U.S. patent application number 13/292300 was filed with the patent office on 2012-03-08 for anti- integrin antibodies, compositions, methods and uses.
Invention is credited to Jill Giles-Komar, Marian T. Nakada, Linda Snyder, Mohit Trikha.
Application Number | 20120058128 13/292300 |
Document ID | / |
Family ID | 38119015 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120058128 |
Kind Code |
A1 |
Giles-Komar; Jill ; et
al. |
March 8, 2012 |
ANTI- INTEGRIN ANTIBODIES, COMPOSITIONS, METHODS AND USES
Abstract
The present invention relates to at least one novel anti-alpha-V
subunit antibodies, including isolated nucleic acids that encode at
least one anti-alpha-V subunit antibody, alpha-V subunit, vectors,
host cells, transgenic animals or plants, and methods of making and
using thereof, including therapeutic compositions, methods and
devices.
Inventors: |
Giles-Komar; Jill;
(Bowningtown, PA) ; Snyder; Linda; (Pottstown,
PA) ; Trikha; Mohit; (Paoli, PA) ; Nakada;
Marian T.; (Malvern, PA) |
Family ID: |
38119015 |
Appl. No.: |
13/292300 |
Filed: |
November 9, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11931533 |
Oct 31, 2007 |
8071729 |
|
|
13292300 |
|
|
|
|
11598411 |
Nov 13, 2006 |
7550142 |
|
|
11931533 |
|
|
|
|
10720323 |
Nov 24, 2003 |
7163681 |
|
|
11598411 |
|
|
|
|
09920267 |
Aug 1, 2001 |
7288390 |
|
|
10720323 |
|
|
|
|
60223363 |
Aug 7, 2000 |
|
|
|
Current U.S.
Class: |
424/172.1 ;
435/375 |
Current CPC
Class: |
A61P 25/18 20180101;
C07K 2317/21 20130101; A01K 2267/01 20130101; A61P 17/06 20180101;
A61P 35/00 20180101; G01N 33/5029 20130101; C07K 16/2848 20130101;
A61P 9/00 20180101; A61P 37/02 20180101; A61P 11/06 20180101; A61P
37/00 20180101; A61K 2039/505 20130101; A61P 23/02 20180101; A61P
25/02 20180101; G01N 2333/70546 20130101; A61P 29/00 20180101; C07K
16/4258 20130101; A61P 5/04 20180101; A61P 25/20 20180101; Y02A
50/412 20180101; C07K 2317/92 20130101; A61P 23/00 20180101; Y02A
50/30 20180101; A61P 5/00 20180101; G01N 33/5008 20130101; C07K
2317/76 20130101; A61P 21/02 20180101; A61P 31/00 20180101; A61P
43/00 20180101; C07K 2317/565 20130101; A61P 5/44 20180101; A61P
25/24 20180101; A61P 25/00 20180101; A01K 2217/05 20130101; C07K
16/2839 20130101; G01N 33/68 20130101; Y02A 50/58 20180101; A61P
37/04 20180101; A61P 37/06 20180101 |
Class at
Publication: |
424/172.1 ;
435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C12N 5/071 20100101
C12N005/071 |
Claims
1. A method for modulating angiogenesis in a cell, tissue, organ or
animal, comprising administering a composition comprising an
effective amount of an isolated human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof, to said cell, tissue,
organ or animal
2. The method of claim 1 wherein the isolated human antibody or
antigen-binding fragment binds to human .alpha..sub.v integrin
subunit with a Kd of 10.sup.-8 M or less.
3. The method of claim 1 wherein the isolated human anti-.alpha.V
subunit antibody or antigen-binding fragment thereof, wherein the
heavy chain variable region sequences is derived from the heavy
chain CNTO 95 variable region sequence of SEQ ID NO.7 and the light
chain variable region sequences is derived from the light chain
CNTO 95 variable region sequence of SEQ ID NO.8.
4. The method of claim 1 wherein the isolated human antibody or
antigen-binding fragment comprises human heavy chain CDR sequences
of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and human light chain
CDR sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
5. The method of claim 1 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof completely inhibits
M21 cell adhesion to vitronectin.
6. The method of claim 1 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises a human IgG
heavy chain and a human kappa light chain.
7. The method of claim 1 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises an IgG1 or
IgG3 heavy chain.
8. A method for inhibiting microcapillary formation in a cell,
tissue, organ or animal, comprising administering a composition
comprising an effective amount of an isolated human anti-.alpha.V
subunit antibody or antigen-binding fragment thereof, to said cell,
tissue, organ or animal.
9. The method of claim 8 wherein the isolated human antibody or
antigen-binding fragment binds to human .alpha..sub.v integrin
subunit with a Kd of 10.sup.-8 M or less.
10. The method of claim 8 wherein the isolated human anti-.alpha.V
subunit antibody or antigen-binding fragment thereof, wherein the
heavy chain variable region sequences is derived from the heavy
chain CNTO 95 variable region sequence of SEQ ID NO.7 and the light
chain variable region sequences is derived from the light chain
CNTO 95 variable region sequence of SEQ ID NO.8.
11. The method of claim 8 wherein the isolated human antibody or
antigen-binding fragment comprises human heavy chain CDR sequences
of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and human light chain
CDR sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
12. The method of claim 8 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof completely inhibits
M21 cell adhesion to vitronectin.
13. The method of claim 8 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises a human IgG
heavy chain and a human kappa light chain.
14. The method of claim 8 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises an IgG1 or
IgG3 heavy chain.
15. A method for treating malignant disease in an animal in need
thereof, comprising administering a composition comprising an
effective amount of an isolated human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof, to said cell, tissue,
organ or animal
16. The method of claim 15 wherein the isolated human antibody or
antigen-binding fragment binds to human .alpha..sub.v integrin
subunit with a Kd of 10.sup.-8 M or less.
17. The method of claim 15 wherein the isolated human antibody or
antigen-binding fragment comprises human heavy chain CDR sequences
of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and human light chain
CDR sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
18. The method of claim 15 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof completely inhibits
M21 cell adhesion to vitronectin.
19. The method of claim 15 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises a human IgG
heavy chain and a human kappa light chain.
20. The method of claim 15 wherein the human anti-.alpha.V subunit
antibody or antigen-binding fragment thereof comprises an IgG1 or
IgG3 heavy chain.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/931,533, filed 31 Oct. 2007, currently allowed, which is a
continuation of U.S. application Ser. No. 11/598,411, filed 13 Nov.
2006, which is a divisional of U.S. application Ser. No. 10/720,323
filed Nov. 24, 2003, now issued under U.S. Pat. No. 7,163,681,
which is a continuation-in-part of U.S. application Ser. No.
09/920,267, filed 1 Aug. 2001, which claims priority to U.S.
provisional application 60/223,363 filed 7 Aug. 2000, each of which
are entirely incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies which bind to
the alpha-V subunit of the integrin family of cell adhesion
receptors, including specified portions or variants thereof The
antibodies of the invention are specific for at least one alpha-V
subunit of a heterodimeric integrin receptor, such as an
alpha-V-beta-1, alpha-V-beta-3, alpha-V-beta-5, alpha-V-beta-6, or
alpha V-beta-8 heterodimeric integrin protein or fragment thereof.
The invention also relates to nucleic acids encoding such
anti-alpha-V subunit antibodies, complementary nucleic acids,
vectors, host cells, and methods of making and using thereof,
including therapeutic formulations, administration and devices.
RELATED ART
[0003] Integrins are a superfamily of cell adhesion receptors,
which exist as heterodimeric transmembrane glycoproteins. They are
part of a large family of cell adhesion receptors which are
involved in cell-extracellular matrix and cell-cell interactions.
Integrins play critical roles in cell adhesion to the extracellular
matrix (ECM) which, in turn, mediates cell survival, proliferation
and migration through intracellular signaling. The receptors
consist of two subunits that are non-covalently bound. Those
subunits are called alpha and beta. The alpha subunits all have
some homology to each other, as do the beta subunits. The receptors
always contain one alpha chain and one beta chain and are thus
called heterodimeric. Both of the subunits contribute to the
binding of ligand. Eighteen alpha subunits and eight beta subunits
have been identified, which heterodimerize to form at least 24
distinct integrin receptors.
[0004] Among the variety of alpha chain subunits is a protein chain
referred to as alpha V. The ITAGV gene encodes integrin alpha chain
V (alphaV). The I-domain containing integrin alpha V undergoes
post-translational cleavage to yield disulfide-linked heavy and
light chains, that combine with multiple integrin beta chains to
form different integrins. Alternative splicing of the gene yields 7
different transcripts; a, b, c, e, f, h, j altogether encoding 6
different protein isoforms of alphaV. Among the known associating
beta chains (beta chains 1,3,5,6, and 8; `ITGB1`, `ITGB3`, `ITGB5`,
`ITGB6`, and `ITGB8`), each can interact with extracellular matrix
ligands. The alpha V beta 3 integrin, perhaps the most studied of
these, is referred to as the vitronectin receptor (VNR). In
addition to providing for cell attachment to other cells or to
extracellular proteins such as vitronectin (alphaVbeta3) and
fibronectin (alphaVbeta6), the integrins are capable of
intracellular signaling which provides clues for cell migration and
secretion of or elaboration of other proteins involved in cell
motility and invasion and angiogenesis. The alpha V integrin
subfamily of integrins recognize the ligand motif arg-gly-asp (RGD)
present in fibronection, vitronection, VonWillebrand factor, and
fibrinogen.
[0005] It has been established that integrins which are alpha-V
containing heterodimers, particularly alpha-V/beta-6, the receptor
for fibronectin, are involved in adhesion of carcinoma cells to
fibronectin and vitronectin. This is especially true for carcinoma
cells arising from the malignant progression of colon cancer
(Lehmann, M. et al. Cancer Res 1994, 54(8), 2102-7. Furthermore,
integrin expression in colon cancer cells is regulated by the
cytoplasmic domain of the beta-6 integrin subunit which signals
through the ERK2 pathway (Niu, J. et al. Int. J. Cancer 2002,
99(4), 529-537) and beta6 expression is associated with secretion
of gelatinase B, an enzyme involved in tumor cell invasion and
metastatic mechanisms (Agrez, et al. Int. J. Cancer 1999, 81(1),
90-97).
[0006] There is now considerable evidence that progressive tumor
growth is dependent upon angiogenesis, the formation of new blood
vessels, to provide tumors with nutrients and oxygen, to carry away
waste products and to act as conduits for the metastasis of tumor
cells to distant sites (Gastl et al., Oncol. 54:177-184). Recent
studies have further defined the roles of integrins in the
angiogenic process. During angiogenesis, a number of integrins that
are expressed on the surface of activated endothelial cells
regulate critical adhesive interactions with a variety of ECM
proteins to regulate distinct biological events such as cell
migration, proliferation and differentiation. Specifically, the
closely related but distinct integrins .alpha.V.beta.3 and
.alpha.V.beta.5 have been shown to mediate independent pathways in
the angiogenic process. An antibody generated against
.alpha.V.beta.3 blocked basic fibroblast growth factor (bFGF)
induced angiogenesis, whereas an antibody specific to
.alpha.V.beta.5 inhibited vascular endothelial growth factor (VEGF)
induced angiogenesis (Eliceiri, et al., J. Clin. Invest.
103:1227-1230 (1999); Friedlander et al., Science 270:1500-1502
(1995)). Therefore, integrins and especially the alpha V subunit
containing integrins, are reasonable therapeutic targets for
diseases that involve angiogenesis such as disease of the eye and
neoplastic disease, tissue remodeling such as restenosis, and
proliferation of certain cells types particularly epithelial and
squamous cell carcinomas.
[0007] Non-human mammalian, chimeric, polyclonal (e.g., anti-sera)
and/or monoclonal antibodies (Mabs) and fragments (e.g.,
proteolytic digestion or fusion protein products thereof) are
potential therapeutic agents that are being investigated in some
cases to attempt to treat certain diseases. However, such
antibodies or fragments can elicit an immune response when
administered to humans. Such an immune response can result in an
immune complex-mediated clearance of the antibodies or fragments
from the circulation, and make repeated administration unsuitable
for therapy, thereby reducing the therapeutic benefit to the
patient and limiting the readministration of the antibody or
fragment. For example, repeated administration of antibodies or
fragments comprising non-human portions can lead to serum sickness
and/or anaphalaxis. In order to avoid these and other problems, a
number of approaches have been taken to reduce the immunogenicity
of such antibodies and portions thereof, including chimerization
and humanization, as well known in the art. These and other
approaches, however, still can result in antibodies or fragments
having some immunogenicity, low affinity, low avidity, or with
problems in cell culture, scale up, production, and/or low yields.
Thus, such antibodies or fragments can be less than ideally suited
for manufacture or use as therapeutic proteins.
[0008] Accordingly, there is a need to provide human antibodies to
anti-integrin alpha-V subunit antibodies or fragments thereof that
overcome one or more of these problems, as well as improvements
over known antibodies or fragments thereof.
SUMMARY OF THE INVENTION
[0009] The present invention provides isolated human anti-integrin
alpha-V subunit antibodies, immunoglobulins, cleavage products and
other specified portions and variants thereof, as well as
anti-alpha-V subunit antibody compositions, encoding or
complementary nucleic acids, vectors, host cells, compositions,
formulations, devices, transgenic animals, transgenic plants, and
methods of making and using thereof, as described and enabled
herein, in combination with what is known in the art. The
antibodies of the invention bind the various forms of the alpha V
subunit of the integrin receptor with particular affinity and
specificity regardless of the various beta subunits of the integrin
heterodimer to which the alpha V subunit is paired. Accordingly,
the antibodies can be used in a variety of methods for diagnosing,
treating, and/or preventing diseases involving cell adhesion
mediated by the integrin receptor, particularly diseases involving
alpha V integrin mediated angiogenesis, such as prostate cancer,
colon cancer, and renal carcinoma.
[0010] Thus, in one embodiment, the present invention provides at
least one isolated anti-integrin alpha-V subunit antibody as
described herein. In one embodiment, the antibody according to the
present invention includes any protein or peptide containing
molecule that comprises at least a portion of a complementarity
determining region (CDR) of a heavy or light chain or a ligand
binding portion thereof derived from the antibody designated CNTO
95, in combination with a heavy chain or light chain variable
region, a heavy chain or light chain constant region, a framework
region, or any portion thereof, that can be incorporated into an
antibody of the present invention. The antibody CNTO 95 described
herein is a human anti-alpha V antibody derived from immunization
of a transgenic mouse containing genes for the expression of human
immunoglobulins. Thus, in one embodiment, the invention is directed
to antibodies containing at least one CDR region or variable region
derived from the CNTO 95 antibody. An antibody of the invention can
include or be derived from any mammal, such as but not limited to a
human, a mouse, a rabbit, a rat, a rodent, a primate, or any
combination thereof, and the like, or the antibody can be derived
from a synthetic source, such as a synthetic phage display
library.
[0011] Particular therapeutic antibodies of the invention include
human monoclonal antibody CNTO 95, and functionally equivalent
antibodies which have the human heavy chain and human light chain
amino acid sequences in their variable regions as set forth in SEQ
ID NO: 7 and SEQ ID NO: 8 respectively, and conservative
modifications thereof.
[0012] Still other particular human antibodies of the invention
include those which comprise a CDR domain having a human heavy and
light chain CDR1 region, a human heavy and light chain CDR2 region,
and a human heavy and light chain CDR3 region, wherein (a) the
CDRI, CDR2, and CDR3 of the human heavy chain regions comprise an
amino acid sequence selected from the group consisting of the amino
acid sequences of the CDR1, CDR2, and CDR3 regions shown in SEQ ID
NOs: 1, 2 and 3, and conservative sequence modifications thereof,
and (b) the CDRI, CDR2, and CDR3 of the human light chain regions
comprise an amino acid sequence selected from the group consisting
of the amino acid sequences of the CDRI, CDR2, and CDR3 regions
shown in SEQ ID Nos: 3, 4 and 5, and conservative sequence
modifications thereof The antibody amino acid sequence can further
optionally comprise at least one specified substitution, insertion
or deletion as described herein or as known in the art.
[0013] Other particular antibodies of the invention include human
monoclonal antibodies which bind to an epitope defined by antibody
CNTO 95, and/or which compete for binding to the alpha V integrin
subunit with antibody CNTO 95, or which have other functional
binding characteristics exhibited by antibody CNTO 95. Such
antibodies include, for example, those which bind to alpha V with a
dissociation constant (KD) Of 10.sup.-7 M or less, such as of
10.sup.-8 M or less, 10.sup.-9 M or less,10.sup.-10M or less, or
even lower (e.g.,10-'' M or less). Such antibodies include those
which competitively inhibit binding of the CNTO 95 antibody to
human alpha-V integrin. Such antibodies further include those which
exhibit no cross reactivity with murine anti alpha V antibodies
LM609, P1F6, or VNR139.
[0014] Isolated human antibodies of the invention include a variety
of antibody isotypes, such as IgG1, (e.g., IgG1k), IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. The antibodies can be
full-length antibodies (e.g., IgG1 or IgG3) or can include only an
antigen-binding portion (e.g., a Fab, F(ab')2, Fv, or a single
chain Fv fragment).
[0015] At least one antibody of the invention binds at least one
specified epitope specific to at least one integrin alpha-V subunit
protein, fragment, portion or any combination thereof The epitope
can comprise at least one antibody binding region that comprises at
least one portion of said protein, which epitope is preferably
comprised of at least 1-5 amino acids of at least one portion of an
alpha-V subunit, such as but not limited to, (a) 29-48, 58-63,
69-79, 82-85, 88-134, 140-157, 161-183, 186-190, 192-198, 202-212,
215-217, 223-237, 240-244 248-255, 259-268, 287-301, 313-322,
326-328, 332-344, 348-351, 354-365, 376-387, 393-401, 407-414,
417-419, 422-433, 443-451, 458-461, 465-469, 472, (b) 32-41, 46-47,
53-55, 58-69, 72-74, 77-79, 85-88, 91-94, 96-105, 110-113, 117-125,
129-142, 145-153, 155-159, 161-163, 166-170, 172-174, 184-197,
200-209, 215-218, 221-225, 184-197, 200-209, 215-218, 221-225,
227-250, 259-261, 263-267, 269-270, 275-281; and (c) 29-35, 43-45,
48-63, 67-69, 72-74, 80-82, 84-87, 95-105, 108-113, 117-142,
145-163, 166-170, 172-176, 184-186, 191-201, 204-206, 216-219,
224-226, 229-251, 260-262, 264-268, 276-282, 286-288, 294-299,
301-318, 323-325, 328-330, 338-342, 345-349, 353-358, of SEQ ID NO:
9, 16, and 17, respectively thereof, or such as but not limited to,
at least one functional, extracellular, soluble, hydrophilic,
external or cytoplasmic domain of said alpha-V subunit protein, or
any portion thereof. Particularly preferred are antibodies which
bind to substantially the same epitope on the alpha V integrin
subunit defined by the epitope of CNTO 95, and/or which compete for
binding to alpha V integrin with antibody CNTO 95, or which have
other functional binding characteristics exhibited by antibody CNTO
95.
[0016] The present invention also provides at least one isolated
anti-alpha-V subunit antibody as described herein, wherein the
antibody has at least one activity, such as, but not limited to
inhibition of vitronectin binding, inhibition of binding of alpha-V
beta-3 to at least one of an alpha-V beta3 ligand or receptor,
inhibition of binding of alpha-V beta-5 to at least one of an
alpha-V beta-5 ligand or receptor, inhibition of binding of alpha-V
beta-6 to at least one of an alpha-V beta-6 ligand or receptor,
angiogenesis modulation, binding to alpha-V subunit or single
integrin expressing cells. A(n) anti-alpha-V subunit antibody can
thus be screened for a corresponding activity according to known
methods, such as but not limited to, competition with the CNTO 95
antibody for at least one biological activity towards an integrin
alpha-V subunit protein.
[0017] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, or hybridizing
to, a polynucleotide encoding the specific anti-integrin alpha-V
subunit antibodies described herein. Such nucleic acid molecules
include those encoding all or a portion of a human monoclonal
anti-alpha V antibody as described herein (e.g., which encode at
least one light or heavy chain CDR of the antibody), as well as
recombinant expression vectors which include such nucleic acids,
and host cells transfected with such vectors. Methods of producing
the antibodies by culturing such host cells are also encompassed by
the invention. Particular nucleic acids provided by the invention
comprise the nucleotide sequences shown in SEQ ID NOs: 10, 11 and
12 and SEQ ID NOs: 13, 14 and 15, which encode the heavy and light
chains CDRs, respectively, of human anti-alpha V antibody CNTO 95
and the nucleic acids which encode the complete variable region of
the heavy or light chain, respectively, as shown in SEQ ID Nos: 18
and 19. The present invention further provides recombinant vectors
comprising said anti-integrin alpha-V subunit antibody nucleic acid
molecules, host cells containing such nucleic acids and/or
recombinant vectors, as well as methods of making and/or using such
antibody nucleic acids, vectors and/or host cells.
[0018] The present invention also provides at least one method for
expressing at least one anti-alpha-V subunit antibody as described
herein, or alpha-V subunit anti-idiotype antibody as described
herein, in a host cell, comprising culturing a host cell as
described herein under conditions wherein at least one anti-alpha-V
subunit antibody is expressed in detectable and/or recoverable
amounts.
[0019] The present invention also provides at least one composition
comprising (a) an isolated anti-alpha-V subunit antibody encoding
nucleic acid and/or antibody as described herein; and (b) a
suitable carrier or diluent. The carrier or diluent can optionally
be pharmaceutically acceptable, according to known carriers or
diluents. The composition can optionally further comprise at least
one further compound, protein or composition.
