gaaaaccatc tcc 33 60 27 DNA Artificial synthetic primer
60 ctggggggac cggacgtctt cctcttc 27 61 27 DNA Artificial synthetic
primer 61 aaagccctcc cactgcccga ggagaaa 27 62 360 DNA Artificial
recombinant antibody variable region 62 caaatgcagc tggtgcagtc
tgggcctgag gtgaagaagc ctgggacctc agtgaaggtc 60 tcctgcaagg
cttctggatt cacctttgac gattactcca tgaactgggt gcgacaggct 120
cgtggacaac gccttgagtg gataggattt attagaaaca aagctaatga ctacacaaca
180 gagtacgctg actctgtgaa gggtagagtc accattacca gggacatgtc
cacgagcaca 240 gcctacatgg agctgagcag cctgagatcc gaggacacgg
ccgtgtatta ctgtgcgaga 300 taccctaggc atcatgctat ggactcctgg
ggccaaggaa cctcggtcac cgtctcctca 360 63 321 DNA Artificial
recombinant antibody variable region 63 gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
gggccagcca aagtattagc aacaacctac actggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatcaagtat gccttccagt ccatctctgg ggtcccatca
180 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag
cctgcagcct 240 gaagattttg caacatatta ctgtcaacag gccaacagct
ggccgctcac gttcggcgga 300 gggaccaagg tggagatcaa a 321 64 120 PRT
Artificial recombinant antibody variable region 64 Gln Met Gln Leu
Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30
Ser Met Asn Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile 35
40 45 Gly Phe Ile Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ala
Asp 50 55 60 Ser Val Lys Gly Arg Val Thr Ile Thr Arg Asp Met Ser
Thr Ser Thr 65 70 75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Tyr Pro Arg His His
Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser
Ser 115 120 65 107 PRT Artificial recombinant antibody variable
region 65 Asp 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 Ser
Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Phe Gln Ser Ile Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Trp Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 66 361 DNA Artificial
recombinant antibody variable region 66 gaggtgcagc tggtggagtc
tgggggaggt gtggtacggc ctggggggtc cctgagactc 60 tcctgtgcag
cctctgggtt caccgtcagt gattactcca tgaactgggt ccgccaggct 120
ccagggaagg gcctggagtg gattgggttt attagaaaca aagctaatgc ctacacaaca
180 gagtacagtg catctgtgaa gggtagattc accatctcaa gagatgattc
aaaaaacacg 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacag
ccgtgtatta ctgtaccaca 300 taccctaggt atcatgctat ggactcctgg
ggccagggca ccatggtcac cgtctcctca 360 g 361 67 321 DNA Artificial
recombinant antibody variable region 67 gccatccagt tgactcagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgca
gggccagcca aagtattagc aacaacctac actggtacct gcagaagcca 120
gggcagtctc cacagctcct gatctattat ggcttccagt ccatctctgg ggtcccatca
180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
tctgcaacct 240 gaagattttg caacttacta ctgtcaacag gccaacagct
ggccgctcac gttcggcgga 300 gggaccaagc tggagatcaa a 321 68 120 PRT
Artificial recombinant antibody variable region 68 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Phe Ile Arg Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser
Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Tyr Pro Arg Tyr His
Ala Met Asp Ser Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser
Ser 115 120 69 107 PRT Artificial recombinant antibody variable
region 69 Ala Ile Gln Leu 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 Ser
Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Tyr Gly Phe Gln Ser Ile 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 Ala Asn Ser Trp Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 70 5 PRT Artificial
recombinant antibody CDR 70 Ser Tyr Asp Met Ser 1 5 71 17 PRT
Artificial recombinant antibody CDR 71 Lys Val Ser Ser Gly Gly Gly
Ser Thr Tyr Tyr Leu Asp Thr Val Gln 1 5 10 15 Gly 72 8 PRT
Artificial recombinant antibody CDR 72 His Leu His Gly Ser Phe Ala
Ser 1 5 73 11 PRT Artificial recombinant antibody CDR 73 Gln Ala
Ser Gln Ser Ile Ser Asn Phe Leu His 1 5 10 74 7 PRT Artificial
recombinant antibody CDR 74 Thr Arg Ser Gln Ser Ile Ser 1 5 75 9
PRT Artificial recombinant antibody CDR 75 Gln Gln Ser Gly Ser Tyr
Pro Leu Thr 1 5
* * * * *