[0020] The present invention further provides at least one
anti-alpha-V subunit antibody method or composition, for
administering a therapeutically effective amount to modulate or
treat at least one alpha-V subunit related condition in a cell,
tissue, organ, animal or patient and/or, prior to, subsequent to,
or during a related condition, as known in the art and/or as
described herein. The compositions include, for example,
pharmaceutical and diagnostic compositions/kits, comprising a
pharmaceutically acceptable carrier and at least one human
anti-alpha V antibody, or an antigen-binding portion thereof. In
one embodiment, the composition comprises a combination of human
antibodies or antigen-binding portions thereof, preferably each of
which binds to a distinct epitope. For example, a pharmaceutical
composition comprising a human monoclonal antibody that mediates
highly effective killing of target cells in the presence of
effector cells can be combined with the human monoclonal antibody
hereof that inhibits the growth of cells expressing alpha V
integrin. Thus, the combination provides multiple therapies
tailored to provide the maximum therapeutic benefit. Compositions,
e.g., pharmaceutical compositions, comprising a combination of at
least one human anti alpha V-antibody, or antigen-binding portion
thereof, and at least one bispecific or multispecific molecule of
the invention, are also within the scope of the invention.
[0021] In yet another aspect of the invention, the human anti-alpha
V antibodies are derivatized, linked to or co-expressed with
another functional molecule, e.g., another peptide or protein
(e.g., an Fab' fragment). For example, an antibody or
antigen-binding portion of the invention can be functionally linked
(e.g., by chemical coupling, genetic fusion, noncovalent
association or otherwise) to one or more other molecular entities,
such as another antibody (e.g., to produce a bispecific or a
multispecific antibody), a cytotoxin, a cellular ligand or an
antigen. Accordingly, present invention encompasses a large variety
of antibody conjugates, bi- and multispecific molecules, and fusion
proteins, all of which bind to alpha V expressing cells and which
target other molecules to the cells, or which bind to alpha V and
to other molecules or cells.
[0022] Alternatively, human antibodies of the invention can be
co-administered with such therapeutic and cytotoxic agents, but not
linked to them. They can be coadministered simultaneously with such
agents (e.g., in a single composition or separately) or can be
administered before or after administration of such agents. Such
agents can include chemotherapeutic agents, such as dacarbazine,
doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine,
chlorambucil, cyclophosphamide hydroxyurea and combinations
thereof. Human antibodies of the invention also can be administered
in conjunction with radiation therapy.
[0023] In yet another embodiment, the present invention provides a
method for inhibiting the proliferation and/or growth of a cell
expressing alpha V integrin, and/or inducing killing of a cell
expressing alpha V integrin, by contacting the cells with (e.g.,
administering to a subject) one or more human antibodies of the
invention and/or related therapeutic compositions, derivatives etc.
containing the antibodies as described above. In a particular
embodiment, the method comprises contacting cells expressing alpha
V integrin either in vitro or in vivo with one or a combination of
human anti-alpha V antibodies of the invention in the presence of a
human effector cell. The method can be employed in culture, e.g. in
vitro or ex vivo (e.g., cultures comprising cells expressing alpha
V and effector cells). For example, a sample containing cells
expressing alpha V and effector cells can be cultured in vitro, and
combined with an antibody of the invention.
[0024] Alternatively, the method can be performed in a subject,
e.g., as part of an in vivo (e.g., therapeutic or prophylactic)
protocol. For use in in vivo treatment and prevention of alpha V
mediated diseases, human antibodies of the present invention are
administered to patients (e.g., human subjects) at therapeutically
effective dosages (e.g., to inhibit, eliminate or prevent growth of
cells expressing alpha V or to inhibit angiogenesis and thus
inhibit the growth of cells where growth is mediated by
angiogenesis) using any suitable route of administration for
antibody-based clinical products as are well known in the art, such
as by injection or infusion.
[0025] Accordingly, human antibodies of the present invention can
be used to treat and/or prevent a variety of alpha V integrin
mediated diseases by administering a suitable dosage (or series of
dosages) of the antibodies to patients suffering from such
diseases. Exemplary diseases that can be treated (e.g.,
ameliorated) or prevented using the methods and compositions of the
invention include, but are not limited to, cancers, such as
metastatic melanoma, prostate cancer, colon cancer, and renal
carcinoma.
[0026] In a particular embodiment of the invention, the patient can
be additionally treated with a chemotherapeutic agent, radiation,
or an agent that modulates, e.g., enhances, the expression or
activity of an Fc receptor, such as a cytokine. Typical cytokines
for administration during treatment include granulocyte
colony-stimulating fact or (G-CSF), granulocytemacrophage
colony-stimulating factor (GM-CSF), interferon-y (IFN-y), and tumor
necrosis factor (TNF). Typical therapeutic agents include, among
others, anti-neoplastic agents such as dacarbazine, doxorubicin
(adriamycin), cisplatin, bleomycin sulfate, carmustine,
chlorambucil, cyclophosphamide, and hydroxyurea.
[0027] In yet another aspect, the present invention provides a
transgenic nonhuman animal, such as a transgenic mouse (also
referred to herein as a "HuMAb mouse"), which expresses a fully
human monoclonal antibody that binds to alpha V. In a particular
embodiment, the transgenic nonhuman animal is a transgenic mouse
having a genome comprising a human heavy chain transgene and a
human light chain transgene encoding all or a portion of an
anti-alpha V antibody of the invention. To generate human
anti-alpha V antibodies, the transgenic nonhuman animal can be
immunized with a purified or enriched preparation of alpha V
antigen and/or cells expressing alpha V. Preferably, the transgenic
nonhuman animal, e.g., the transgenic mouse, is capable of
producing multiple isotypes of human monoclonal antibodies to Alpha
V (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and
isotype switching. Isotype switching may occur by, e.g., classical
or non-classical isotype switching.
[0028] Accordingly, in another embodiment, the invention provides
isolated cells derived from a transgenic nonhuman animal as
described above, e.g., a transgenic mouse, which express human
anti-alpha V antibodies. The isolated B-cells can then be
immortalized by fusion to an immortalized cell to provide a source
(e.g., a hybridoma) of human anti-alpha V antibodies. Such
hybridomas (i.e., which produce human anti-ALPHA V antibodies) are
also included within the scope of the invention.
[0029] As exemplified herein, human anti-alpha V antibodies can be
obtained directly from hybridomas which express the antibody, or
can be cloned and recombinantly expressed in a host cell, such as a
transfectoma (e.g., a transfectoma consisting of immortalized CHO
cells or lymphocytic cells). Accordingly, the present invention
provides methods for producing human monoclonal antibodies which
bind to human alpha V. In a particular embodiment, the method
includes immunizing a transgenic nonhuman animal, e.g., a
transgenic mouse, as previously described (e.g., having a genome
comprising a human heavy chain transgene and a human light chain
transgene encoding all or a portion of an anti-alpha V antibody),
with a purified or enriched preparation of human alpha V antigen
and/or cells expressing human alpha V. B cells (e.g., splenic B
cells) of the animal are then obtained and fused with myeloma cells
to form immortal, hybridoma cells that secrete human monoclonal
antibodies against alpha V.
[0030] The present invention further provides at least one
anti-alpha-V subunit antibody method or composition, for diagnosing
at least one alpha-V subunit related condition in a cell, tissue,
organ, animal or patient and/or, prior to, subsequent to, or during
a related condition, as known in the art and/or as described
herein.
[0031] The present invention further provides at least one alpha-V
subunit anti-idiotype antibody to at least one alpha-V subunit
antibody of the present invention. The anti-idiotype antibody
includes any protein or peptide containing molecule that comprises
at least a portion of an immunoglobulin molecule, such as but not
limited to at least one complementarity determinng region (CDR) of
a heavy or light chain or a ligand binding portion thereof, a heavy
chain or light chain variable region, a heavy chain or light chain
constant region, a framework region, or any portion thereof, that
can be incorporated into an antibody of the present invention. An
anti-idiotype antibody of the invention can include or be derived
from any mammal, such as but not limited to a human, a mouse, a
rabbit, a rat, a rodent, a primate, and the like.
[0032] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, or hybridizing
to, a polynucleotide encoding at least one alpha-V subunit
anti-idiotype antibody, comprising at least one specified sequence,
domain, portion or variant thereof. The present invention further
provides recombinant vectors comprising said alpha-V subunit
anti-idiotype antibody encoding nucleic acid molecules, host cells
containing such nucleic acids and/or recombinant vectors, as well
as methods of making and/or using such anti-idiotype antiobody
nucleic acids, vectors and/or host cells.
DESCRIPTION OF THE FIGURES
[0033] FIG. 1 shows a graph of doubling dilutions of
anti-.alpha.V.beta.3 Mabs which were incubated on .alpha.V.beta.3
coated plates for 1 hour at RT. Plates were washed twice and probed
with HRP labeled goat anti-human IgG kappa specific antibody for 1
hour at RT. Plates were again washed, developed with OPD substrate
and OD's measured at 490 nm.
[0034] FIG. 2 shows a graph of calcein-labeled M21 cells which were
preincubated with antibody samples in the absence or presence of
P1F6, anti-.alpha.V.beta.5 ascites for 30 minutes, then added to
vitronectin coated plates for 45 minutes. Non-bound M21 cells were
removed with two 150 .mu.L/well washes with HBSS with calcium.
Plate was read on a fluorometer at 485-538 nm.
[0035] FIG. 3 shows a graph of cell adhesion where MDA MB 435L2
cells were harvested and pre-incubated with various concentrations
of CNTO95 for 10 minutes. Tumor cells were then added to
vitronectin coated Linbro plates and incubated at 37.degree. C. for
one hour. Wells were washed three times and the MTT based Cell
Titer AQ dye was added to each well. Cell adhesion was determined
in an ELISA plate reader where OD490 nm is directly proportional to
cell adhesion. Cell adhesion to BSA coated wells served as negative
control (data not shown). Each data point is the mean of triplicate
determinations.
[0036] FIGS. 4A-D show graphs of antibody binding to
.alpha.v.beta.3 where this ligand was preincubated in doubling
dilutions starting at 10 ug/mL with 50 mM EDTA in 1% BSA-HBSS (in
the absence of Ca++) or with 1% BSA-HBSS (with Ca++) for 30 min,
37EC. Mixtures added to plates coated with CNTO 95, C372, c7E3 or
LM609 IgG and incubated for 1 hour, 37.degree. C. LM609 or CNTO 95
added at 20 :g/mL in appropriate buffer (+/-Ca++) for 30 min,
37.degree. C. Plates probed with goat anti-mouse IgG Fc, HRP or
goat anti-human IgG Fc, HRP.
[0037] FIGS. 4E-G show graphs of antibody binding to a alphaVbeta5,
where this ligand was preincubated in doubling dilutions starting
at 10 ug/mL with 50 mM EDTA in 1% BSA-HBSS (in the absence of Ca++)
or with 1% BSA-HBSS (with Ca++) for 30 min, 37.degree. C. Mixtures
added to plates coated with CNTO 95, C372, c7E3 IgG and incubated
for 1 hour, 37.degree. C. VNR139 was added at 10 mg/mL in
appropriate buffer (+/-Ca++) for 30 min, 37.degree. C. Plates
probed with goat anti-mouse IgG Fc, HRP.
[0038] FIGS. 5A-B shows a graph of saturation binding curve of CNTO
95 (FIG. 5A) and abciximab (an anti-gpIIb/IIIa/.alpha.vB3 antibody)
(FIG. 5B) on .alpha.v.beta.3 coated plates.
[0039] FIGS. 6A-B shows a graph of saturation binding curve of CNTO
95 (FIG. 5A) and abciximab (FIG. 5B) on .alpha.v.beta.5 coated
plates.
[0040] FIGS. 7A-C shows saturation binding curves with graphs
binding to (FIG. 7A): A375S2; (FIG. 7B): HT-29; (FIG. 7C): M21.
Cells were plated 2 days prior to experiment, and 1.times.10.sup.5
cells/well at the time of study. 125-I CNTO 95 (1 .mu.Ci/.mu.g) was
added in 1% growth media and incubated on cells for 1.5 h,
37.degree. C. Nonspecific binding was determined using 100.times.
cold mAb in media. Cells were washed 3.times. and counted for bound
radioactivity. Each curve represents 4-5 separate studies, and each
data point in an experiment was the mean of triplicate samples.
[0041] FIGS. 8A-C shows saturation binding curves with graphs
binding to (FIG. 8A): A375S2; (FIG. 8B): HT-29; (FIG. 8C): M21.
Cells were plated 2 days prior to the experiment, and
1.times.10.sup.5 cells/well at the time of study. 125-I abciximab(1
.mu.Ci/.mu.g) was added in 1% growth media and incubated on cells
for 1.5 h, 37.degree. C. Nonspecific binding was determined using
100.times. cold mAb in media. Cells were washed 3.times. and
counted for bound radioactivity. Each curve represents 4-5 separate
studies, and each data point in an experiment was the mean of
triplicate samples.
[0042] FIG. 9 shows a representation of microcapillary formation of
endothelial cells from MC beads cultured in fibrin gels. Objective
lens: 40X. The assay was performed as described in Methods of
Example 4.
[0043] FIG. 10 shows a graph of quantification of capillary
formation in a fibrin gel in media containing 30 ng/ml bFGF
dissolved in 0.1% serum. The number of microcapillary sprouts were
quantified as described in Methods of Example 4. Control indicates
vehicle control, mouse (M) and human (H) IgG served as negative
controls. LM-P1 F6 is a combination of both LM609 and P1F6. Each
bar represents the mean of 6 wells (+/-SD).
[0044] FIG. 11 shows a graph of quantification of capillary
formation in a fibrin gel in complete media. The number of
microcapillary sprouts were quantified as described in Methods of
Example 4. Control indicates vehicle control. Mouse (m) and human
(h)-IgG served as negative controls. LM-P1F6 is a combination of
both LM609 and P1F6. Each bar represents the mean of 6 wells
(+/-SD).
[0045] FIG. 12. HT29 cells (FIGS. 12A, B and C) express
.alpha.v.beta.5, but not .alpha.v.beta.3 integrin on their surface.
HUVEC (FIGS. 12D, E and F) and A375S.2 (FIGS. 12G, H and I) cells
express .alpha.v.beta.5 and .alpha.v.beta.3 integrin on their
surface. Tumor cells and endothelial cells were stained by
immunofluorescence and analyzed by flow cytometry. The histogram on
the left represents background fluorescence in the presence of
isotype matched antibody. The histogram on the right indicates
positive staining. A, D, G, LM609 (mAb directed to .alpha.v.beta.3,
10 .mu.g/ml); B, E, H, PIF6 (mAb directed to .alpha.v.beta.5, 10
.mu.g/ml); and C, F, I, GenO95 (10 .mu.g/ml).
[0046] FIG. 13. Adhesion of HUVECS to matrix protein-coated plates.
Adhesion assay was performed as described in Methods of Example 5.
Plate was read on a fluorometer at 485-538 nm. Cell adhesion to BSA
coated wells served as a negative control. In FIG. 13, the extent
of cell adhesion in the presence of various concentrations of
antibody was plotted as a percent of cell adhesion in the absence
of antibody that was considered as 100%. Each data point is the
mean of triplicate determinations (+/-SD).
[0047] FIG. 14. Adhesion of human melanoma cells to matrix
protein-coated plates. Adhesion assay was performed as described in
Methods of Example 5. Cell adhesion to BSA coated wells served as a
negative control. In FIG. 14 the extent of cell adhesion in the
presence of various concentrations of antibody was plotted as a
percent of cell adhesion in the absence of antibody that was
considered as 100%. Each data point is the mean of triplicate
determinations (+/-SD).
[0048] FIG. 15. Adhesion of human colon carcinoma HT29 cells to
vitronectin. The adhesion assay was performed as described in the
Examples. Cell adhesion to BSA coated wells served as a negative
control. Data in FIG. 15 are plotted as percent of maximum binding
(absence of antibody), and are the mean of triplicate
determinations (+/-SD).
[0049] FIGS. 16A-D. Migration of HUVECS toward 2 .mu.g/ml
vitronectin. The assay was performed as described in Methods and
cells were allowed to migrate for 6 h. Photomicrographs are
representative fields (10.times. objective lens) of cell migration
in FIG. 16A, absence of antibody, (16B), CNTO 95 (5 .mu.g/ml),
(16C), CNTO 95 (40 .mu.g/ml). FIG. 16D is graphical representation
of cell migration in the presence of varying concentrations of
GenO95. The data were normalized to percent of control (no
antibody) which was considered as 100%, and each point is the mean
of three transwell filters (+/-SD).
[0050] FIG. 17. Migration of HUVECS toward 2 .mu.g/ml vitronectin
in the presence of antibodies to .alpha.v.beta.3 and
.alpha.v.beta.5. The migration assay was performed as described in
Methods of Example 5, and cells were allowed to migrate for 6
hours. LM609 and P1F6 are mAbs directed to .alpha.v.beta.3 and
.alpha.v.beta.5, respectively. The data shown in FIG. 17 were
normalized to percent of control (no antibody) which was considered
as 100%, and each bar is the mean of three transwell filters
(+/-SD). BSA, mouse IgG and human IgG served as negative controls.
LM609-PIF6 represents combinations of both antibodies. The
antibodies and BSA were used at a concentration of 10 .mu.g/ml.
[0051] FIGS. 18A-E. Migration of HUVECS towards 2% FBS. Migration
assay was allowed to proceed for 4 h and the data was captured as
described in the Methods of Example 5. FIG. 18(A) is a graphical
representation of cell migration in the presence of LM609, P1F6,
combination of LM609+P1F6, isotype matched control antibodies
(human and mouse). The antibodies and proteins were used at a
concentration of 10 .mu.g/ml. FIG. 18(B) is a graphical
representation of cell migration in the presence of ReoPro and
GenO95. Photomicrographs are representative fields (10.times.
objective lens) of cell migration in FIG. 18(C), the absence of
antibody, FIG. 18(D), GenO95 (5 .mu.g/ml), and FIG. 18(E), GenO95
(20 .mu.g/ml). The data were normalized to percent of control (no
antibody) which was considered as 100%, and each point is the mean
of three transwell filters (+/-SD).
[0052] FIGS. 19A-E. Migration of A375S.2 cells toward 10% FBS.
Migration assay was allowed to proceed for 4 h and the data was
captured as described in the Methods of Example 5. Antibodies were
used at a concentration of 10 .mu.g/ml. FIG. 19(A) is a graphical
representation of cell migration in the presence of varying
concentrations of GenO95. FIG. 19(B) is a graphical representation
of cell migration in the presence of LM609, P1F6, combination of
LM609+P1F6, isotype matched control antibodies (human and mouse).
The data were normalized to percent of control, which was
considered as 100%, and each point is the mean of three transwell
filters (+/-SD). Photomicro-graphs are representative fields
(10.times. objective lens) of cell migration in FIG. 19(C), absence
of antibody, FIG. 19(D), GenO95 (5 .mu.g/ml), and FIG. 19(E),
GenO95 (20 .mu.g/ml).
[0053] FIGS. 20A-E. Migration of HUVECS towards vitronectin in the
presence of bFGF. The undersides of migration chamber filters were
coated with 2 .mu.g/ml vitronectin, and the assay was performed as
described in the Methods of Example 5. Cells were allowed to
migrate for 6 h. In FIGS. 20A-E, each data point is the mean of 3
transwell filters (+/-SD). FIG. 20(A), bFGF; FIG. 20(B), CNTO 95 (5
.mu.g/ml); FIG. 20 (C), CNTO 95 (40 .mu.g/ml); FIG. 20 (D),
no-bFGF. FIG. 20 (E), Inhibition of cell migration in the presence
of various antibodies is shown graphically.
[0054] FIGS. 21A-D. Invasion of A375S.2 cells through a fibrin gel
(5 mg/ml). Invasion assay was allowed to proceed for 24 h and data
was captured as described in the Methods of Example 5.
Photomicrographs are representative fields (4.times. objective
lens) of cell invasion in FIG. 21(A) the absence of antibodies,
FIG. 21(B) CNTO 95 (10 .mu.g/ml), FIGS. 21(C) and (D) are graphical
representation of cell invasion in presence of CNTO 95, 10E5
F(ab').sub.2, LM609, P1F6, LM-PIF6 (LM609+P1F6), human and mouse
IgGs (H-IgG and M-IgG). Graph FIG. 21(D): The concentration of all
antibodies and proteins is 10 .mu.g/ml. The data were normalized to
percent of control (no antibody) which was considered as 100%, and
each point is the mean of three transwell filters (+/-SD).
[0055] FIGS. 22A-D. are histograms from flow cytometric analysis of
HEK cells transfected with various integrin DNA and
immunoflurescently stained as noted. FIG. 22(A): cells stained with
antibodies for specific subunits. FIG. 22(B): mock transfected and
avb6 transfected cells were analyzed for expression of
.alpha.v.beta.3, .alpha.v.beta.5, and .beta.1 integrins. FIG.
22(C): HEK 293 cells were transfected with .alpha.V, .beta.6, or
.alpha.v.beta.6 cDNA and CNTO 95 binding measured. The vertical
line serves as a reference marker and indicates the fluorescence
intensity at which mock transfectants were <2% positive. FIG.
22(D): analysis of mock transfected (A, B, C) or .alpha.v.beta.6
transfected (C, D, E) HEK 293 cells for CNTO 95 immunoreactivity.
Cells were stained with anti-avb6 (A, D) or CNTO 95 (B, E). Double
staining was used to detect cells which were immunoreactive with
both antibodies simultaneously (C, F). The upper-right quadrant in
F indicates that cells which stained intensely for avb6 also
stained intensely for CNTO 95.
[0056] FIG. 23 is a graph showing the number of microvessels
sprouting from rat aortic rings treated as described. BSA (20
ug/ml) and irrelevant human IgG (20 ug/ml) were used as negative
controls. Data points represent one rat aorta, with mean values for
each group indicated by lines. P values were determined by
comparing the antibody-treated groups with the BSA-treated group
using a two-tailed unpaired t-test.
[0057] FIG. 24 are graphs of the data on length FIG. 24(A) and
number FIG. 24(B) of blood vessels found in bFGF-impregnated
Matrigel plugs in nude rats examined on day 7. Vessel number and
length were assessed by microscopy (2.times.) aided by image
analysis software (Phase 3 Image System). Each point represents
average per view from one Matrigel sample (2 plugs/animal), the
line represents the group mean. A two-tailed unpaired t-test
indicated P<0.0001 for all four CNTO 95 dose groups compared to
the control IgG group.
[0058] FIGS. 25A-C are graphs of the data on length Figure (A),
number Figure (B) and density FIG. 25(C) of blood vessels found in
bFGF-impregnated Matrigel plugs in monkeys examined on day 7. For
FIG. 25(A) and FIG. 25(B) each point represents average per
2.times. field from one Matrigel sample (4 Matrigel plugs/animal).
Horizontal lines indicate mean. Matrigel alone, bFGF-PBS, 5 ug/ml
bFGF. bFGF-control IgG, 5 ug/ml bFGF, 10 ug/kg control IgG i.v.
bFGF-CNTO 95, 5 ug/ml bFGF, 10 mg/kg CNTO 95 i.v. A two-tailed
unpaired t-test analysis indicated P<0.001 for Matrigel alone
and bFGF-CNTO 95 groups compared to the bFGF-PBS and bFGF-control
IgG groups. There was no difference between bFGF-CNTO 95 and
Matrigel alone groups (P>0.05). For FIG. 25(C), the percentage
of Matrigel cross-sectional area occupied by vessels was calculated
using computer-assisted image analysis. Each point represents the
average density per Matrigel sample (4 samples/animal). A
two-tailed unpaired t-test analysis indicated P<0.01 for
Matrigel alone and bFGF-CNTO 95 groups compared to the bFGF-HBSS
and bFGF-control IgG groups. There was no difference between
bFGF-CNTO 95 and Matrigel alone groups (P>0.05).
[0059] FIGS. 26A and B are graphs of the data on length FIG. 26(A)
and number FIG. 26(B) of blood vessels found in bFGF-impregnated
Matrigel plugs in nude rats. Each point represents average per view
from one Matrigel plug and the line is the mean from 10 plugs (2
plugs/per animal). Inside refers to CNTO 95 (40 mg/ml) that was
included in the Matrigel solution prior to injection. IV refers to
CNTO 95 (10 mg/kg) that was injected intravenously as a bolus after
the Matrigel solution was injected into the rats. Inside +IV refers
to CNTO 95 that was mixed with the Matrigel solution and injected
intravenously. A two-tailed unpaired t-test analysis indicated
P<0.001 for all three CNTO 95 groups compared to the control IgG
group. The three CNTO 95 groups were not statistically different
from each other.
[0060] FIG. 27. is a graph showing the change volume over time of a
human melanoma tumor in nude mice and the effect of administering
CNTO 95. Mice were inoculated subcutaneously with A375.S2 cells
(3.times.106), and dosing with CNTO 95 or control was initiated
three days later. Mice were treated with CNTO 95 or vehicle three
times per week at a dose of 10 mg/kg i.p. Each data point is the
mean tumor volume from 10 tumor-bearing animals (.+-.SEM). CNTO 95
given three times per week significantly inhibited growth of tumors
when compared to control treated animals at day 26 (P=0.0005).
[0061] FIG. 28. is a graph showing the change volume over time of a
human melanoma tumor in nude rats and the effect of administering
CNTO 95. Rats were inoculated subcutaneously with A375.S2 cells
(3.times.106), and therapy with CNTO 95 or control was initiated
three days later. Rats were treated with CNTO 95 or vehicle once
per week at a dose of 10 mg/kg i.v. Each data point is the mean
tumor volume from 9 tumor-bearing animals (.+-.SEM).
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention provides isolated, recombinant and/or
synthetic anti-alpha-V subunit human monoclonal antibodies and
alpha-V subunit anti-idiotype antibodies thereto, as well as
compositions and encoding nucleic acid molecules comprising at
least one polynucleotide encoding at least one anti-alpha-V subunit
antibody or anti-idiotype antibody. The present invention further
includes, but is not limited to, methods of making and using such
nucleic acids and antibodies and anti-idiotype antibodies,
including diagnostic and therapeutic compositions, methods and
devices. Therapies of the invention employ isolated human
monoclonal antibodies and/or related compositions containing the
antibodies which bind to an epitope present on the alpha V integrin
subunit and is capable of blocking the binding of various alpha V
containing integrins, regardless of the beta subunit to which it is
associated. In a particular embodiment exemplified herein, the
human antibodies are produced in a nonhuman transgenic animal,
e.g., a transgenic mouse. Accordingly, aspects of the invention
include not only antibodies, antibody fragments, and pharmaceutical
compositions thereof, but also nonhuman transgenic animals, B-cells
and hybridomas which produce monoclonal antibodies. Methods of
using the antibodies of the invention to detect a cell expressing
alpha V, or to inhibit growth, differentiation and/or motility of a
cell expressing alpha V, either in vitro or in vivo, are also
encompassed by the invention.
[0063] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0064] The term "alpha V integrin", "alpha V subunit integrin", and
"alpha V subunit containing integrin" are used interchangeably
herein to mean Alpha V transmembrane glycoprotein subunits of a
functional integrins heterodimer and include all of the variants,
isoforms and species homologs of alpha V. Accordingly, human
antibodies of the invention may, in certain cases, cross-react with
alpha V from species other than human, or other proteins which are
structurally related to human alpha V (e.g., human alpha V
homologs). In other cases, the antibodies may be completely
specific for human alpha V and not exhibit species or other types
of cross-reactivity.
[0065] As used herein, an "antibody" includes whole antibodies and
any antigen binding fragment or a single chain thereof. Thus the
antibody includes any protein or peptide containing molecule that
comprises at least a portion of an immunoglobulin molecule, such as
but not limited to at least one complementarity determining region
(CDR) of a heavy or light chain or a ligand binding portion
thereof, a heavy chain or light chain variable region, a heavy
chain or light chain constant region, a framework (FR) region, or
any portion thereof, or at least one portion of a binding protein,
which can be incorporated into an antibody of the present
invention. An "alpha V antibody", "alpha V subunit antibody" or
"alpha V integrin antibody" is an antibody that affects the alpha V
ligand, such as but not limited to where such antibody modulates,
decreases, increases, antagonizes, angonizes, mitigates, aleviates,
blocks, inhibits, abrogates and/or interferes with at least one
alpha-V subunit activity or binding, or with alpha-V subunit
receptor activity or binding, in vitro, in situ and/or in vivo. As
a non-limiting example, a suitable anti-alpha-V subunit antibody,
specified portion or variant of the present invention can bind at
least one alpha-V subunit, or specified portions, variants or
domains thereof. A suitable anti-alpha-V subunit antibody,
specified portion, or variant can also optionally affect at least
one of alpha-V subunit activity or function, such as but not
limited to, RNA, DNA or protein synthesis, alpha-V subunit release,
alpha-V subunit receptor signaling, membrane alpha-V subunit
cleavage, alpha-V subunit activity, alpha-V subunit production
and/or synthesis.
[0066] The term "antibody" is further intended to encompass
antibodies, digestion fragments, specified portions and variants
thereof, including antibody mimetics or comprising portions of
antibodies that mimic the structure and/or function of an antibody
or specified fragment or portion thereof, including single chain
antibodies and fragments thereof Functional fragments include
antigen-binding fragments that bind to a mammalian alpha-V subunit.
Examples of binding fragments encompassed within the term "antigen
binding portion" of an antibody include (i) a Fab fragment, a
monovalent fragment consisting of the VL, VH, CL and CH, domains;
(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH, domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(I 988) Science 242:423-426,-and Huston et al. (1988) Proc. Natl.
Acad Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies.
[0067] Such fragments can be produced by enzymatic cleavage,
synthetic or recombinant techniques, as known in the art and/or as
described herein. Antibodies can also be produced in a variety of
truncated forms using antibody genes in which one or more stop
codons have been introduced upstream of the natural stop site. For
example, a combination gene encoding a F(ab').sub.2 heavy chain
portion can be designed to include DNA sequences encoding the
CH.sub.1 domain and/or hinge region of the heavy chain. The various
portions of antibodies can be joined together chemically by
conventional techniques, or can be prepared as a contiguous protein
using genetic engineering techniques.
[0068] The term "epitope" means a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. The term "native
conformational epitope" or "native protein epitope" are used
interchangeably herein, and include protein epitopes resulting from
conformational folding of the integrin molecule which arise when
amino acids from differing portions of the linear sequence of the
integrin molecule come together in close proximity in 3 dimensional
space. Such conformational epitopes are distributed on the
extracellular side of the plasma membrane.
[0069] The term "bispecific molecule" is intended to include any
agent, e.g., a protein, peptide, or protein or peptide complex,
which has two different binding specificities. For example, the
molecule may bind to, or interact with, (a) a cell surface antigen
and (b) an Fc receptor on the surface of an effector cell. The term
"multispecific molecule" or "heterospecific molecule" is intended
to include any agent, e.g. a protein, peptide, or protein or
peptide complex, which has more than two different binding
specificities. For example, the molecule may bind to, or interact
with, (a) a cell surface antigen, (b) an Fc receptor on the surface
of an effector cell, and (c) at least one other component.
Accordingly, the invention includes, but is not limited to,
bispecific, trispecific, tetraspecific, and other multispecific
molecules which are directed to cell surface antigens, such as
alpha V, and to other targets, such as Fc receptors on effector
cells.
[0070] The term "bispecific antibodies" also includes diabodies.
Diabodies are bivalent, bispecific antibodies in which the VH and
VL domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad Sci. USA 90:6444-6448; Poljak, R. J., et al. (I 994) Structure
2:1121-1123). Bispecific, heterospecific, heteroconjugate or
similar antibodies can also be used that are monoclonal, preferably
human or humanized, antibodies that have binding specificities for
at least two different antigens. In the present case, one of the
binding specificities is for at least one alpha-V subunit protein,
the other one is for any other antigen. Methods for making
bispecific antibodies are known in the art. Traditionally, the
recombinant production of bispecific antibodies is based on the
co-expression of two immunoglobulin heavy chain-light chain pairs,
where the two heavy chains have different specificities (Milstein
and Cuello, Nature 305:537 (1983)). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. The 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, e.g., in WO 93/08829, U.S. Pat. Nos. 6,210,668,
6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902,
5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333,
5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980,
WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBO J.
10:3655 (1991), Suresh et al., Methods in Enzymology 121:210
(1986), each entirely incorporated herein by reference.
[0071] As used herein, the term "heteroantibodies" refers to two or
more antibodies, antibody binding fragments (e.g., Fab),
derivatives therefrom, or antigen binding regions linked together,
at least two of which have different specificities. These different
specificities include a binding specificity for an Fc receptor on
an effector cell, and a binding specificity for an antigen or
epitope on a target cell, e.g., a tumor cell.
[0072] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences.
Thus as used herein, the term "human antibody" refers to an
antibody in which substantially every part of the protein (e.g.,
CDR, framework, C.sub.L, C.sub.H domains (e.g., C.sub.H1, C.sub.H2,
C.sub.H3), hinge, (V.sub.L, V.sub.H)) is substantially
non-immunogenic in humans, with only minor sequence changes or
variations. Similarly, antibodies designated primate (monkey,
baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig,
hamster, and the like) and other mammals designate such species,
sub-genus, genus, sub-family, family specific antibodies. Further,
chimeric antibodies include any combination of the above. Such
changes or variations optionally and preferably retain or reduce
the immunogenicity in humans or other species relative to
non-modified antibodies. Thus, a human antibody is distinct from a
chimeric or humanized antibody. It is pointed out that a human
antibody can be produced by a non-human animal or prokaryotic or
eukaryotic cell that is capable of expressing functionally
rearranged human immunoglobulin (e.g., heavy chain and/or light
chain) genes. Further, when a human antibody is a single chain
antibody, it can comprise a linker peptide that is not found in
native human antibodies. For example, an Fv can comprise a linker
peptide, such as two to about eight glycine or other amino acid
residues, which connects the variable region of the heavy chain and
the variable region of the light chain. Such linker peptides are
considered to be of human origin.
[0073] As used herein, a human antibody is "derived from" a
particular germline sequence if the antibody is obtained from a
system using human immunoglobulin sequences, e.g., by immunizing a
transgenic mouse carrying human immunoglobulin genes or by
screening a human immunoglobulin gene library. A human antibody
that is "derived from" a human germline immunoglobulin sequence can
be identified as such by comparing the amino acid sequence of the
human antibody to the amino acid sequence of human germline
immunoglobulins. A selected human antibody typically is at least
90% identical in amino acids sequence to an amino acid sequence
encoded by a human germline immunoglobulin gene and contains amino
acid residues that identify the human antibody as being human when
compared to the gemiline immunoglobulin amino acid sequences of
other species (e.g., murine germline sequences). In certain cases,
a human antibody may be at least 95%, or even at least 96%, 97%,
98%, or 99% identical in amino acid sequence to the amino acid
sequence encoded by the germline immunoglobulin gene. Typically, a
human antibody derived from a particular human germline sequence
will display no more than 10 amino acid differences from the amino
acid sequence encoded by the human germline immunoglobulin gene. In
certain cases, the human antibody may display no more than 5, or
even no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germline inimunoglobulin
gene.
[0074] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions derived from
human germline immunoglobulin sequences.
[0075] In one embodiment, the human monoclonal antibodies are
produced by a hybridoma which includes a B cell obtained from a
transgenic nonhuman animal, e.g., a transgenic mouse, having a
genome comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0076] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom (described further in Section I, below), (b)
antibodies isolated from a host cell transformed to express the
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies can be subjected to in vitro
mutagenesis (or, when an animal I 0 transgenic for human Ig
sequences is used, in vivo somatic mutagenesis) and thus the amino
acid sequences of the VH and VL regions of the recombinant
antibodies are sequences that, while derived from and related to
human germline VH and VL sequences, may not naturally exist within
the human antibody germline repertoire in vivo.
[0077] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to Alpha V is substantially free
of antibodies that specifically bind antigens other than Alpha V).
An isolated antibody that specifically binds to an epitope, isoform
or variant of human Alpha V may, however, have cross-reactivity to
other related antigens, e.g., from other species (e.g., Alpha V
species homologs). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals. In
one embodiment of the invention, a combination of "isolated"
monoclonal antibodies having different specificities are combined
in a well defined composition.
[0078] As used herein, "specific binding" refers to antibody
binding to a predetermined antigen. Typically, the antibody binds
with a dissociation constant (K.sub.D) of 10-7 M or less, and binds
to the predetermined antigen with a K.sub.D that is at least
twofold less than its K.sub.D for binding to a non-specific antigen
(e.g., BSA, casein) other than the predetermined antigen or a
closely-related antigen. The phrases "an antibody recognizing an
antigen" and " an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen".
[0079] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D Of 10.sup.-8 M or less, more
preferably 10.sup.-9 M or less and even more preferably 10.sup.-10
M or less. However, "high affinity" binding can vary for other
antibody isotypes. For example, "high affinity" binding for an IgM
isotype refers to an antibody having a K.sub.D of 10.sup.-7 M or
less, more preferably 10.sup.-8 M or less. The term "Kassoc" or
"Ka", as used herein, is intended to refer to the association rate
of a particular antibody-antigen interaction, whereas the term
"Kdis" or "Kd," as used herein, is intended to refer to the
dissociation rate of a particular antibody-antigen interaction, The
term "K.sub.D", as used herein, is intended to refer to the
dissociation constant, which is obtained from the ratio of Kd to Ka
(i.e. Kd/Ka) and is expressed as a molar concentration (M).
[0080] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes.
[0081] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA. The term "isolated nucleic acid molecule,"
as used herein in reference to nucleic acids encoding antibodies or
antibody portions (e.g., VH, VL, CDR3) that bind to Alpha V, is
intended to refer to a nucleic acid molecule in which the
nucleotide sequences encoding the antibody or antibody portion are
free of other nucleotide sequences encoding antibodies or antibody
portions that bind antigens other than Alpha V, which other
sequences may naturally flank the nucleic acid in human genomic
DNA. In one embodiment, the human anti-Alpha V antibody, or portion
thereof, includes the nucleotide or amino acid sequence of CNTO 95,
as well as heavy chain (VH) and light chain (VL) variable regions
having the amino acid sequences shown in SEQ ID NOs: 7 and 8,
respectively, and nucleotide sequences encoding them, including SEQ
ID Nos: 18 and 19.
[0082] As disclosed and claimed herein, the sequences set forth in
SEQ ID NOs. 1-8 and 10-15 include "conservative sequence
modifications", i.e., nucleotide and amino acid sequence
modifications which do not significantly affect or alter the
binding characteristics of the antibody encoded by the nucleotide
sequence or containing the amino acid sequence. Such conservative
sequence modifications include nucleotide and amino acid
substitutions, additions and deletions. Modifications can be
introduced into SEQ ID NOs: 1-8 and 10-15 by standard techniques
known in the art, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Conservative amino acid substitutions
include ones in which the amino acid residue is replaced with an
amino acid residue having a similar side chain. Families of amino
acid residues having similar side chains have been defined in the
art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in a
human anti-Alpha V antibody is preferably replaced with another
amino acid residue from the same side chain family.
[0083] Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of a anti-Alpha V antibody
coding sequence, such as by saturation mutagenesis, and the
resulting modified anti-Alpha V antibodies can be screened for
binding activity.
[0084] Accordingly, antibodies encoded by the (heavy and light
chain variable region) nucleotide sequences disclosed herein and/or
containing the (heavy and light chain variable region) amino acid
sequences disclosed herein (i.e., SEQ ID NOs: 1-8) include
substantially similar antibodies encoded by or containing similar
sequences which have been conservatively modified. Further
discussion as to how such substantially similar antibodies can be
generated based on the partial (i.e., heavy and light chain
variable regions) sequences disclosed herein as SEQ ID NOs: 1-8 is
provided below. For nucleic acids, the term "substantial homology"
indicates that two nucleic acids, or designated sequences thereof,
when optimally aligned and compared, are identical, with
appropriate nucleotide insertions or deletions, in at least about
80% of the nucleotides, usually at least about 90% to 95%, and more
preferably at least about 98% to 99.5% of the nucleotides.
Alternatively, substantial homology exists when the sequences
hybridize under selective hybridization conditions, to the
complement of segments with the strand. The percent identity
between two sequences is a function of the number of identical
positions shared by the sequences (i.e., % homology=# of identical
positions/total # of positions.times.100), taking into account the
number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm, as described in the non-limiting examples below.
[0085] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package
(available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap
weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,
5, or 6. The percent identity between two nucleotide or amino acid
sequences can also determined using the algorithm of E. Meyers and
W. Miller (Comput. AppL Biosci., 4:11-17 (1988)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM 1 20
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid
sequences can be determined using the Needleman and Wunsch (J. Mol.
Biol. 48:444-453 (1970)) algorithm which has been incorporated into
the GAP program in the GCG software package (available at
www.gcg.com), using either a Blossurn 62 matrix or a PAM2 5 0
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. The nucleic acid and protein
sequences of the present invention can further be used as a "query
sequence" to perform a search against public databases to, for
example, identify related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (I 990) J Mol. Biol. 215.403-1 0. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389 When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See
www.ncbi.nlm.nih.gov.
[0086] The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0087] The nucleic acid compositions of the present invention,
while often in a native sequence (except for modified restriction
sites and the like), from either cDNA, genomic or mixtures thereof,
may be mutated in accordance with standard techniques to provide
gene sequences. For coding sequences, these mutations, may affect
amino acid sequence as desired. In particular, DNA sequences
substantially homologous to or derived from native V, D, J,
constant, switches and other such sequences described herein are
contemplated (where "derived" indicates that a sequence is
identical or modified from another sequence).
[0088] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence. With
respect to transcription regulatory sequences, operably linked
means that the DNA sequences being linked are contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. For switch sequences, operably linked indicates that
the sequences are capable of effecting switch recombination.
[0089] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasinid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genorne. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0090] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, CHO cells and
lymphocytic cells.
[0091] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and nonmammals, such as nonhuman
primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
[0092] The terms "transgenic, nonhuman animal" refers to a nonhuman
animal having a genome comprising one or more human heavy and/or
light chain transgenes or transchromosomes (either integrated or
non-integrated into the animal's natural genomic DNA) and which is
capable of expressing fully human antibodies. For example, a
transgenic mouse can have a human light chain transgene and either
a human heavy chain transgene or human heavy chain transchromosome,
such that the mouse produces human anti-Alpha V antibodies when
immunized with alpha V antigen and/or cells expressing Alpha V. The
human heavy chain transgene can be integrated into the chromosomal
DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice,
or the human heavy chain transgene can be maintained
extrachromosomally, as is the case for transchromosomal (e.g., KM)
mice as described in WO 02/43478. Such transgenic and
transchromosomal mice are capable of producing multiple isotypes of
human monoclonal antibodies to Alpha V (e.g., IgG, IgA and/or IgE)
by undergoing V-D-J recombination and isotype switching.
[0093] Anti-alpha-V subunit antibodies (also termed alpha-V subunit
antibodies) useful in the methods and compositions of the present
invention are characterized by high affinity binding to alpha-V
subunit and preferably having low toxicity. In particular, an
antibody, specified fragment or variant of the invention, where the
individual components, such as the variable region, constant region
and framework, individually and/or collectively, optionally and
preferably possess low immunogenicity, are useful in the present
invention. The antibodies that can be used in the invention are
optionally characterized by their ability to treat patients for
extended periods with measurable alleviation of symptoms and low
and/or acceptable toxicity. Low or acceptable immunogenicity and/or
high affinity, as well as other suitable properties, can contribute
to the therapeutic results achieved. "Low immunogenicity" is
defined herein as raising significant HAHA, HACA or HAMA responses
in less than about 75%, or preferably less than about 50% of the
patients treated and/or raising low titres in the patient treated
(less than about 300, preferably less than about 100 measured with
a double antigen enzyme immunoassay) (Elliott et al., Lancet
344:1125-1127 (1994), entirely incorporated herein by
reference).
CITATIONS
[0094] All publications or patents cited herein are entirely
incorporated herein by reference as they show the state of the art
at the time of the present invention and/or to provide description
and enablement of the present invention. Publications refer to any
scientific or patent publications, or any other information
available in any media format, including all recorded, electronic
or printed formats. The following references are entirely
incorporated herein by reference: Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,
N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow
and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.
(1989); Colligan, et al., eds., Current Protocols in Immunology,
John Wiley & Sons, Inc., NY (1994-2001); Colligan et al.,
Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001).
1. Production of Antibodies
[0095] Anti-alpha-V subunit antibodies of the present invention can
be optionally produced by a variety of techniques, including
conventional monoclonal antibody techniques, e.g., the standard
somatic cell hybridization technique of Kohler and Milstein (1975)
Nature 256:495. A variety of cell lines, mixed cell lines, an
immortalized cell or clonal population of immortalized cells, can
be used, as well known in the art. See, e.g., Ausubel, et al., ed.,
Current Protocols in Molecular Biology, John Wiley & Sons,
Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A
Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y.
(1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold
Spring Harbor, N.Y. (1989); Colligan, et al., eds., Current
Protocols in Immunology, John Wiley & Sons, Inc., NY
(1994-2001); Colligan et al., Current Protocols in Protein Science,
John Wiley & Sons, NY, N.Y., (1997-2001), each entirely
incorporated herein by reference.
[0096] Human antibodies that are specific for human alpha-V subunit
proteins or fragments thereof can be raised against an appropriate
immunogenic antigen, such as isolated and/or alpha-V subunit
protein or a portion thereof (including synthetic molecules, such
as synthetic peptides). Other specific or general mammalian
antibodies can be similarly raised. Preparation of immunogenic
antigens, and monoclonal antibody production can be performed using
any suitable technique.
[0097] In one approach, a hybridoma is produced by fusing a
suitable immortal cell line (e.g., a myeloma cell line such as, but
not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5,
>243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937,
MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH
3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O or the
like, or heteromyelomas, fusion products thereof, or any cell or
fusion cell derived therefrom, or any other suitable cell line as
known in the art. See, e.g., www.atcc.org, www.lifetech.com., and
the like, with antibody producing cells, such as, but not limited
to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or
other immune or B cell containing cells, or any other cells
expressing heavy or light chain constant or variable or framework
or CDR sequences, either as endogenous or heterologous nucleic
acid, as recombinant or endogenous, viral, bacterial, algal,
prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent,
equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA,
rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA,
mRNA, tRNA, single, double or triple stranded, hybridized, and the
like or any combination thereof. See, e.g., Ausubel, supra, and
Colligan, Immunology, supra, chapter 2, entirely incorporated
herein by reference. Antibody producing cells can also be obtained
from the peripheral blood or, preferably the spleen or lymph nodes,
of humans or other suitable animals that have been immunized with
the antigen of interest. Any other suitable host cell can also be
used for expressing heterologous or endogenous nucleic acid
encoding an antibody, specified fragment or variant thereof, of the
present invention. The fused cells (hybridomas) or recombinant
cells can be isolated using selective culture conditions or other
suitable known methods, and cloned by limiting dilution or cell
sorting, or other known methods. Cells which produce antibodies
with the desired specificity can be selected by a suitable assay
(e.g., ELISA).
[0098] Other suitable methods of producing or isolating antibodies
of the requisite specificity can be used, including, but not
limited to, methods that select recombinant antibody from a peptide
or protein library (e.g., but not limited to, a bacteriophage,
ribosome, oligonucleotide, RNA, cDNA, or the like, display library;
e.g., as available from Cambridge antibody Technologies,
Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation,
Aberdeen, Scotland, UK; BioInvent, Lund, Sweden; Dyax Corp., Enzon,
Affymax/Biosite; Xoma, Berkeley, Calif.; Ixsys. See, e.g., EP
368,684, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240;
PCT/GB92/00883; PCT/GB93/00605; U.S. Ser. No. 08/350,260 (May 12,
1994); PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835; (CAT/MRC);
WO90/14443; WO90/14424; WO90/14430; PCT/US94/1234; WO92/18619;
WO96/07754; (Scripps); EP 614 989 (MorphoSys); WO95/16027
(Biolnvent); WO88/06630; WO90/3809 (Dyax); U.S. Pat. No. 4,704,692
(Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371 998; EP 550
400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); or stochastically
generated peptides or proteins--U.S. Pat. Nos. 5,723,323,
5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803,
EP 590 689 (Ixsys, now Applied Molecular Evolution (AME), each
entirely incorporated herein by reference) or that rely upon
immunization of transgenic animals (e.g., SCID mice, Nguyen et al.,
Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.
Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161
(1998), each entirely incorporated by reference as well as related
patents and applications) that are capable of producing a
repertoire of human antibodies, as known in the art and/or as
described herein. Such techniques, include, but are not limited to,
ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA,
94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,
95:14130-14135 (Nov. 1998)); single cell antibody producing
technologies (e.g., selected lymphocyte antibody method ("SLAM")
(U.S. Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892
(1987); Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848
(1996)); gel microdroplet and flow cytometry (Powell et al.,
Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, Mass.;
Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,
Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et
al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress
Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technology,
Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam,
Netherlands (1988)). Methods for engineering or humanizing
non-human or human antibodies can also be used and are well known
in the art. Generally, a humanized or engineered antibody has one
or more amino acid residues from a source which is non-human, e.g.,
but not limited to mouse, rat, rabbit, non-human primate or other
mammal. These human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable, constant or other domain of a known human sequence. Known
human Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez/query.fcgi;
www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uniheidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH05/kuby05.htm;
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/mikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immunology.html.; www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.ac.jp/.about.yasuhito/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/links.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.unimarburg.de/.about.rek/AEPStart.html;baserv.uci.kun.n1/.abou-
t.jraats/links1.html; www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;
www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/ www.abgen.html;
www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.about.fmolina/Web-pages/Pept/spottech.html;
www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html and in
Kabat et al., Sequences of Proteins of Immunological Interest, U.S.
Dept. Health (1983), each entirely incorporated herein by
reference.
[0099] Such imported sequences can be used to reduce immunogenicity
or reduce, enhance or modify binding, affinity, on-rate, off-rate,
avidity, specificity, half-life, or any other suitable
characteristic, as known in the art. Generally part or all of the
non-human or human CDR sequences are maintained while the non-human
sequences of the variable and constant regions are replaced with
human or other amino acids. Antibodies can also optionally be
humanized with retention of high affinity for the antigen and other
favorable biological properties. To achieve this goal, humanized
antibodies can be optionally prepared by a process of analysis of
the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR (framework) residues can be
selected and combined from the consensus and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding. Humanization or engineering of
antibodies of the present invention can be performed using any
known method, such as but not limited to those described in, Winter
(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature
332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et
al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol.
196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S.
Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476,
5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370,
5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT/:
US98/16280, US96/18978, US91/09630, US91/05939, US94/01234,
GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424,
WO90/14430, EP 229246, each entirely incorporated herein by
reference, included references cited therein.
[0100] The anti-alpha-V subunit antibody can also be optionally
generated by immunization of a transgenic animal (e.g., mouse, rat,
hamster, non-human primate, and the like) capable of producing a
repertoire of human antibodies, as described herein and/or as known
in the art. Cells that produce a human anti-alpha-V subunit
antibody can be isolated from such animals and immortalized using
suitable methods, such as the methods described herein.
[0101] Transgenic mice that can produce a repertoire of human
antibodies that bind to human antigens can be produced by known
methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428,
5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016
and 5,789,650 issued to Lonberg et al.; Jakobovits et al. WO
98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO
98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,
Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151
B1, Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.
5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438
474 B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440
A, Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int.
Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21
(1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et
al., Nucleic Acids Research 20 (23):6287-6295 (1992), Tuaillon et
al., Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al.,
Int Rev Immunol 13(1):65-93 (1995) and Fishwald et al., Nat
Biotechnol 14(7):845-851 (1996), which are each entirely
incorporated herein by reference). Generally, these mice comprise
at least one transgene comprising DNA from at least one human
immunoglobulin locus that is functionally rearranged, or which can
undergo functional rearrangement. The endogenous immunoglobulin
loci in such mice can be disrupted or deleted to eliminate the
capacity of the animal to produce antibodies encoded by endogenous
genes.
[0102] To generate fully human monoclonal antibodies to Alpha V,
HuMAb mice can be immunized with a purified or enriched preparation
of Alpha V antigen and/or cells expressing Alpha V, as described by
Lonberg, N. et al. (1994) Nature 3 68(6474) 856.859; Fishwild, D.
et al. (1996) Nature Biotechnology 14:845-851 and WO 98/24884.
Preferably, the mice will be 6-16 weeks of age upon the first
infusion. For example, a purified or enriched preparation (5-20 pg)
of Alpha V antigen (e.g., purified from Alpha V-expressing LNCaP
cells) can be used to immunize the HuMAb mice intraperitoneally. In
the event that immunizations using a purified or enriched
preparation of Alpha V antigen do not result in antibodies, mice
can also be immunized 1 5 with cells expressing Alpha V, e.g., a
tumor cell line, to promote immune responses. Cumulative experience
with various antigens has shown that the HuMAb transgenic mice
typically respond best when initially immunized intraperitoneally
(IP) with antigen in complete Freund's adjuvant, followed by every
other week i.p. immunizations (up to a total of 6) with antigen in
incomplete Freund's adjuvant, followed by every other week IP/SC
immunizations (up to a total of 10) with antigen in incomplete
Freund's adjuvant. The immune response can be monitored over the
course of the immunization protocol with plasma samples being
obtained by retroorbital bleeds. The plasma can be screened by
ELISA (as described below), and mice with sufficient titers of
anti-Alpha V human immunoglobulin can be used for fusions. Mice can
be boosted intravenously with antigen 3 days before sacrifice and
removal of the spleen. It is expected that 2-3 fusions for each
antigen may need to be performed. Several mice will be immunized
for each antigen.
[0103] To generate hybridomas producing human monoclonal antibodies
to Alpha V, splenocytes and lymph node cells from immunized mice
can be isolated and fused to an appropriate immortalized cell line,
such as a mouse myeloma cell line. The resulting hybridomas can be
screened for the production of antigen-specific antibodies.
[0104] Human antibodies of the invention also can be produced in a
host cell transfectorma using, for example, a combination of
recombinant DNA techniques and gene transfection methods as is well
known in the art (e.g., Morrison, S. (1985) Science 229:1202).
[0105] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification, site directed mutagenesis) and
can be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the VH segment is
operatively linked to the CH segment(s) within the vector and the
VI, segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino tem-finus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0106] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss, M. A. and Wood,
C. R. (I 985) Immunology Today 6:12-13).
[0107] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfrCHO cells, described in Urlaub and
Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:42164220, used with a
DHFR selectable marker, e.g., as described in R. J. Kaufman and P.
A. Sharp (I 982) Mol. Biol. 159:601-62 1), NSO myeloma cells, COS
cells and SP2 cells. In particular, for use with NSO myeloma cells,
another preferred expression system is the GS gene expression
system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
[0108] Screening antibodies for specific binding to similar
proteins or fragments can also be conveniently achieved using
peptide display libraries. This method involves the screening of
large collections of peptides for individual members having the
desired function or structure. antibody screening of peptide
display libraries is well known in the art. The displayed peptide
sequences can be from 3 to 5000 or more amino acids in length,
frequently from 5-100 amino acids long, and often from about 8 to
25 amino acids long. In addition to direct chemical synthetic
methods for generating peptide libraries, several recombinant DNA
methods have been described. One type involves the display of a
peptide sequence on the surface of a bacteriophage or cell. Each
bacteriophage or cell contains the nucleotide sequence encoding the
particular displayed peptide sequence. Such methods are described
in PCT Patent Publication Nos. 91/17271, 91/18980, 91/19818, and
93/08278. Other systems for generating libraries of peptides have
aspects of both in vitro chemical synthesis and recombinant
methods. See, PCT Patent Publication Nos. 92/05258, 92/14843, and
96/19256. See also, U.S. Pat. Nos. 5,658,754; and 5,643,768.
Peptide display libraries, vector, and screening kits are
commercially available from such suppliers as Invitrogen (Carlsbad,
Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK).
See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778,
5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733,
5,767,260, 5,856,456, assigned to Enzon; U.S. Pat. Nos. 5,223,409,
5,403,484, 5,571,698, 5,837,500, assigned to Dyax, U.S. Pat. Nos.
5,427,908, 5,580,717, assigned to Affymax; U.S. Pat. No. 5,885,793,
assigned to Cambridge antibody Technologies; U.S. Pat. No.
5,750,373, assigned to Genentech, U.S. Pat. Nos. 5,618,920,
5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to
Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra, each of
the above patents and publications entirely incorporated herein by
reference.
[0109] Antibodies of the present invention can also be prepared
using at least one anti-alpha-V subunit antibody encoding nucleic
acid to provide transgenic animals or mammals, such as goats, cows,
horses, sheep, and the like, that produce such antibodies in their
milk. Such animals can be provided using known methods. See, e.g.,
but not limited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316;
5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of
which is entirely incorporated herein by reference.
[0110] Antibodies of the present invention can additionally be
prepared using at least one anti-alpha-V subunit antibody encoding
nucleic acid to provide transgenic plants and cultured plant cells
(e.g., but not limited to tobacco, maize, and duckweed) that
produce such antibodies, specified portions or variants in the
plant parts or in cells cultured therefrom. As a non-limiting
example, transgenic tobacco leaves expressing recombinant proteins
have been successfully used to provide large amounts of recombinant
proteins, e.g., using an inducible promoter. See, e.g., Cramer et
al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references
cited therein. Also, transgenic maize have been used to express
mammalian proteins at commercial production levels, with biological
activities equivalent to those produced in other recombinant
systems or purified from natural sources. See, e.g., Hood et al.,
Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited
therein. antibodies have also been produced in large amounts from
transgenic plant seeds including antibody fragments, such as single
chain antibodies (scFv's), including tobacco seeds and potato
tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109
(1998) and reference cited therein. Thus, antibodies of the present
invention can also be produced using transgenic plants, according
to know methods. See also, e.g., Fischer et al., Biotechnol. Appl.
Biochem. 30:99-108 (October, 1999), Ma et al., Trends Biotechnol.
13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995);
Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994); and
references cited therein. See, also generally for plant expression
of antibodies, but not limited to, Each of the above references is
entirely incorporated herein by reference.
2. Nucleic Acid Molecules
[0111] Using the information provided herein, such as the
nucleotide sequences encoding at least 70-100% of the contiguous
amino acids of at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8,
specified fragments, variants or consensus sequences thereof, or a
deposited vector comprising at least one of these sequences, a
nucleic acid molecule of the present invention encoding at least
one anti-alpha-V subunit antibody can be obtained using methods
described herein or as known in the art.
[0112] Nucleic acid molecules of the present invention can be in
the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in
the form of DNA, including, but not limited to, cDNA and genomic
DNA obtained by cloning or produced synthetically, or any
combinations thereof. The DNA can be triple-stranded,
double-stranded or single-stranded, or any combination thereof. Any
portion of at least one strand of the DNA or RNA can be the coding
strand, also known as the sense strand, or it can be the non-coding
strand, also referred to as the anti-sense strand.
[0113] Isolated nucleic acid molecules of the present invention can
include nucleic acid molecules comprising an open reading frame
(ORF), optionally with one or more introns, e.g., but not limited
to, at least one specified portion of at least one CDR, as CDR1,
CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS:
1-3) or light chain (e.g., SEQ ID NOS: 4-6); nucleic acid molecules
comprising the coding sequence for an anti-alpha-V subunit antibody
or variable region (e.g., SEQ ID NOS: 7, 8) including but not
limited to SEQ ID Nos; 18 and 19; and nucleic acid molecules which
comprise a nucleotide sequence substantially different from those
described above but which, due to the degeneracy of the genetic
code, still encode at least one anti-alpha-V subunit antibody as
described herein and/or as known in the art. Of course, the genetic
code is well known in the art. Thus, it would be routine for one
skilled in the art to generate such degenerate nucleic acid
variants that code for specific anti-alpha-V subunit antibodies of
the present invention. See, e.g., Ausubel, et al., supra, and such
nucleic acid variants are included in the present invention.
Non-limiting examples of isolated nucleic acid molecules of the
present invention include SEQ ID NOS: 10, 11, 12, 13, 14, 15, 18,
and 19 corresponding to non-limiting examples of a nucleic acid
encoding, respectively, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC
CDR2, LC CDR3, HC variable region and LC variable region.
[0114] In another aspect, the invention provides isolated nucleic
acid molecules encoding a(n) anti-alpha-V subunit antibody having
an amino acid sequence as encoded by the nucleic acid contained in
the plasmid designated clone C371A.
[0115] As indicated herein, nucleic acid molecules of the present
invention which comprise a nucleic acid encoding an anti-alpha-V
subunit antibody can include, but are not limited to, those
encoding the amino acid sequence of an antibody fragment, by
itself; the coding sequence for the entire antibody or a portion
thereof; the coding sequence for an antibody, fragment or portion,
as well as additional sequences, such as the coding sequence of at
least one signal leader or fusion peptide, with or without the
aforementioned additional coding sequences, such as at least one
intron, together with additional, non-coding sequences, including
but not limited to, non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and
polyadenylation signals (for example--ribosome binding and
stability of mRNA); an additional coding sequence that codes for
additional amino acids, such as those that provide additional
functionalities. Thus, the sequence encoding an antibody can be
fused to a marker sequence, such as a sequence encoding a peptide
that facilitates purification of the fused antibody comprising an
antibody fragment or portion.
3. Polynucleotides Which Selectively Hybridize to a Polynucleotide
as Described Herein
[0116] The present invention provides isolated nucleic acids that
hybridize under selective hybridization conditions to a
polynucleotide disclosed herein. Thus, the polynucleotides of this
embodiment can be used for isolating, detecting, and/or quantifying
nucleic acids comprising such polynucleotides. For example,
polynucleotides of the present invention can be used to identify,
isolate, or amplify partial or full-length clones in a deposited
library. In some embodiments, the polynucleotides are genomic or
cDNA sequences isolated, or otherwise complementary to, a cDNA from
a human or mammalian nucleic acid library.
[0117] Preferably, the cDNA library comprises at least 80%
full-length sequences, preferably at least 85% or 90% full-length
sequences, and more preferably at least 95% full-length sequences.
The cDNA libraries can be normalized to increase the representation
of rare sequences. Low or moderate stringency hybridization
conditions are typically, but not exclusively, employed with
sequences having a reduced sequence identity relative to
complementary sequences. Moderate and high stringency conditions
can optionally be employed for sequences of greater identity. Low
stringency conditions allow selective hybridization of sequences
having about 70% sequence identity and can be employed to identify
orthologous or paralogous sequences.
[0118] Optionally, polynucleotides of this invention will encode at
least a portion of an antibody encoded by the polynucleotides
described herein. The polynucleotides of this invention embrace
nucleic acid sequences that can be employed for selective
hybridization to a polynucleotide encoding an antibody of the
present invention. See, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by reference.
4. Construction of Nucleic Acids
[0119] The isolated nucleic acids of the present invention can be
made using (a) recombinant methods, (b) synthetic techniques, (c)
purification techniques, or combinations thereof, as well-known in
the art.
[0120] The nucleic acids can conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites can be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences can
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the coding sequence--is optionally
a vector, adapter, or linker for cloning and/or expression of a
polynucleotide of the present invention.
[0121] Additional sequences can be added to such cloning and/or
expression sequences to optimize their function in cloning and/or
expression, to aid in isolation of the polynucleotide, or to
improve the introduction of the polynucleotide into a cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well
known in the art. (See, e.g., Ausubel, supra; or Sambrook,
supra)
5. Recombinant Methods for Constructing Nucleic Acids
[0122] The isolated nucleic acid compositions of this invention,
such as RNA, cDNA, genomic DNA, or any combination thereof, can be
obtained from biological sources using any number of cloning
methodologies known to those of skill in the art. In some
embodiments, oligonucleotide probes that selectively hybridize,
under stringent conditions, to the polynucleotides of the present
invention are used to identify the desired sequence in a cDNA or
genomic DNA library. The isolation of RNA, and construction of cDNA
and genomic libraries, is well known to those of ordinary skill in
the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
6. Nucleic Acid Screening and Isolation Methods
[0123] A cDNA or genomic library can be screened using a probe
based upon the sequence of a polynucleotide of the present
invention, such as those disclosed herein. Probes can be used to
hybridize with genomic DNA or cDNA sequences to isolate homologous
genes in the same or different organisms. Those of skill in the art
will appreciate that various degrees of stringency of hybridization
can be employed in the assay; and either the hybridization or the
wash medium can be stringent. As the conditions for hybridization
become more stringent, there must be a greater degree of
complementarity between the probe and the target for duplex
formation to occur. The degree of stringency can be controlled by
one or more of temperature, ionic strength, pH and the presence of
a partially denaturing solvent such as formamide. For example, the
stringency of hybridization is conveniently varied by changing the
polarity of the reactant solution through, for example,
manipulation of the concentration of formamide within the range of
0% to 50%. The degree of complementarity (sequence identity)
required for detectable binding will vary in accordance with the
stringency of the hybridization medium and/or wash medium. The
degree of complementarity will optimally be 100%, or 70-100%, or
any range or value therein. However, it should be understood that
minor sequence variations in the probes and primers can be
compensated for by reducing the stringency of the hybridization
and/or wash medium.
[0124] Methods of amplification of RNA or DNA are well known in the
art and can be used according to the present invention without
undue experimentation, based on the teaching and guidance presented
herein.
[0125] Known methods of DNA or RNA amplification include, but are
not limited to, polymerase chain reaction (PCR) and related
amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; U.S. Pat. Nos.
4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat. No. 5,142,033 to
Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S. Pat. No.
5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, et al;
U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370
to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.
4,656,134 to Ringold) and RNA mediated amplification that uses
anti-sense RNA to the target sequence as a template for
double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et
al, with the tradename NASBA), the entire contents of which
references are incorporated herein by reference. (See, e.g.,
Ausubel, supra; or Sambrook, supra.)
[0126] For instance, polymerase chain reaction (PCR) technology can
be used to amplify the sequences of polynucleotides of the present
invention and related genes directly from genomic DNA or cDNA
libraries. PCR and other in vitro amplification methods can also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of the desired mRNA in samples, for
nucleic acid sequencing, or for other purposes. Examples of
techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, supra, Sambrook, supra,
and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No.
4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to
Methods and Applications, Eds., Academic Press Inc., San Diego,
Calif. (1990). Commercially available kits for genomic PCR
amplification are known in the art. See, e.g., Advantage-GC Genomic
PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein
(Boehringer Mannheim) can be used to improve yield of long PCR
products.
7. Synthetic Methods for Constructing Nucleic Acids
[0127] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by known methods (see,
e.g., Ausubel, et al., supra). Chemical synthesis generally
produces a single-stranded oligonucleotide, which can be converted
into double-stranded DNA by hybridization with a complementary
sequence, or by polymerization with a DNA polymerase using the
single strand as a template. One of skill in the art will recognize
that while chemical synthesis of DNA can be limited to sequences of
about 100 or more bases, longer sequences can be obtained by the
ligation of shorter sequences.
8. Recombinant Expression Cassettes
[0128] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence of the present invention, for
example a cDNA or a genomic sequence encoding an antibody of the
present invention, can be used to construct a recombinant
expression cassette that can be introduced into at least one
desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the present invention operably linked
to transcriptional initiation regulatory sequences that will direct
the transcription of the polynucleotide in the intended host cell.
Both heterologous and non-heterologous (i.e., endogenous) promoters
can be employed to direct expression of the nucleic acids of the
present invention.
[0129] In some embodiments, isolated nucleic acids that serve as
promoter, enhancer, or other elements can be introduced in the
appropriate position (upstream, downstream or in intron) of a
non-heterologous form of a polynucleotide of the present invention
so as to up or down regulate expression of a polynucleotide of the
present invention. For example, endogenous promoters can be altered
in vivo or in vitro by mutation, deletion and/or substitution.
[0130] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to includes promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV), Simian Virus 40
(SV40), adenovirus, (e.g., the adenovirus major late promoter
(AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences
may be used, such as the ubiquitin promoter or P-globin
promoter.
[0131] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4, 399,216, 4,634,665,
and 5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells
with 10 methotrexate selection/amplification) and the neo gene (for
G418 selection).
9. Vectors and Host Cells
[0132] The present invention also relates to vectors that include
isolated nucleic acid molecules of the present invention, host
cells that are genetically engineered with the recombinant vectors,
and the production of at least one anti-alpha-V subunit antibody by
recombinant techniques, as is well known in the art. See, e.g.,
Sambrook, et al., supra; Ausubel, et al., supra, each entirely
incorporated herein by reference.
[0133] The polynucleotides can optionally be joined to a vector
containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as
a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it can be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0134] The DNA insert should be operatively linked to an
appropriate promoter. The expression constructs will further
contain sites for transcription initiation, termination and, in the
transcribed region, a ribosome binding site for translation. The
coding portion of the mature transcripts expressed by the
constructs will preferably include a translation initiating at the
beginning and a termination codon (e.g., UAA, UGA or UAG)
appropriately positioned at the end of the mRNA to be translated,
with UAA and UAG preferred for mammalian or eukaryotic cell
expression.
[0135] Expression vectors will preferably but optionally include at
least one selectable marker. Such markers include, e.g., but not
limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S.
Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636;
5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or
glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359;
5,827,739) resistance for eukaryotic cell culture, and tetracycline
or ampicillin resistance genes for culturing in E. coli and other
bacteria or prokaryotics (the above patents are entirely
incorporated hereby by reference). Appropriate culture mediums and
conditions for the above-described host cells are known in the art.
Suitable vectors will be readily apparent to the skilled artisan.
Introduction of a vector construct into a host cell can be effected
by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other known methods.
Such methods are described in the art, such as Sambrook, supra,
Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15,
16.
[0136] At least one antibody of the present invention can be
expressed in a modified form, such as a fusion protein, and can
include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of
additional amino acids, particularly charged amino acids, can be
added to the N-terminus of an antibody to improve stability and
persistence in the host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties can be
added to an antibody of the present invention to facilitate
purification. Such regions can be removed prior to final
preparation of an antibody or at least one fragment thereof Such
methods are described in many standard laboratory manuals, such as
Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel,
supra, Chapters 16, 17 and 18.
[0137] Those of ordinary skill in the art are knowledgeable in the
numerous expression systems available for expression of a nucleic
acid encoding a protein of the present invention.
[0138] Alternatively, nucleic acids of the present invention can be
expressed in a host cell by turning on (by manipulation) in a host
cell that contains endogenous DNA encoding an antibody of the
present invention. Such methods are well known in the art, e.g., as
described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and
5,733,761, entirely incorporated herein by reference.
[0139] Illustrative of cell cultures useful for the production of
the antibodies, specified portions or variants thereof, are
mammalian cells. Mammalian cell systems often will be in the form
of monolayers of cells although mammalian cell suspensions or
bioreactors can also be used. A number of suitable host cell lines
capable of expressing intact glycosylated proteins have been
developed in the art, and include the COS-1 (e.g., ATCC CRL 1650),
COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO
(e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines,
Cos-7 cells, PerC.6 cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14,
293 cells, HeLa cells and the like, which are readily available
from, for example, American Type Culture Collection, Manassas, Va.
(www.atcc.org). Preferred host cells include cells of lymphoid
origin such as myeloma and lymphoma cells.
[0140] Expression vectors for these cells can include one or more
of the following expression control sequences, such as, but not
limited to an origin of replication; a promoter (e.g., late or
early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062;
5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase)
promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at
least one human immunoglobulin promoter; an enhancer, and/or
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site), and transcriptional terminator sequences. See,
e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells
useful for production of nucleic acids or proteins of the present
invention are known and/or available, for instance, from the
American Type Culture Collection Catalogue of Cell Lines and
Hybridomas (www.atcc.org) or other known or commercial sources.
[0141] When eukaryotic host cells are employed, polyadenlyation or
transcription terminator sequences are typically incorporated into
the vector. An example of a terminator sequence is the
polyadenlyation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript can also be
included. An example of a splicing sequence is the VP1 intron from
SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally,
gene sequences to control replication in the host cell can be
incorporated into the vector, as known in the art. Also, to avoid
high surface expression of heavy chain molecules, it may be
necessary to use an expression vector that eliminates transmembrane
domain variant splices.
10. Purification of an Antibody
[0142] An anti-alpha-V subunit antibody can be recovered and
purified from recombinant cell cultures by well-known methods
including, but not limited to, protein A purification, ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be employed for
purification. See, e.g., Colligan, Current Protocols in Immunology,
or Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely
incorporated herein by reference.
[0143] Antibodies of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a eukaryotic host,
including, for example, yeast, higher plant, insect and mammalian
cells. Depending upon the host employed in a recombinant production
procedure, the antibody of the present invention can be
glycosylated or can be non-glycosylated, with glycosylated
preferred. Such methods are described in many standard laboratory
manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel,
supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein
Science, supra, Chapters 12-14, all entirely incorporated herein by
reference.
11. Anti-Alpha-V Subunit Antibodies of the Invention
[0144] Since it is well known in the art that antibody heavy and
light chain CDR3 domains play a particularly important role in the
binding specificity/affinity of an antibody for an antigen, the
recombinant antibodies of the invention prepared as set forth above
preferably comprise the heavy and light chain CDR3s of CNTO 95. The
antibodies further can comprise the CDR2s of CNTO 95.The antibodies
further can comprise the CDRIs of CNTO 95. Accordingly, the
invention further provides anti-alpha V antibodies comprising: (1)
human heavy chain framework regions, a human heavy chain CDRI
region, a human heavy chain CDR2 region, and a human heavy chain
CDR3 region, wherein the human heavy chain CDR3 region is selected
from the CDR3s of CNTO 95 as shown in SEQ ID NO: 3, and (2) human
light chain framework regions, a human light chain CDR1 region, a
human light chain CDR2 region, and a human light chain CDR3 region,
wherein the human light chain CDR3 region is selected from the
CDR3s of CNTO 95 as shown in SEQ ID NO: 6, wherein the antibody
binds Alpha V integrin. The antibody may further comprise the heavy
chain CDR2 and/or the light chain CDR2 of CNTO 95. The antibody may
further comprise the heavy chain CDR1 and/or the light chain CDR1
of CNTO 95.
[0145] As a non-limiting example, the antibody or antigen-binding
portion or variant can comprise at least one of the heavy chain
CDR3 having the amino acid sequence of SEQ ID NO:3, and/or a light
chain CDR3 having the amino acid sequence of SEQ ID NO:6. In a
particular embodiment, the antibody or antigen-binding fragment can
have an antigen-binding region that comprises at least a portion of
at least one heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having
the amino acid sequence of the corresponding CDRs 1, 2 and/or 3
(e.g., SEQ ID NOS: 1, 2, and/or 3). In another particular
embodiment, the antibody or antigen-binding portion or variant can
have an antigen-binding region that comprises at least a portion of
at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3) having
the amino acid sequence of the corresponding CDRs 1, 2 and/or 3
(e.g., SEQ ID NOS: 4, 5, and/or 6). In a preferred embodiment the
three heavy chain CDRs and the three light chain CDRs of the
anitbody or antigen-binding fragment have the amino acid sequence
of the corresponding CDR of at least one of mAb CNTO 95, Gen0101,
CNTO 95, C372A, as described herein. Such antibodies can be
prepared by chemically joining together the various portions (e.g.,
CDRs, framework) of the antibody using conventional techniques, by
preparing and expressing a (i.e., one or more) nucleic acid
molecule that encodes the antibody using conventional techniques of
recombinant DNA technology or by using any other suitable
method.
[0146] Preferably, the CDR1, 2, and/or 3 of the engineered
antibodies described above comprise the exact amino acid
sequence(s) as those of CNTO 95 disclosed herein. However, the
ordinarily skilled artisan will appreciate that some deviation from
the exact CDR sequences of CNTO 95 may be possible while still
retaining the ability of the antibody to bind Alpha V effectively
(e.g., conservative substitutions). Accordingly, in another
embodiment, the engineered antibody may be composed of one or more
CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to one
or more CDRs of CNTO 95. In addition to simply binding Alpha V,
engineered antibodies such as those described above may be selected
for their retention of other functional properties of antibodies of
the invention, such as:
[0147] 1). binding to live cells expressing human Alpha V; 2)
binding to human Alpha V with a K.sub.D of 10.sup.-8 M or less
(e.g., 10.sup.-9 M or 10.sup.-10 M or less); 3) binding to a unique
epitope on Alpha V (to eliminate the possibility that monoclonal
antibodies with complimentary activities when used in combination
would compete for binding to the same epitope); 4) inhibition of
angiogenesis resulting in growth inhibition of tumor cells in
vivo.
[0148] Human monoclonal antibodies of the invention can be tested
for binding to Alpha V by, for example, standard ELISA.
[0149] To determine if the selected human anti-alpha V monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Competition studies using unlabeled monoclonal
antibodies and biotinylated monoclonal antibodies can be performed
using alpha V coated-ELISA plates. Biotinylated mAb binding can be
detected with a strep-avidin-alkaline phosphatase probe.
[0150] To determine the isotype of purified antibodies, isotype
ELISAs can be performed. In order to demonstrate binding of
monoclonal antibodies to live cells expressing the alpha V, flow
cytometry can be used. Anti-alpha V human IgGs can be further
tested for reactivity with alpha V antigen by Western blotting.
[0151] In another aspect of the invention, the structural features
of an human anti-alpha V antibodies of the invention, CNTO 95, are
used to create structurally related human anti-Alpha V antibodies
that retain at least one functional property of the antibodies of
the invention, such as binding to Alpha V. More specifically, one
or more CDR regions of CNTO 95 can be combined recombinantly with
known human framework regions and CDRs to create additional,
recombinantly-engineered, human anti-Alpha V antibodies of the
invention.
[0152] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-Alpha V antibody comprising: preparing
an antibody comprising (1) human heavy chain framework regions and
human heavy chain CDRs, wherein at least one of the human heavy
chain CDRs comprises an amino acid sequence selected from the amino
acid sequences of CDRs shown in SEQ ID NOs: 1-3; and (2) human
light chain framework regions and human light chain CDRs, wherein
at least one of the human heavy chain CDRs comprises an amino
acid-sequence selected from the amino acid sequences of CDRs shown
in SEQ ID NOs: 4-6; wherein the antibody retains the ability to
bind to Alpha V. The ability of the antibody to bind Alpha V can be
determined using standard binding assays, such as those set forth
in the Examples (e.g., an ELISA).
[0153] The antibodies of the invention can bind human alpha-V
subunit with a wide range of affinities (K.sub.D). In a preferred
embodiment, at least one human mAb of the present invention can
optionally bind human alpha-V subunit with high affinity. For
example, a human mAb can bind human alpha-V subunit with a K.sub.D
equal to or less than about 10.sup.-7 M, such as but not limited
to, 0.1-9.9 (or any range or value therein).times.10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12,
10.sup.-13 M or any range or value therein.
[0154] The affinity or avidity of an antibody for an antigen can be
determined experimentally using any suitable method. (See, for
example, Berzofsky, et al., "Antibody-Antigen Interactions," In
Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York,
N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New
York, N.Y. (1992); and methods described herein). The measured
affinity of a particular antibody-antigen interaction can vary if
measured under different conditions (e.g., salt concentration, pH).
Thus, measurements of affinity and other antigen-binding parameters
(e.g., K.sub.D, K.sub.a, K.sub.d) are preferably made with
standardized solutions of antibody and antigen, and a standardized
buffer, such as the buffer described herein.
[0155] Preferably, the human antibody or antigen-binding fragment
of the invention binds human alpha-V subunit and, thereby partially
or substantially neutralizes at least one biological activity of
the protein. An antibody, or specified portion or variant thereof,
that partially or preferably substantially neutralizes at least one
biological activity of at least one alpha-V subunit protein or
fragment can bind the protein or fragment and thereby inhibit
activities mediated through the binding of alpha-V subunit to its
ligand or through other alpha-V subunit-dependent or mediated
mechanisms. As used herein, the term "neutralizing antibody" refers
to an antibody that can inhibit an alpha-V subunit-dependent
activity by about 20-120%, preferably by at least about 10, 20, 30,
40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100% or more depending on the assay. The capacity of an
anti-alpha-V subunit antibody to inhibit an alpha-V
subunit-dependent activity is preferably assessed by at least one
suitable alpha-V subunit protein or receptor assay, as described
herein and/or as known in the art. A human antibody of the
invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or
isotype and can comprise a kappa or lambda light chain. In one
embodiment, the human antibody comprises an IgG heavy chain or
defined fragment, for example, at least one of isotypes, IgG1,
IgG2, IgG3 or IgG4. Antibodies of this type can be prepared by
employing a transgenic mouse or other trangenic non-human mammal
comprising at least one human light chain (e.g., IgG, IgA and IgM
(e.g., .gamma.1,.gamma.2, .gamma.3, .gamma.4) transgenes as
described herein and/or as known in the art. In another embodiment,
the anti-human alpha-V subunit human antibody comprises an IgG1
heavy chain and a IgG1, light chain.
[0156] At least one antibody of the invention binds at least one
specified epitope specific to at least one alpha-V subunit protein,
subunit, fragment, portion or any combination thereof. The at least
one epitope can comprise at least one antibody binding region that
comprises at least one portion of said protein, which epitope is
preferably comprised of at least one extracellular, soluble,
hydrophillic, external or cytoplasmic portion of said protein. The
at least one specified epitope can comprise any combination of at
least one amino acid sequence of at least 1-3 amino acids to the
entire specified portion of contiguous amino acids of the SEQ ID
NOS:9, 16 or 17.
[0157] As previously stated, the invention also relates to
antibodies, antigen-binding fragments, immunoglobulin chains and
CDRs comprising amino acids in a sequence that is substantially the
same as an amino acid sequence described herein. Preferably, such
antibodies or antigen-binding fragments and antibodies comprising
such chains or CDRs can bind human alpha-V subunit with high
affinity (e.g., K.sub.D less than or equal to about 10.sup.-9 M).
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions. A conservative amino acid substitution
refers to the replacement of a first amino acid by a second amino
acid that has chemical and/or physical properties (e.g, charge,
structure, polarity, hydrophobicity/hydrophilicity) that are
similar to those of the first amino acid. Conservative
substitutions include replacement of one amino acid by another
within the following groups: lysine (K), arginine (R) and histidine
(H); aspartate (D) and glutamate (E); asparagine (N), glutamine
(Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E;
alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),
phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and
glycine (G); F, W and Y; C, S and T.
[0158] An anti-alpha-V subunit antibody of the present invention
can include one or more amino acid substitutions, deletions or
additions, either from natural mutations or human manipulation, as
specified herein.
[0159] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions, insertions or deletions for any given anti-alpha-V
subunit antibody will not be more than 40, 30, 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or
any range or value therein, as specified herein.
[0160] Amino acids in an anti-alpha-V subunit antibody of the
present invention that are essential for function can be identified
by methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15;
Cunningham and Wells, Science 244:1081-1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity, such as, but not limited to at least one
alpha-V subunit neutralizing activity. Sites that are critical for
antibody binding can also be identified by structural analysis such
as crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos, et al., Science 255:306-312 (1992)).
[0161] Anti-alpha-V subunit antibodies of the present invention can
include, but are not limited to, at least one portion, sequence or
combination selected from 5 to all of the contiguous amino acids of
at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6.
[0162] A(n) anti-alpha-V subunit antibody can further optionally
comprise a polypeptide of at least one of 70-100% of the contiguous
amino acids of at least one of SEQ ID NOS: 7, 8.
[0163] In one embodiment, the amino acid sequence of an
immunoglobulin chain, or portion thereof (e.g., variable region,
CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the
amino acid sequence of the corresponding chain of at least one of
SEQ ID NOS:7, 8. For example, the amino acid sequence of a light
chain variable region can be compared with the sequence of SEQ ID
NO:8, or the amino acid sequence of a heavy chain CDR3 can be
compared with SEQ ID NO:7. Preferably, 70-100% amino acid identity
(i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or
value therein) is determined using a suitable computer algorithm,
as known in the art.
[0164] Exemplary heavy chain and light chain variable regions
sequences are provided in SEQ ID NOS: 7, 8. The antibodies of the
present invention, or specified variants thereof, can comprise any
number of contiguous amino acid residues from an antibody of the
present invention, wherein that number is selected from the group
of integers consisting of from 10-100% of the number of contiguous
residues in an anti-alpha-V subunit antibody. Optionally, this
subsequence of contiguous amino acids is at least about 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in
length, or any range or value therein. Further, the number of such
subsequences can be any integer selected from the group consisting
of from 1 to 20, such as at least 2, 3, 4, or 5.
[0165] As those of skill will appreciate, the present invention
includes at least one biologically active antibody of the present
invention. Biologically active antibodies have a specific activity
at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or
70%, and most preferably at least 80%, 90%, or 95%-100% of that of
the native (non-synthetic), endogenous or related and known
antibody. Methods of assaying and quantifying measures of enzymatic
activity and substrate specificity, are well known to those of
skill in the art.
[0166] In another aspect, the invention relates to human antibodies
and antigen-binding fragments, as described herein, which are
modified by the covalent attachment of an organic moiety. Such
modification can produce an antibody or antigen-binding fragment
with improved pharmacokinetic properties (e.g., increased in vivo
serum half-life). The organic moiety can be a linear or branched
hydrophilic polymeric group, fatty acid group, or fatty acid ester
group. In particular embodiments, the hydrophilic polymeric group
can have a molecular weight of about 800 to about 120,000 Daltons
and can be a polyalkane glycol (e.g., polyethylene glycol (PEG),
polypropylene glycol (PPG)), carbohydrate polymer, amino acid
polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid
ester group can comprise from about eight to about forty carbon
atoms.
[0167] The modified antibodies and antigen-binding fragments of the
invention can comprise one or more organic moieties that are
covalently bonded, directly or indirectly, to the antibody. Each
organic moiety that is bonded to an antibody or antigen-binding
fragment of the invention can independently be a hydrophilic
polymeric group, a fatty acid group or a fatty acid ester group. As
used herein, the term "fatty acid" encompasses mono-carboxylic
acids and di-carboxylic acids. A "hydrophilic polymeric group," as
the term is used herein, refers to an organic polymer that is more
soluble in water than in octane. For example, polylysine is more
soluble in water than in octane. Thus, an antibody modified by the
covalent attachment of polylysine is encompassed by the invention.
Hydrophilic polymers suitable for modifying antibodies of the
invention can be linear or branched and include, for example,
polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol
(mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose,
oligosaccharides, polysaccharides and the like), polymers of
hydrophilic amino acids (e.g., polylysine, polyarginine,
polyaspartate and the like), polyalkane oxides (e.g., polyethylene
oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
Preferably, the hydrophilic polymer that modifies the antibody of
the invention has a molecular weight of about 800 to about 150,000
Daltons as a separate molecular entity. For example PEG.sub.5000
and PEG.sub.20,000, wherein the subscript is the average molecular
weight of the polymer in Daltons, can be used. The hydrophilic
polymeric group can be substituted with one to about six alkyl,
fatty acid or fatty acid ester groups. Hydrophilic polymers that
are substituted with a fatty acid or fatty acid ester group can be
prepared by employing suitable methods. For example, a polymer
comprising an amine group can be coupled to a carboxylate of the
fatty acid or fatty acid ester, and an activated carboxylate (e.g.,
activated with N,N-carbonyl diimidazole) on a fatty acid or fatty
acid ester can be coupled to a hydroxyl group on a polymer.
[0168] Fatty acids and fatty acid esters suitable for modifying
antibodies of the invention can be saturated or can contain one or
more units of unsaturation. Fatty acids that are suitable for
modifying antibodies of the invention include, for example,
n-dodecanoate (C.sub.12, laurate), n-tetradecanoate (C.sub.14,
myristate), n-octadecanoate (C.sub.18, stearate), n-eicosanoate
(C.sub.20, arachidate), n-docosanoate (C.sub.22, behenate),
n-triacontanoate (C.sub.30), n-tetracontanoate (C.sub.40),
cis-.DELTA.9-octadecanoate (C.sub.18, oleate), all
cis-.DELTA.5,8,11,14-eicosatetraenoate (C.sub.20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid,
docosanedioic acid, and the like. Suitable fatty acid esters
include mono-esters of dicarboxylic acids that comprise a linear or
branched lower alkyl group. The lower alkyl group can comprise from
one to about twelve, preferably one to about six, carbon atoms.
[0169] The modified human antibodies and antigen-binding fragments
can be prepared using suitable methods, such as by reaction with
one or more modifying agents. A "modifying agent" as the term is
used herein, refers to a suitable organic group (e.g., hydrophilic
polymer, a fatty acid, a fatty acid ester) that comprises an
activating group. An "activating group" is a chemical moiety or
functional group that can, under appropriate conditions, react with
a second chemical group thereby forming a covalent bond between the
modifying agent and the second chemical group. For example,
amine-reactive activating groups include electrophilic groups such
as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),
N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups
that can react with thiols include, for example, maleimide,
iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic
acid thiol (TNB-thiol), and the like. An aldehyde functional group
can be coupled to amine- or hydrazide-containing molecules, and an
azide group can react with a trivalent phosphorous group to form
phosphoramidate or phosphorimide linkages. Suitable methods to
introduce activating groups into molecules are known in the art
(see for example, Hermanson, G. T., Bioconjugate Techniques,
Academic Press: San Diego, Calif. (1996)). An activating group can
be bonded directly to the organic group (e.g., hydrophilic polymer,
fatty acid, fatty acid ester), or through a linker moiety, for
example a divalent C.sub.1-C.sub.12 group wherein one or more
carbon atoms can be replaced by a heteroatom such as oxygen,
nitrogen or sulfur. Suitable linker moieties include, for example,
tetraethylene glycol, --(CH.sub.2).sub.3--,
--NH--(CH.sub.2).sub.6--NH--, --(CH.sub.2).sub.2--NH-- and
--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH--NH--.
Modifying agents that comprise a linker moiety can be produced, for
example, by reacting a mono-Boc-alkyldiamine (e.g.,
mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid
in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) to form an amide bond between the free amine and the fatty
acid carboxylate. The Boc protecting group can be removed from the
product by treatment with trifluoroacetic acid (TFA) to expose a
primary amine that can be coupled to another carboxylate as
described, or can be reacted with maleic anhydride and the
resulting product cyclized to produce an activated maleimido
derivative of the fatty acid. (See, for example, Thompson, et al.,
WO 92/16221 the entire teachings of which are incorporated herein
by reference.)
[0170] The modified antibodies of the invention can be produced by
reacting a human antibody or antigen-binding fragment with a
modifying agent. For example, the organic moieties can be bonded to
the antibody in a non-site specific manner by employing an
amine-reactive modifying agent, for example, an NHS ester of PEG.
Modified human antibodies or antigen-binding fragments can also be
prepared by reducing disulfide bonds (e.g., intra-chain disulfide
bonds) of an antibody or antigen-binding fragment. The reduced
antibody or antigen-binding fragment can then be reacted with a
thiol-reactive modifying agent to produce the modified antibody of
the invention. Modified human antibodies and antigen-binding
fragments comprising an organic moiety that is bonded to specific
sites of an antibody of the present invention can be prepared using
suitable methods, such as reverse proteolysis (Fisch et al.,
Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate
Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.
6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68
(1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463
(1997)), and the methods described in Hermanson, G. T.,
Bioconjugate Techniques, Academic Press: San Diego, Calif.
(1996).
12. Anti-Idiotype Antibodies to Anti-Alpha-V Subunit Antibody
Compositions
[0171] In addition to monoclonal or chimeric anti-alpha-V subunit
antibodies, the present invention is also directed to an
anti-idiotypic (anti-Id) antibody specific for such antibodies of
the invention. An anti-Id antibody is an antibody which recognizes
unique determinants generally associated with the antigen-binding
region of another antibody. The anti-Id can be prepared by
immunizing an animal of the same species and genetic type (e.g.
mouse strain) as the source of the Id antibody with the antibody or
a CDR containing region thereof. The immunized animal will
recognize and respond to the idiotypic determinants of the
immunizing antibody and produce an anti-Id antibody. The anti-Id
antibody may also be used as an "immunogen" to induce an immune
response in yet another animal, producing a so-called anti-anti-Id
antibody.
13. Anti-Alpha-V Subunit Antibody Compositions
[0172] The present invention also provides at least one
anti-alpha-V subunit antibody composition comprising at least one,
at least two, at least three, at least four, at least five, at
least six or more anti-alpha-V subunit antibodies thereof, as
described herein and/or as known in the art that are provided in a
non-naturally occurring composition, mixture or form. Such
compositions comprise non-naturally occurring compositions
comprising at least one or two full length, C- and/or N-terminally
deleted variants, domains, fragments, or specified variants, of the
anti-alpha-V subunit antibody amino acid sequence selected from the
group consisting of 70-100% of the contiguous amino acids of SEQ ID
NOS: 1, 2, 3, 4, 5, 6, 7, 8, or specified fragments, domains or
variants thereof Preferred anti-alpha-V subunit antibody
compositions include at least one or two full length, fragments,
domains or variants as at least one CDR or LBR containing portions
of the anti-alpha-V subunit antibody sequence of 70-100% of SEQ ID
NOS:1, 2, 3, 4, 5, 6, or specified fragments, domains or variants
thereof. Further preferred compositions comprise 40-99% of at least
one of 70-100% of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or specified
fragments, domains or variants thereof. Such composition
percentages are by weight, volume, concentration, molarity, or
molality as liquid or dry solutions, mixtures, suspension,
emulsions or colloids, as known in the art or as described
herein.
[0173] Anti-alpha-V subunit antibody compositions of the present
invention can further comprise at least one of any suitable and
effective amount of a composition or pharmaceutical composition
comprising at least one anti-alpha-V subunit antibody to a cell,
tissue, organ, animal or patient in need of such modulation,
treatment or therapy, optionally further comprising at least one
selected from at least one TNF antagonist (e.g., but not limited to
a TNF antibody or fragment, a soluble TNF receptor or fragment,
fusion proteins thereof, or a small molecule TNF antagonist), an
antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,
azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteroid, (dexamethasone), an anabolic
steroid (testosterone), a diabetes related agent, a mineral, a
nutritional, a thyroid agent, a vitamin, a calcium related hormone,
an antidiarrheal, an antitussive, an antiemetic, an antiulcer, a
laxative, an anticoagulant, an erythropoietin (e.g., epoetin
alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim
(GM-CSF, Leukine), an immunization, an immunoglobulin (rituximab),
an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab),
a growth hormone, a hormone antagonist, a reproductive hormone
antagonist (flutamide, nilutamide), a hormone release modulator
(leuprolide, goserelin), a hormone replacement drug, an estrogen
receptor modulator (tamoxifen), a retinoid (tretinoin), a
topoisomerase inhibitor (etoposide, irinotecan), a cytoxin
(doxorubicin, dacarbazine), a mydriatic, a cycloplegic, an
alkylating agent (carboplatin), a nitrogen mustard (melphalen,
chlorabucil), a nitrosourea (carmustine, estramustine) an
antimetabolite (methotrexate, cytarabine, fluorouracil), a mitotic
inhibitor (vincristine, taxol), a radiopharmaceutical
(Iodine131-tositumomab), a radiosensitizer (misonidazole,
tirapazamine) an antidepressant, antimanic agent, an antipsychotic,
an anxiolytic, a hypnotic, a sympathomimetic, a stimulant,
donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled steroid, a leukotriene inhibitor, a methylxanthine, a
cromolyn, an epinephrine or analog, dornase alpha (Pulmozyme), a
cytokine (interferon alpha-2, IL2) or a cytokine antagonist
(infliximab). Non-limiting examples of such cytokines include, but
are not limted to, any of IL-1 to IL-23, IL-6, anti-tumor
antibodies, chemotherapeutic agents or radiation therapies.
Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and
Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which references are entirely
incorporated herein by reference.
[0174] Such anti-cancer can also include toxin molecules that are
associated, bound, co-formulated or co-administered with at least
one antibody of the present invention. The toxin can optionally act
to selectively kill the pathologic cell or tissue. The pathologic
cell can be a cancer or other cell. Such toxins can be, but are not
limited to, purified or recombinant toxin or toxin fragment
comprising at least one functional cytotoxic domain of toxin, e.g.,
selected from at least one of ricin, diphtheria toxin, a venom
toxin, or a bacterial toxin. The term toxin also includes both
endotoxins and exotoxins produced by any naturally occurring,
mutant or recombinant bacteria or viruses which may cause any
pathological condition in humans and other mammals, including toxin
shock, which can result in death. Such toxins may include, but are
not limited to, enterotoxigenic E. coli heat-labile enterotoxin
(LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas
enterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal
enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal
enterotoxins and the like. Such bacteria include, but are not
limited to, strains of a species of enterotoxigenic E. coli (ETEC),
enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),
Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus
pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella
flexneri, Shigella boydii, and Shigella sonnei), Salmonella species
(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella
enteritidis), Clostridium species (e.g., Clostridium perfringens,
Clostridium dificile, Clostridium botulinum), Camphlobacter species
(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter
species, (e.g., Heliobacter pylori), Aeromonas species (e.g.,
Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae),
Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species
(e.g., Vibrios cholerae, Vibrios parahemolyticus), Klebsiella
species, Pseudomonas aeruginosa, and Streptococci. See, e.g.,
Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and
Co., Boston, (1990); Evans et al., eds., Bacterial Infections of
Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum
Medical Book Co., New York (1991); Mandell et al, Principles and
Practice of Infectious Diseases, 3d. Ed., Churchill Livingstone,
New York (1990); Berkow et al, eds., The Merck Manual, 16th
edition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMS
Microbiology Immunology, 76:121-134 (1991); Marrack et al, Science,
248:705-711 (1990), the contents of which references are
incorporated entirely herein by reference.
[0175] Anti-alpha-V subunit antibody compounds, compositions or
combinations of the present invention can further comprise at least
one of any suitable auxiliary, such as, but not limited to,
diluent, binder, stabilizer, buffers, salts, lipophilic solvents,
preservative, adjuvant or the like. Pharmaceutically acceptable
auxiliaries are preferred. Non-limiting examples of, and methods of
preparing such sterile solutions are well known in the art, such
as, but limited to, Gennaro, Ed., Remington's Pharmaceutical
Sciences, 18.sup.th Edition, Mack Publishing Co. (Easton, Pa.)
1990. Pharmaceutically acceptable carriers can be routinely
selected that are suitable for the mode of administration,
solubility and/or stability of the anti-alpha-V subunit antibody,
fragment or variant composition as well known in the art or as
described herein.
[0176] Pharmaceutical excipients and additives useful in the
present composition include but are not limited to proteins,
peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides;
derivatized sugars such as alditols, aldonic acids, esterified
sugars and the like; and polysaccharides or sugar polymers), which
can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein
excipients include serum albumin such as human serum albumin (HSA),
recombinant human albumin (rHA), gelatin, casein, and the like.
Representative amino acid/antibody components, which can also
function in a buffering capacity, include alanine, glycine,
arginine, betaine, histidine, glutamic acid, aspartic acid,
cysteine, lysine, leucine, isoleucine, valine, methionine,
phenylalanine, aspartame, and the like. One preferred amino acid is
glycine.
[0177] Carbohydrate excipients suitable for use in the invention
include, for example, monosaccharides such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol
(glucitol), myoinositol and the like. Preferred carbohydrate
excipients for use in the present invention are mannitol,
trehalose, and raffinose.
[0178] Anti-alpha-V subunit antibody compositions can also include
a buffer or a pH adjusting agent; typically, the buffer is a salt
prepared from an organic acid or base. Representative buffers
include organic acid salts such as salts of citric acid, ascorbic
acid, gluconic acid, carbonic acid, tartaric acid, succinic acid,
acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or
phosphate buffers. Preferred buffers for use in the present
compositions are organic acid salts such as citrate.
[0179] Additionally, anti-alpha-V subunit antibody compositions of
the invention can include polymeric excipients/additives such as
polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates
(e.g., cyclodextrins, such as 2-hydroxypropyl- -cyclodextrin),
polyethylene glycols, flavoring agents, antimicrobial agents,
sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and
chelating agents (e.g., EDTA).
[0180] These and additional known pharmaceutical excipients and/or
additives suitable for use in the anti-alpha-V subunit antibody,
portion or variant compositions according to the invention are
known in the art, e.g., as listed in "Remington: The Science &
Practice of Pharmacy", 19.sup.th ed., Williams & Williams,
(1995), and in the "Physician's Desk Reference", 52.sup.nd ed.,
Medical Economics, Montvale, N.J. (1998), the disclosures of which
are entirely incorporated herein by reference. Preferrred carrier
or excipient materials are carbohydrates (e.g., saccharides and
alditols) and buffers (e.g., citrate) or polymeric agents.
14. Formulations
[0181] As noted above, the invention provides for stable
formulations, which is preferably a phosphate buffer with saline or
a chosen salt, as well as preserved solutions and formulations
containing a preservative as well as multi-use preserved
formulations suitable for pharmaceutical or veterinary use,
comprising at least one anti-alpha-V subunit antibody in a
pharmaceutically acceptable formulation. Preserved formulations
contain at least one known preservative or optionally selected from
the group consisting of at least one phenol, m-cresol, p-cresol,
o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride
(e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and
the like), benzalkonium chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous
diluent. Any suitable concentration or mixture can be used as known
in the art, such as 0.001-5%, or any range or value therein, such
as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02,
0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range
or value therein. Non-limiting examples include, no preservative,
0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3%
benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%),
0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g.,
0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s)
(e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01,
0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and
the like.
[0182] As noted above, the invention provides an article of
manufacture, comprising packaging material and at least one vial
comprising a solution of at least one anti-alpha-V subunit antibody
with the prescribed buffers and/or preservatives, optionally in an
aqueous diluent, wherein said packaging material comprises a label
that indicates that such solution can be held over a period of 1,
2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72
hours or greater. The invention further comprises an article of
manufacture, comprising packaging material, a first vial comprising
lyophilized at least one anti-alpha-V subunit antibody, and a
second vial comprising an aqueous diluent of prescribed buffer or
preservative, wherein said packaging material comprises a label
that instructs a patient to reconstitute the at least one
anti-alpha-V subunit antibody in the aqueous diluent to form a
solution that can be held over a period of twenty-four hours or
greater.
[0183] The at least one anti-alpha-V subunit antibody used in
accordance with the present invention can be produced by
recombinant means, including from mammalian cell or transgenic
preparations, or can be purified from other biological sources, as
described herein or as known in the art.
[0184] The range of at least one anti-alpha-V subunit antibody in
the product of the present invention includes amounts yielding upon
reconstitution, if in a wet/dry system, concentrations from about
1.0 .mu.g/ml to about 1000 mg/ml, although lower and higher
concentrations are operable and are dependent on the intended
delivery vehicle, e.g., solution formulations will differ from
transdermal patch, pulmonary, transmucosal, or osmotic or micro
pump methods.
[0185] Preferably, the aqueous diluent optionally further comprises
a pharmaceutically acceptable preservative. Preferred preservatives
include those selected from the group consisting of phenol,
m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
alkylparaben (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal, or mixtures thereof. The concentration of
preservative used in the formulation is a concentration sufficient
to yield an anti-microbial effect. Such concentrations are
dependent on the preservative selected and are readily determined
by the skilled artisan.
[0186] Other excipients, e.g. isotonicity agents, buffers,
antioxidants, preservative enhancers, can be optionally and
preferably added to the diluent. An isotonicity agent, such as
glycerin, is commonly used at known concentrations. A
physiologically tolerated buffer is preferably added to provide
improved pH control. The formulations can cover a wide range of
pHs, such as from about pH 4 to about pH 10, and preferred ranges
from about pH 5 to about pH 9, and a most preferred range of about
6.0 to about 8.0. Preferably the formulations of the present
invention have pH between about 6.8 and about 7.8. Preferred
buffers include phosphate buffers, most preferably sodium
phosphate, particularly phosphate buffered saline (PBS).
[0187] Other additives, such as a pharmaceutically acceptable
solubilizers like Tween 20 (polyoxyethylene (20) sorbitan
monolaurate), Tween 40 (polyoxyethylene (20) sorbitan
monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan
monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block
copolymers), and PEG (polyethylene glycol) or non-ionic surfactants
such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic.RTM.
polyls, other block co-polymers, and chelators such as EDTA and
EGTA can optionally be added to the formulations or compositions to
reduce aggregation. These additives are particularly useful if a
pump or plastic container is used to administer the formulation.
The presence of pharmaceutically acceptable surfactant mitigates
the propensity for the protein to aggregate.
[0188] The formulations of the present invention can be prepared by
a process which comprises mixing at least one anti-alpha-V subunit
antibody and a preservative selected from the group consisting of
phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
alkylparaben, (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal or mixtures thereof in an aqueous diluent. Mixing
the at least one anti-alpha-V subunit antibody and preservative in
an aqueous diluent is carried out using conventional dissolution
and mixing procedures. To prepare a suitable formulation, for
example, a measured amount of at least one anti-alpha-V subunit
antibody in buffered solution is combined with the desired
preservative in a buffered solution in quantities sufficient to
provide the protein and preservative at the desired concentrations.
Variations of this process would be recognized by one of ordinary
skill in the art. For example, the order the components are added,
whether additional additives are used, the temperature and pH at
which the formulation is prepared, are all factors that can be
optimized for the concentration and means of administration
used.
[0189] The claimed formulations can be provided to patients as
clear solutions or as dual vials comprising a vial of lyophilized
at least one anti-alpha-V subunit antibody that is reconstituted
with a second vial containing water, a preservative and/or
excipients, preferably a phosphate buffer and/or saline and a
chosen salt, in an aqueous diluent. Either a single solution vial
or dual vial requiring reconstitution can be reused multiple times
and can suffice for a single or multiple cycles of patient
treatment and thus can provide a more convenient treatment regimen
than currently available.
[0190] The present claimed articles of manufacture are useful for
administration over a period of immediately to twenty-four hours or
greater. Accordingly, the presently claimed articles of manufacture
offer significant advantages to the patient. Formulations of the
invention can optionally be safely stored at temperatures of from
about 2 to about 40.degree. C. and retain the biologically activity
of the protein for extended periods of time, thus, allowing a
package label indicating that the solution can be held and/or used
over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.
If preserved diluent is used, such label can include use up to 1-12
months, one-half, one and a half, and/or two years.
[0191] The solutions of at least one anti-alpha-V subunit antibody
in the invention can be prepared by a process that comprises mixing
at least one antibody in an aqueous diluent. Mixing is carried out
using conventional dissolution and mixing procedures. To prepare a
suitable diluent, for example, a measured amount of at least one
antibody in water or buffer is combined in quantities sufficient to
provide the protein and optionally a preservative or buffer at the
desired concentrations. Variations of this process would be
recognized by one of ordinary skill in the art. For example, the
order the components are added, whether additional additives are
used, the temperature and pH at which the formulation is prepared,
are all factors that can be optimized for the concentration and
means of administration used.
[0192] The claimed products can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized at
least one anti-alpha-V subunit antibody that is reconstituted with
a second vial containing the aqueous diluent. Either a single
solution vial or dual vial requiring reconstitution can be reused
multiple times and can suffice for a single or multiple cycles of
patient treatment and thus provides a more convenient treatment
regimen than currently available.
[0193] The claimed products can be provided indirectly to patients
by providing to pharmacies, clinics, or other such institutions and
facilities, clear solutions or dual vials comprising a vial of
lyophilized at least one anti-alpha-V subunit antibody that is
reconstituted with a second vial containing the aqueous diluent.
The clear solution in this case can be up to one liter or even
larger in size, providing a large reservoir from which smaller
portions of the at least one antibody solution can be retrieved one
or multiple times for transfer into smaller vials and provided by
the pharmacy or clinic to their customers and/or patients.
[0194] Recognized devices comprising these single vial systems
include those pen-injector devices for delivery of a solution such
as BD Pens, BD Autojector.RTM., Humaject.RTM., NovoPen.RTM.,
B-D.RTM.Pen, AutoPen.RTM., and OptiPen.RTM., GenotropinPen.RTM.,
Genotronorm Pen.RTM., Humatro Pen.RTM., Reco-Pen.RTM., Roferon
Pen.RTM., Biojector.RTM., iject.RTM., J-tip Needle-Free
Injector.RTM., Intraject.RTM., Medi-Ject.RTM., e.g., as made or
developed by Becton Dickensen (Franklin Lakes, N.J.,
www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,
www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com);
National Medical Products, Weston Medical (Peterborough, UK,
www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,
www.mediject.com). Recognized devices comprising a dual vial system
include those pen-injector systems for reconstituting a lyophilized
drug in a cartridge for delivery of the reconstituted solution such
as the HumatroPen.RTM..
[0195] The products presently claimed include packaging material.
The packaging material provides, in addition to the information
required by the regulatory agencies, the conditions under which the
product can be used. The packaging material of the present
invention provides instructions to the patient to reconstitute the
at least one anti-alpha-V subunit antibody in the aqueous diluent
to form a solution and to use the solution over a period of 2-24
hours or greater for the two vial, wet/dry, product. For the single
vial, solution product, the label indicates that such solution can
be used over a period of 2-24 hours or greater. The presently
claimed products are useful for human pharmaceutical product
use.
[0196] The formulations of the present invention can be prepared by
a process that comprises mixing at least one anti-alpha-V subunit
antibody and a selected buffer, preferably a phosphate buffer
containing saline or a chosen salt. Mixing the at least one
antibody and buffer in an aqueous diluent is carried out using
conventional dissolution and mixing procedures. To prepare a
suitable formulation, for example, a measured amount of at least
one antibody in water or buffer is combined with the desired
buffering agent in water in quantities sufficient to provide the
protein and buffer at the desired concentrations. Variations of
this process would be recognized by one of ordinary skill in the
art. For example, the order the components are added, whether
additional additives are used, the temperature and pH at which the
formulation is prepared, are all factors that can be optimized for
the concentration and means of administration used.
[0197] The claimed stable or preserved formulations can be provided
to patients as clear solutions or as dual vials comprising a vial
of lyophilized at least one anti-alpha-V subunit antibody that is
reconstituted with a second vial containing a preservative or
buffer and excipients in an aqueous diluent. Either a single
solution vial or dual vial requiring reconstitution can be reused
multiple times and can suffice for a single or multiple cycles of
patient treatment and thus provides a more convenient treatment
regimen than currently available.
[0198] At least one anti-alpha-V subunit antibody in either the
stable or preserved formulations or solutions described herein, can
be administered to a patient in accordance with the present
invention via a variety of delivery methods including SC or IM
injection; transdermal, pulmonary, transmucosal, implant, osmotic
pump, cartridge, micro pump, or other means appreciated by the
skilled artisan, as well-known in the art.
15. Therapeutic Applications
[0199] The anti-alpha-V subunit antibodies of the present invention
or specified variants thereof can be used to measure or effect in
an cell, tissue, organ or animal (including mammals and humans), to
diagnose, monitor, modulate, treat, alleviate, help prevent the
incidence of, or reduce the symptoms of, at least one condition
mediated, affected or modulated by alpha V integrins. Such
conditions are selected from, but not limited to, diseases or
conditions mediated by cell adhesion and/or angiogenesis. Such
diseases or conditions include an immune disorder or disease, a
cardiovascular disorder or disease, an infectious, malignant,
and/or neurologic disorder or disease, or other known or specified
alpha-V integrin subunit related conditions. In particular, the
antibodies are useful for the treatment of diseases that involve
angiogenesis such as disease of the eye and neoplastic disease,
tissue remodeling such as restenosis, and proliferation of certain
cells types particularly epithelial and squamous cell carcinomas.
Particular indications include use in the treatment of
atherosclerosis, restenosis, cancer metastasis, rheumatoid
arthritis, diabetic retinopathy and macular degeneration. The
neutralizing antibodies of the invention are also useful to prevent
or treat unwanted bone resorption or degradation, for example as
found in osteoporosis or resulting from PTHrP overexpression by
some tumors. The antibodies may also be useful in the treatment of
various fibrotic diseases such as idiopathic pulmonary fibrosis,
diabetic nephropathy, hepatitis, and cirrhosis.
[0200] Thus, the present invention provides a method for modulating
or treating at least one alpha-V subunit related disease, in a
cell, tissue, organ, animal, or patient, as known in the art or as
described herein, using at least one alpha-V subunit antibody of
the present invention. Particular indications are discussed
below:
[0201] Malignant Disease
[0202] The present invention also provides a method for modulating
or treating at least one malignant disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: leukemia, acute leukemia, acute lymphoblastic leukemia
(ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),
chromic myelocytic leukemia (CML), chronic lymphocytic leukemia
(CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a
lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's
lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma,
colorectal carcinoma, pancreatic carcinoma, renal cell carcinoma,
breast cancer, nasopharyngeal carcinoma, malignant histiocytosis,
paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors,
adenocarcinomas, squamous cell carcinomas, sarcomas, malignant
melanoma, particularly metastatic melanoma, hemangioma, metastatic
disease, cancer related bone resorption, cancer related bone pain,
and the like.
[0203] Immune Related Disease
[0204] The present invention also provides a method for modulating
or treating at least one immune related disease, in a cell, tissue,
organ, animal, or patient including, but not limited to, at least
one of rheumatoid arthritis, juvenile rheumatoid arthritis,
systemic onset juvenile rheumatoid arthritis, psoriatic arthritis,
ankylosing spondilitis, gastric ulcer, seronegative arthropathies,
osteoarthritis, inflammatory bowel disease, ulcerative colitis,
systemic lupus erythematosis, antiphospholipid syndrome,
iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary
fibrosis, systemic vasculitis/wegener's granulomatosis,
sarcoidosis, orchitis/vasectomy reversal procedures,
allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic contact dermatitis, allergic conjunctivitis,
hypersensitivity pneumonitis, transplants, organ transplant
rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis,
neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage,
burns, ionizing radiation exposure, acute pancreatitis, adult
respiratory distress syndrome, rheumatoid arthritis,
alcohol-induced hepatitis, chronic inflammatory pathologies,
sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes,
nephrosis, atopic diseases, hypersensitity reactions, allergic
rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis,
pernicious anemia, hemolytic disesease, thrombocytopenia, graft
rejection of any organ or tissue, kidney translplant rejection,
heart transplant rejection, liver transplant rejection, pancreas
transplant rejection, lung transplant rejection, bone marrow
transplant (BMT) rejection, skin allograft rejection, cartilage
transplant rejection, bone graft rejection, small bowel transplant
rejection, fetal thymus implant rejection, parathyroid transplant
rejection, xenograft rejection of any organ or tissue, allograft
rejection, anti-receptor hypersensitivity reactions, Graves
disease, Raynoud's disease, type B insulin-resistant diabetes,
asthma, myasthenia gravis, antibody-meditated cytotoxicity, type
III hypersensitivity reactions, systemic lupus erythematosus, POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, skin changes
syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed
connective tissue disease, idiopathic Addison's disease, diabetes
mellitus, chronic active hepatitis, primary billiary cirrhosis,
vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV
hypersensitivity, contact dermatitis, hypersensitivity pneumonitis,
allograft rejection, granulomas due to intracellular organisms,
drug sensitivity, metabolic/idiopathic, Wilson's disease,
hemachromatosis, alpha-1-antitrypsin deficiency, diabetic
retinopathy, hashimoto's thyroiditis, osteoporosis,
hypothalamic-pituitary-adrenal axis evaluation, primary biliary
cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic
fibrosis, neonatal chronic lung disease, chronic obstructive
pulmonary disease (COPD), familial hematophagocytic
lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,
nephrotic syndrome, nephritis, glomerular nephritis, acute renal
failure, hemodialysis, uremia, toxicity, preeclampsia, OKT3
therapy, anti-CD3 therapy, cytokine therapy, chemotherapy,
radiation therapy (e.g., including but not limited toasthenia,
anemia, cachexia, and the like), chronic salicylate intoxication,
and the like. See, e.g., the Merck Manual, 12th-17th Editions,
Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992,
1999), Pharmacotherapy Handbook, Wells et al., eds., Second
Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each
entirely incorporated by reference.
[0205] Cardiovascular Disease
[0206] The present invention also provides a method for modulating
or treating at least one cardiovascular disease in a cell, tissue,
organ, animal, or patient, including, but not limited to, at least
one of cardiac stun syndrome, myocardial infarction, congestive
heart failure, stroke, ischemic stroke, hemorrhage,
arteriosclerosis, atherosclerosis, restenosis, diabetic
ateriosclerotic disease, hypertension, arterial hypertension,
renovascular hypertension, syncope, shock, syphilis of the
cardiovascular system, heart failure, cor pulmonale, primary
pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats,
atrial flutter, atrial fibrillation (sustained or paroxysmal), post
perfusion syndrome, cardiopulmonary bypass inflammation response,
chaotic or multifocal atrial tachycardia, regular narrow QRS
tachycardia, specific arrythmias, ventricular fibrillation, His
bundle arrythmias, atrioventricular block, bundle branch block,
myocardial ischemic disorders, coronary artery disease, angina
pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart
diseases, endocarditis, pericardial disease, cardiac tumors, aordic
and peripheral aneuryisms, aortic dissection, inflammation of the
aorta, occulsion of the abdominal aorta and its branches,
peripheral vascular disorders, occulsive arterial disorders,
peripheral atherlosclerotic disease, thromboangitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous
thrombosis, varicose veins, arteriovenous fistula, lymphederma,
lipedema, unstable angina, reperfusion injury, post pump syndrome,
ischemia-reperfusion injury, and the like. Such a method can
optionally comprise administering an effective amount of a
composition or pharmaceutical composition comprising at least one
anti-alpha-V subunit antibody to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy.
[0207] Neurologic Disease
[0208] The present invention also provides a method for modulating
or treating at least one neurologic disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: neurodegenerative diseases, multiple sclerosis, migraine
headache, AIDS dementia complex, demyelinating diseases, such as
multiple sclerosis and acute transverse myelitis; extrapyramidal
and cerebellar disorders' such as lesions of the corticospinal
system; disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs which block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; Progressive
supranucleo Palsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia, telangiectasia, and mitochondrial
multi.system disorder); demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit' such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease; and
Dementia pugilistica, and the like. Such a method can optionally
comprise administering an effective amount of a composition or
pharmaceutical composition comprising at least one TNF antibody or
specified portion or variant to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy. See,
e.g., the Merck Manual, 16.sup.th Edition, Merck & Company,
Rahway, N.J. (1992).
[0209] The present invention also provides a method for modulating
or treating at least one infectious disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: acute or chronic bacterial infection, acute and chronic
parasitic or infectious processes, including bacterial, viral and
fungal infections, HIV infection/HIV neuropathy, meningitis,
hepatitis (A,B or C, or the like), septic arthritis, peritonitis,
pneumonia, epiglottitis, e. coli 0157:h7, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue
hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome,
streptococcal myositis, gas gangrene, mycobacterium tuberculosis,
mycobacterium avium intracellulare, pneumocystis carinii pneumonia,
pelvic inflammatory disease, orchitis/epidydimitis, legionella,
lyme disease, influenza a, epstein-barr virus, vital-associated
hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and
the like.
[0210] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising at least one anti-alpha-V
subunit antibody to a cell, tissue, organ, animal or patient in
need of such modulation, treatment or therapy. Such a method can
optionally further at least one selected from at least one TNF
antagonist (e.g., but not limited to a TNF antibody or fragment, a
soluble TNF receptor or fragment, fusion proteins thereof, or a
small molecule TNF antagonist), an antirheumatic (e.g.,
methotrexate, auranofin, aurothioglucose, azathioprine, etanercept,
gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide,
sulfasalzine), a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anethetic, a neuromuscular blocker, an
antimicrobial (e.g., aminoglycoside, an antifungal, an
antiparasitic, an antiviral, a carbapenem, cephalosporin, a
flurorquinolone, a macrolide, a penicillin, a sulfonamide, a
tetracycline, another antimicrobial), an antipsoriatic, a
corticosteriod (dexamethasone), an anabolic steroid (testosterone),
a diabetes related agent, a mineral, a nutritional, a thyroid
agent, a vitamin, a calcium related hormone, an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an
anticoagulant, an erythropoietin (e.g., epoetin alpha), a
filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an immunization, an immunoglobulin (rituximab), an
immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a
growth hormone, a hormone antagonist, a reproductive hormone
antagonist (flutamide, nilutamide), a hormone release modulator
(leuprolide, goserelin), a hormone replacement drug, an estrogen
receptor modulator (tamoxifen), a retinoid (tretinoin), a
topoisomerase inhibitor (etoposide, irinotecan), a cytoxin
(doxorubicin), a mydriatic, a cycloplegic, an alkylating agent
(carboplatin), a nitrogen mustard (melphalen, chlorabucil), a
nitrosourea (carmustine, estramustine) an antimetabolite
(methotrexate, cytarabine, fluorouracil), a mitotic inhibitor
(vincristine, taxol), a radiopharmaceutical
(Iodine131-tositumomab), a radiosensitizer (misonidazole,
tirapazamine) an antidepressant, antimanic agent, an antipsychotic,
an anxiolytic, a hypnotic, a sympathomimetic, a stimulant,
donepezil, tacrine, an asthma medication, a beta agonist, an
inhaled steroid, a leukotriene inhibitor, a methylxanthine, a
cromolyn, an epinephrine or analog, dornase alpha (Pulmozyme), a
cytokine (interferon alpha-2, IL2) or a cytokine antagonist
(inflixamab). Suitable dosages are well known in the art. See,
e.g., Wells et al., eds., Pharmacotherapy Handbook, 2.sup.nd
Edition, Appleton and Lange, Stamford, Conn. (2000); PDR
Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition,
Tarascon Publishing, Loma Linda, Calif. (2000), each of which
references are entirely incorporated herein by reference.
[0211] Particular combinations for treatment of neoplastic diseases
comprise co-administration or combination therapy by administering,
before concurrently, and/or after, an antineplastic agent such as
an alkylating agent, a nitrogen mustard, a nitrosurea, an
antibiotic, an anti-metabolite, a hormonal agonist or antagonist,
an immunomodulator, and the like. For use in metastatic melanoma
and other neoplastic diseases,a preferred combination is to
co-administer the antibody with dacarbazine, interferon alpha,
interleukin-2, temozolomide, cisplatin, vinblastine, Imatinib
Mesylate, carmustine, paclitaxel and the like. For metastatic
melanoma, dacarbazine is preferred.
[0212] Therapeutic Treatments
[0213] Typically, treatment of pathologic conditions is effected by
administering an effective amount or dosage of at least one
anti-alpha-V subunit antibody composition that total, on average, a
range from at least about 0.01 to 500 milligrams of at least one
anti-alpha-V subunit antibody per kilogram of patient per dose, and
preferably from at least about 0.1 to 100 milligrams
antibody/kilogram of patient per single or multiple administration,
depending upon the specific activity of contained in the
composition. Alternatively, the effective serum concentration can
comprise 0.1-5000 .mu.g/ml serum concentration per single or
multiple adminstration. Suitable dosages are known to medical
practitioners and will, of course, depend upon the particular
disease state, specific activity of the composition being
administered, and the particular patient undergoing treatment. In
some instances, to achieve the desired therapeutic amount, it can
be necessary to provide for repeated administration, i.e., repeated
individual administrations of a particular monitored or metered
dose, where the individual administrations are repeated until the
desired daily dose or effect is achieved.
[0214] Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
and/or 100-500 mg/kg/administration, or any range, value or
fraction thereof, or to achieve a serum concentration of 0.1, 0.5,
0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0,
4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5,
8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9,
13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9,
7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,
11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5,
15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5,
19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000,
4500, and/or 5000 .mu.g/ml serum concentration per single or
multiple administration, or any range, value or fraction
thereof.
[0215] Alternatively, the dosage administered can vary depending
upon known factors, such as the pharmacodynamic characteristics of
the particular agent, and its mode and route of administration;
age, health, and weight of the recipient; nature and extent of
symptoms, kind of concurrent treatment, frequency of treatment, and
the effect desired. Usually a dosage of active ingredient can be
about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily
0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per
administration or in sustained release form is effective to obtain
desired results.
[0216] As a non-limiting example, treatment of humans or animals
can be provided as a one-time or periodic dosage of at least one
antibody of the present invention 0.1 to 100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively or additionally, at least one of
week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or
52, or alternatively or additionally, at least one of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years,
or any combination thereof, using single, infusion or repeated
doses.
[0217] Dosage forms (composition) suitable for internal
administration generally contain from about 0.1 milligram to about
500 milligrams of active ingredient per unit or container. In these
pharmaceutical compositions the active ingredient will ordinarily
be present in an amount of about 0.5-99.999% by weight based on the
total weight of the composition.
[0218] For parenteral administration, the antibody can be
formulated as a solution, suspension, emulsion or lyophilized
powder in association, or separately provided, with a
pharmaceutically acceptable parenteral vehicle. Examples of such
vehicles are water, saline, Ringer's solution, dextrose solution,
and 1-10% human serum albumin. Liposomes and nonaqueous vehicles
such as fixed oils can also be used. The vehicle or lyophilized
powder can contain additives that maintain isotonicity (e.g.,
sodium chloride, mannitol) and chemical stability (e.g., buffers
and preservatives). The formulation is sterilized by known or
suitable techniques.
[0219] Suitable pharmaceutical carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
[0220] Alternative Administration
[0221] Many known and developed modes of can be used according to
the present invention for administering pharmaceutically effective
amounts of at least one anti-alpha-V subunit antibody according to
the present invention. While pulmonary administration is used in
the following description, other modes of administration can be
used according to the present invention with suitable results.
[0222] Alpha-V subunit antibodies of the present invention can be
delivered in a carrier, as a solution, emulsion, colloid, or
suspension, or as a dry powder, using any of a variety of devices
and methods suitable for administration by inhalation or other
modes described here within or known in the art.
[0223] Parenteral Formulations and Administration
[0224] Formulations for parenteral administration can contain as
common excipients sterile water or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes and the like. Aqueous or oily suspensions for
injection can be prepared by using an appropriate emulsifier or
humidifier and a suspending agent, according to known methods.
Agents for injection can be a non-toxic, non-orally administrable
diluting agent such as aquous solution or a sterile injectable
solution or suspension in a solvent. As the usable vehicle or
solvent, water, Ringer's solution, isotonic saline, etc. are
allowed; as an ordinary solvent, or suspending solvent, sterile
involatile oil can be used. For these purposes, any kind of
involatile oil and fatty acid can be used, including natural or
synthetic or semisynthetic fatty oils or fatty acids; natural or
synthetic or semisynthtetic mono- or di- or tri-glycerides.
Parental administration is known in the art and includes, but is
not limited to, conventional means of injections, a gas pressured
needle-less injection device as described in U.S. Pat. No.
5,851,198, and a laser perforator device as described in U.S. Pat.
No. 5,839,446 entirely incorporated herein by reference.
[0225] Alternative Delivery
[0226] The invention further relates to the administration of at
least one anti-alpha-V subunit antibody by parenteral,
subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal
means. At least one anti-alpha-V subunit antibody composition can
be prepared for use for parenteral (subcutaneous, intramuscular or
intravenous) or any other administration particularly in the form
of liquid solutions or suspensions; for use in vaginal or rectal
administration particularly in semisolid forms such as, but not
limited to, creams and suppositories; for buccal, or sublingual
administration such as, but not limited to, in the form of tablets
or capsules; or intranasally such as, but not limited to, the form
of powders, nasal drops or aerosols or certain agents; or
transdermally such as not limited to a gel, ointment, lotion,
suspension or patch delivery system with chemical enhancers such as
dimethyl sulfoxide to either modify the skin structure or to
increase the drug concentration in the transdermal patch
(Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, D. S.,
Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely
incorporated herein by reference), or with oxidizing agents that
enable the application of formulations containing proteins and
peptides onto the skin (WO 98/53847), or applications of electric
fields to create transient transport pathways such as
electroporation, or to increase the mobility of charged drugs
through the skin such as iontophoresis, or application of
ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and
4,767,402) (the above publications and patents being entirely
incorporated herein by reference).
[0227] Pulmonary/Nasal Administration
[0228] For pulmonary administration, preferably at least one
anti-alpha-V subunit antibody composition is delivered in a
particle size effective for reaching the lower airways of the lung
or sinuses. According to the invention, at least one anti-alpha-V
subunit antibody can be delivered by any of a variety of inhalation
or nasal devices known in the art for administration of a
therapeutic agent by inhalation. These devices capable of
depositing aerosolized formulations in the sinus cavity or alveoli
of a patient include metered dose inhalers, nebulizers, dry powder
generators, sprayers, and the like. Other devices suitable for
directing the pulmonary or nasal administration of antibodies are
also known in the art. All such devices can use of formulations
suitable for the administration for the dispensing of antibody in
an aerosol. Such aerosols can be comprised of either solutions
(both aqueous and non aqueous) or solid particles. Metered dose
inhalers like the Ventolin.RTM. metered dose inhaler, typically use
a propellent gas and require actuation during inspiration (See,
e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like
Turbuhaler.TM. (Astra), Rotahaler.RTM. (Glaxo), Diskus.RTM.
(Glaxo), Spiros.TM. inhaler (Dura), devices marketed by Inhale
Therapeutics, and the Spinhaler.RTM. powder inhaler (Fisons), use
breath-actuation of a mixed powder (U.S. Pat. No. 4,668,218 Astra,
EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No.
5,458,135 Inhale, WO 94/06498 Fisons, entirely incorporated herein
by reference). Nebulizers like AERx.TM. Aradigm, the Ultravent.RTM.
nebulizer (Mallinckrodt), and the Acorn II.RTM. nebulizer (Marquest
Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376),
the above references entirely incorporated herein by reference,
produce aerosols from solutions, while metered dose inhalers, dry
powder inhalers, etc. generate small particle aerosols. These
specific examples of commercially available inhalation devices are
intended to be a representative of specific devices suitable for
the practice of this invention, and are not intended as limiting
the scope of the invention. Preferably, a composition comprising at
least one anti-alpha-V subunit antibody is delivered by a dry
powder inhaler or a sprayer. There are a several desirable features
of an inhalation device for administering at least one antibody of
the present invention. For example, delivery by the inhalation
device is advantageously reliable, reproducible, and accurate. The
inhalation device can optionally deliver small dry particles, e.g.
less than about 10 m, preferably about 1-5 m, for good
respirability.
[0229] Administration of Alpha-V Subunit Antibody Compositions as a
Spray
[0230] A spray including alpha-V subunit antibody composition
protein can be produced by forcing a suspension or solution of at
least one anti-alpha-V subunit antibody through a nozzle under
pressure. The nozzle size and configuration, the applied pressure,
and the liquid feed rate can be chosen to achieve the desired
output and particle size. An electrospray can be produced, for
example, by an electric field in connection with a capillary or
nozzle feed. Advantageously, particles of at least one anti-alpha-V
subunit antibody composition protein delivered by a sprayer have a
particle size less than about 10 m, preferably in the range of
about 1 .mu.m to about 5 .mu.m, and most preferably about 2 .mu.m
to about 3 .mu.m.
[0231] Formulations of at least one anti-alpha-V subunit antibody
composition protein suitable for use with a sprayer typically
include antibody composition protein in an aqueous solution at a
concentration of about 0.1 mg to about 100 mg of at least one
anti-alpha-V subunit antibody composition protein per ml of
solution or mg/gm, or any range or value therein, e.g., but not
lmited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100
mg/ml or mg/gm. The formulation can include agents such as an
excipient, a buffer, an isotonicity agent, a preservative, a
surfactant, and, preferably, zinc. The formulation can also include
an excipient or agent for stabilization of the antibody composition
protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk proteins useful in formulating antibody
composition proteins include albumin, protamine, or the like.
Typical carbohydrates useful in formulating antibody composition
proteins include sucrose, mannitol, lactose, trehalose, glucose, or
the like. The antibody composition protein formulation can also
include a surfactant, which can reduce or prevent surface-induced
aggregation of the antibody composition protein caused by
atomization of the solution in forming an aerosol. Various
conventional surfactants can be employed, such as polyoxyethylene
fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty
acid esters. Amounts will generally range between 0.001 and 14% by
weight of the formulation. Especially preferred surfactants for
purposes of this invention are polyoxyethylene sorbitan monooleate,
polysorbate 80, polysorbate 20, or the like. Additional agents
known in the art for formulation of a protein such as alpha-V
subunit antibodies, or specified portions or variants, can also be
included in the formulation.
[0232] Administration of Alpha-V Subunit Antibody Compositions by a
Nebulizer
[0233] Antibody composition protein can be administered by a
nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
Typically, in a jet nebulizer, a compressed air source is used to
create a high-velocity air jet through an orifice. As the gas
expands beyond the nozzle, a low-pressure region is created, which
draws a solution of antibody composition protein through a
capillary tube connected to a liquid reservoir. The liquid stream
from the capillary tube is sheared into unstable filaments and
droplets as it exits the tube, creating the aerosol. A range of
configurations, flow rates, and baffle types can be employed to
achieve the desired performance characteristics from a given jet
nebulizer. In an ultrasonic nebulizer, high-frequency electrical
energy is used to create vibrational, mechanical energy, typically
employing a piezoelectric transducer. This energy is transmitted to
the formulation of antibody composition protein either directly or
through a coupling fluid, creating an aerosol including the
antibody composition protein. Advantageously, particles of antibody
composition protein delivered by a nebulizer have a particle size
less than about 10 .mu.m, preferably in the range of about 1 .mu.m
to about 5 .mu.m, and most preferably about 2 .mu.m to about 3
.mu.m.
[0234] Formulations of at least one anti-alpha-V subunit antibody
suitable for use with a nebulizer, either jet or ultrasonic,
typically include a concentration of about 0.1 mg to about 100 mg
of at least one anti-alpha-V subunit antibody protein per ml of
solution. The formulation can include agents such as an excipient,
a buffer, an isotonicity agent, a preservative, a surfactant, and,
preferably, zinc. The formulation can also include an excipient or
agent for stabilization of the at least one anti-alpha-V subunit
antibody composition protein, such as a buffer, a reducing agent, a
bulk protein, or a carbohydrate. Bulk proteins useful in
formulating at least one anti-alpha-V subunit antibody composition
proteins include albumin, protamine, or the like. Typical
carbohydrates useful in formulating at least one anti-alpha-V
subunit antibody include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The at least one anti-alpha-V subunit
antibody formulation can also include a surfactant, which can
reduce or prevent surface-induced aggregation of the at least one
anti-alpha-V subunit antibody caused by atomization of the solution
in forming an aerosol. Various conventional surfactants can be
employed, such as polyoxyethylene fatty acid esters and alcohols,
and polyoxyethylene sorbital fatty acid esters. Amounts will
generally range between 0.001 and 4% by weight of the formulation.
Especially preferred surfactants for purposes of this invention are
polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate
20, or the like. Additional agents known in the art for formulation
of a protein such as antibody protein can also be included in the
formulation.
[0235] Administration of Alpha-V Subunit Antibody Compositions by a
Metered Dose Inhaler
[0236] In a metered dose inhaler (MDI), a propellant, at least one
anti-alpha-V subunit antibody, and any excipients or other
additives are contained in a canister as a mixture including a
liquefied compressed gas. Actuation of the metering valve releases
the mixture as an aerosol, preferably containing particles in the
size range of less than about 10 .mu.m, preferably about 1 .mu.m to
about 5 .mu.m, and most preferably about 2 .mu.m to about 3 .mu.m.
The desired aerosol particle size can be obtained by employing a
formulation of antibody composition protein produced by various
methods known to those of skill in the art, including jet-milling,
spray drying, critical point condensation, or the like. Preferred
metered dose inhalers include those manufactured by 3M or Glaxo and
employing a hydrofluorocarbon propellant.
[0237] Formulations of at least one anti-alpha-V subunit antibody
for use with a metered-dose inhaler device will generally include a
finely divided powder containing at least one anti-alpha-V subunit
antibody as a suspension in a non-aqueous medium, for example,
suspended in a propellant with the aid of a surfactant. The
propellant can be any conventional material employed for this
purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydro fluoro carbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a
(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the
like. Preferably the propellant is a hydrofluorocarbon. The
surfactant can be chosen to stabilize the at least one anti-alpha-V
subunit antibody as a suspension in the propellant, to protect the
active agent against chemical degradation, and the like. Suitable
surfactants include sorbitan trioleate, soya lecithin, oleic acid,
or the like. In some cases solution aerosols are preferred using
solvents such as ethanol. Additional agents known in the art for
formulation of a protein such as protein can also be included in
the formulation.
[0238] One of ordinary skill in the art will recognize that the
methods of the current invention can be achieved by pulmonary
administration of at least one anti-alpha-V subunit antibody
compositions via devices not described herein.
[0239] Oral Formulations and Administration
[0240] Formulations for oral rely on the co-administration of
adjuvants (e.g., resorcinols and nonionic surfactants such as
polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to
increase artificially the permeability of the intestinal walls, as
well as the co-administration of enzymatic inhibitors (e.g.,
pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and
trasylol) to inhibit enzymatic degradation. The active constituent
compound of the solid-type dosage form for oral administration can
be mixed with at least one additive, including sucrose, lactose,
cellulose, mannitol, trehalose, raffinose, maltitol, dextran,
starches, agar, arginates, chitins, chitosans, pectins, gum
tragacanth, gum arabic, gelatin, collagen, casein, albumin,
synthetic or semisynthetic polymer, and glyceride. These dosage
forms can also contain other type(s) of additives, e.g., inactive
diluting agent, lubricant such as magnesium stearate, paraben,
preserving agent such as sorbic acid, ascorbic acid,
.alpha.-tocopherol, antioxidant such as cysteine, disintegrator,
binder, thickener, buffering agent, sweetening agent, flavoring
agent, perfuming agent, etc.
[0241] Tablets and pills can be further processed into
enteric-coated preparations. The liquid preparations for oral
administration include emulsion, syrup, elixir, suspension and
solution preparations allowable for medical use. These preparations
can contain inactive diluting agents ordinarily used in said field,
e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More
recently, microspheres of artificial polymers of mixed amino acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat.
No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to
deliver biologically active agents orally are known in the art.
[0242] Mucosal Formulations and Administration
[0243] For absorption through mucosal surfaces, compositions and
methods of administering at least one anti-alpha-V subunit antibody
include an emulsion comprising a plurality of submicron particles,
a mucoadhesive macromolecule, a bioactive peptide, and an aqueous
continuous phase, which promotes absorption through mucosal
surfaces by achieving mucoadhesion of the emulsion particles (U.S.
Pat. No. 5,514,670). Mucous surfaces suitable for application of
the emulsions of the present invention can include corneal,
conjunctival, buccal, sublingual, nasal, vaginal, pulmonary,
stomachic, intestinal, and rectal routes of administration.
Formulations for vaginal or rectal administration, e.g.
suppositories, can contain as excipients, for example,
polyalkyleneglycols, vaseline, cocoa butter, and the like.
Formulations for intranasal administration can be solid and contain
as excipients, for example, lactose or can be aqueous or oily
solutions of nasal drops. For buccal administration excipients
include sugars, calcium stearate, magnesium stearate,
pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
[0244] Transdermal Formulations and Administration
[0245] For transdermal administration, the at least one
anti-alpha-V subunit antibody is encapsulated in a delivery device
such as a liposome or polymeric nanoparticles, microparticle,
microcapsule, or microspheres (referred to collectively as
microparticles unless otherwise stated). A number of suitable
devices are known, including microparticles made of synthetic
polymers such as polyhydroxy acids such as polylactic acid,
polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and polyphosphazenes, and natural polymers such as
collagen, polyamino acids, albumin and other proteins, alginate and
other polysaccharides, and combinations thereof (U.S. Pat. Nos.
5,814,599).
[0246] Prolonged Administration and Formulations
[0247] It can be sometimes desirable to deliver the compounds of
the present invention to the subject over prolonged periods of
time, for example, for periods of one week to one year from a
single administration. Various slow release, depot or implant
dosage forms can be utilized. For example, a dosage form can
contain a pharmaceutically acceptable non-toxic salt of the
compounds that has a low degree of solubility in body fluids, for
example, (a) an acid addition salt with a polybasic acid such as
phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic
acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene
mono- or di-sulfonic acids, polygalacturonic acid, and the like;
(b) a salt with a polyvalent metal cation such as zinc, calcium,
bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,
cadmium and the like, or with an organic cation formed from e.g.,
N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c)
combinations of (a) and (b) e.g. a zinc tannate salt. Additionally,
the compounds of the present invention or, preferably, a relatively
insoluble salt such as those just described, can be formulated in a
gel, for example, an aluminum monostearate gel with, e.g. sesame
oil, suitable for injection. Particularly preferred salts are zinc
salts, zinc tannate salts, pamoate salts, and the like. Another
type of slow release depot formulation for injection would contain
the compound or salt dispersed for encapsulated in a slow
degrading, non-toxic, non-antigenic polymer such as a polylactic
acid/polyglycolic acid polymer for example as described in U.S.
Pat. No. 3,773,919. The compounds or, preferably, relatively
insoluble salts such as those described above can also be
formulated in cholesterol matrix silastic pellets, particularly for
use in animals. Additional slow release, depot or implant
formulations, e.g. gas or liquid liposomes are known in the
literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled
Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker,
Inc., N.Y., 1978).
16. Diagnostic and Research Applications
[0248] For diagnostic applications, the antibodies of the invention
typically will be labeled with a detectable moiety. The detectable
moiety can be any one which is capable of producing, either
directly or indirectly, a detectable signal. For example, the
detectable moiety may be a radioisotope, such as .sup.3H, .sup.14C,
.sup.32P, .sup.36S, or .sup.126I, a fluorescent or chemiluminescent
compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin; radioactive isotopic labels, such as, e.g., .sup.125I,
.sup.32P, .sup.14C, technicium, or .sup.3H, or an enzyme, such as
alkaline phosphatase, beta-galactosidase or horseradish
peroxidase.
[0249] Any method known in the art for separately conjugating the
antibody to the detectable moiety may be employed, including those
methods described by Hunter, et al., Nature 144:945 (1962); David,
e at., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth.
40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407
(1982).
[0250] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc., 1987).
[0251] Competitive binding assays rely on the ability of a labeled
standard (which may be alpha.v or an immunologically reactive
portion thereof) to compete with the test sample analyte (alpha.v)
for binding with a limited amount of antibody. The amount of
.alpha.v in the test sample is inversely proportional to the amount
of standard that becomes bound to the antibodies. To facilitate
determining the amount of standard that becomes bound, the
antibodies generally are insolubilized before or after the
competition, so that the standard and analyte that are bound to the
antibodies may conveniently be separated from the standard and
analyte which remain unbound.
[0252] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three part complex. David &
Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0253] The antibodies of the invention also are useful for in vivo
imaging, wherein an antibody labeled with a detectable moiety such
as a radio-opaque agent or radioisotope is administered to, a host,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host is assayed. This imaging technique
is useful in the staging and treatment of neoplasms or bone
disorders. The antibody may be labeled with any moiety that is
detectable in a host, whether by nuclear magnetic resonance,
radiology, or other detection means known in the art.
[0254] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
Cloning and Expression of Alpha-V Subunit Antibody in Mammalian
Cells
[0255] A typical mammalian expression vector contains at least one
promoter element, which mediates the initiation of transcription of
mRNA, the antibody coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pIRES1neo, pRetro-Off,
pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.),
pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-)
(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat
(ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
Mammalian host cells that could be used include human Hela 293, H9
and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV
1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO)
cells.
[0256] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[0257] The transfected gene can also be amplified to express large
amounts of the encoded antibody. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et
al., Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
antibodies.
[0258] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec.
Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer
(Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
contain in addition the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene.
[0259] Cloning and Expression in CHO Cells
[0260] The vector pC4 is used for the expression of alpha-V subunit
antibody. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr
(ATCC Accession No. 37146). The plasmid contains the mouse DHFR
gene under control of the SV40 early promoter. Chinese hamster
ovary- or other cells lacking dihydrofolate activity that are
transfected with these plasmids can be selected by growing the
cells in a selective medium (e.g., alpha minus MEM, Life
Technologies, Gaithersburg, Md.) supplemented with the
chemotherapeutic agent methotrexate. The amplification of the DHFR
genes in cells resistant to methotrexate (MTX) has been well
documented (see, e.g., F. W. Alt, et al., J. Biol. Chem.
253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys.
Acta 1097:107-143 (1990); and M. J. Page and M. A. Sydenham,
Biotechnology 9:64-68 (1991)). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach can be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained that contain the
amplified gene integrated into one or more chromosome(s) of the
host cell.
[0261] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early
gene of human cytomegalovirus (CMV) (Boshart, et al., Cell
41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow integration of
the genes. Behind these cloning sites the plasmid contains the 3'
intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the
expression, e.g., the human b-actin promoter, the SV40 early or
late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On
gene expression systems and similar systems can be used to express
the alpha-V subunit antibody in a regulated way in mammalian cells
(M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551
(1992)). For the polyadenylation of the mRNA other signals, e.g.,
from the human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the
chromosomes can also be selected upon co-transfection with a
selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0262] The plasmid pC4 is digested with restriction enzymes and
then dephosphorylated using calf intestinal phosphatase by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0263] The DNA sequence encoding the complete alpha-V subunit
antibody is used, corresponding to HC and LC variable regions of a
alpha-V subunit antibody of the present invention, according to
known method steps. Isolated nucleic acid encoding a suitable human
constant region (i.e., HC and LC regions) is also used in this.
[0264] The isolated variable and constant region encoding DNA and
the dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
[0265] Chinese hamster ovary (CHO) cells lacking an active DHFR
gene are used for transfection. 5 .mu.g of the expression plasmid
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using
lipofectin. The plasmid pSV2neo contains a dominant selectable
marker, the neo gene from Tn5 encoding an enzyme that confers
resistance to a group of antibiotics including G418. The cells are
seeded in alpha minus MEM supplemented with 1 .mu.g/ml G418. After
2 days, the cells are trypsinized and seeded in hybridoma cloning
plates (Greiner, Germany) in alpha minus MEM supplemented with 10,
25, or 50 ng/ml of methotrexate plus 1 .mu.g/ml G418. After about
10-14 days single clones are trypsinized and then seeded in 6-well
petri dishes or 10 ml flasks using different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained that grow
at a concentration of 100-200 mM. Expression of the desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or
by reverse phase HPLC analysis.
EXAMPLE 2
Method of Making and Characterization of Non-Limiting Example of
Fully Human Alpha-V Subunit Antibody
[0266] Summary. (CBA/J x C57/BL6/J) F.sub.2 hybrid mice (Taylor et
al., International Immunology 6:579-591 (1993); Lonberg et al.,
Nature 368:856-859 (1994); Neuberger, Nature Biotechnology 14:826
(1996); Fishwild et al., Nature Biotechnology 14:845-851 (1996))
containing human variable and constant region antibody transgenes
for both heavy and light chains were immunized with human placental
.alpha.V.beta.3. One fusion yielded 2 totally human .alpha.V.beta.3
reactive IgG1.kappa. monoclonal antibodies, named CNTO 95 and
GenO.101. The totally human anti-.alpha.V.beta.3 antibodies were
further characterized and both were found to be reactive to the
.alpha.V.beta.3 and .alpha.V.beta.5 subunits suggesting specificity
for the shared alpha chain of both molecules. One Mab, CNTO 95,
also known as CNTO 95, inhibits the binding of both .alpha.V.beta.3
and .alpha.V.beta.5 to vitronectin in cell based assays.
[0267] Abbreviations:
[0268] BSA--bovine serum albumin
[0269] CO.sub.2--carbon dioxide
[0270] DMSO--dimethyl sulfoxide
[0271] EIA--enzyme immunoassay
[0272] FBS--fetal bovine serum
[0273] H.sub.2O.sub.2--hydrogen peroxide
[0274] HC--heavy chain
[0275] HRP--horseradish peroxidase
[0276] Ig--immunoglobulin
[0277] IP--intraperitoneal
[0278] IV--intravenous
[0279] Mab--monoclonal antibody
[0280] OD--optical density
[0281] OPD--o-Phenylenediamine dihydrochloride
[0282] PEG--polyethylene glycol
[0283] PSA--penicillin, streptomycin, amphotericin
[0284] RT--room temperature
[0285] SQ--subcutaneous
[0286] TBS--Tris buffered saline
[0287] v/v--volume per volume
[0288] w/v--weight per volume
[0289] Introduction:
[0290] We have utilized transgenic mice that contain human heavy
and light chain immunoglobulin genes to generate totally human
monoclonal antibodies that are specific to the .alpha.V integrins.
These novel antibodies can be used therapeutically to inhibit the
angiogenic process by blocking the binding of .alpha.V integrins to
their respective ECM ligands and provide additional tools in the
treatment of various cancers.
[0291] Materials and Methods
[0292] Animals
[0293] Transgenic mice have been developed by GenPharm
International that express human immunoglobulins but not mouse IgM
or Ig.kappa.. These mice contain human sequence transgenes that
undergo V(D)J joining, heavy-chain class switching and somatic
mutation to generate a repertoire of human sequence immunoglobulins
(Taylor et al., International Immunology 6:579-591 (1993)). The
light chain transgene is derived in part from a yeast artificial
chromosome clone that includes nearly half of the germline human W
region. In addition to several VH genes, the heavy-chain (HC)
transgene encodes both human .mu. and human .gamma.1 (Lonberg et
al., Nature 368:856-859 (1994)) and/or .gamma.3 constant regions. A
mouse derived from the HC012 genotypic lineage was used in the
immunization and fusion process to generate these monoclonal
antibodies.
[0294] Purification of Human .alpha.V.beta.3
[0295] Human placenta (disrupted using a meat grinder) or M21 human
melanoma cells expressing the .alpha.V.beta.3 integrin were
extracted with saline containing 20 mM Tris pH 7.5, 1 mM
CaCl.sub.2, 1 mM MnCl.sub.2, 100 mM Octylthioglucoside (OTG from
Pierce), 0.05% sodium azide and 1 mM phenylmethylsulfonyl fluoride
(Sigma). The mixture was stirred for 1 hr at room temperature and
clarified by centrifugation at 10,000.times.g. The supernatant from
placental extracts was applied to an affinity column consisting of
Mab 10E5 coupled to sepharose (Pharmacia) to remove GPIIb/IIIa and
the flow-through fraction was applied to an affinity column
consisting of Mab c7E3 Fab coupled to sepharose (Pharmacia) to bind
.alpha.V.beta.3. The c7E3 column was washed with PBS containing 1
mM CaCl.sub.2, 1 mM MnCl.sub.2, and 0.1% OTG followed by 0.1M
sodium acetate pH 4.5, 1 mM CaCl.sub.2, 1 mM MnCl.sub.2, and 0.1%
OTG, pH 3.0. The column was eluted with 0.1M glycine, 2% acetic
acid, 1 mM CaCl.sub.2, 1 mM MnCl.sub.2, and 0.1% OTG. The eluate
containing purified .alpha.V.beta.3 was neutralized using 2M Tris
pH 8.5. Purity of the preparations was characterized by SDS-PAGE
analysis and ELISA to rule out GPIIb/IIIa contamination (Wayner, et
al., J. Cell Biol. 113: 919-929 (1991)).
[0296] Immunizations
[0297] A fifteen to 17 week old surgically castrated male mouse
obtained from GenPharm was immunized IP (200 .mu.L) and in 2 sites
SQ (100 .mu.L per site) with a total of 20 .mu.g of placental
.alpha.V.beta.3 (prep V fraction, JG21197) emulsified with an equal
volume of complete Freund's adjuvant (day 0). The mouse was
immunized two weeks later in the same manner with .alpha.V.beta.3
emulsified with an equal volume of incomplete Freund's adjuvant.
Three subsequent 10 .mu.g IP/ 10 .mu.g SQ injections with
incomplete Freund's adjuvant were administered on days 28, 42, and
56. The mouse was then bled on days 42 and 56 by retro-orbital
puncture without anti-coagulant. The blood was allowed to clot at
RT for one hour and the serum was collected and titered using an
.alpha.V.beta.3 solid phase EIA assay. The fusion, named GenO, was
performed when repeated injections did not cause titers to
increase. At that time, the mouse with a specific human IgG titer
of 1:1280 against .alpha.V.beta.3 was given a final IV booster
injection of 10 .mu.g .alpha.V.beta.3 diluted in 100 .mu.L
physiological saline. Three days later, the mouse was euthanized by
cervical dislocation and the spleen was removed aseptically and
immersed in 10 mL of cold phosphate buffered saline (PBS)
containing 100 U/mL penicillin, 100 .mu.g/mL streptomycin, and 0.25
.mu.g/mL amphotericin B (PSA). The splenocytes were harvested by
sterilely perfusing the spleen with PSA-PBS. The cells were washed
once in cold PSA-PBS, counted using Trypan blue dye exclusion and
resuspended in RPMI 1640 media containing 25 mM Hepes.
[0298] Cell Lines
[0299] The non-secreting mouse myeloma fusion partner, SP2/0 was
employed. The cell line was expanded in .alpha.MEM (modified)
medium (JRH Biosciences) supplemented with 10% (v/v) FBS (Cell
Culture Labs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine
(all from JRH Biosciences) and cryopreserved in 95% FBS and 5% DMSO
(Sigma), then stored in a vapor phase liquid nitrogen freezer in
CBS. The cell bank was sterile (Quality Control Centocor, Malvern)
and free of mycoplasma (Bionique Laboratories). Cells were
maintained in log phase culture until fusion. They were washed in
PBS, counted, and viability determined (>95%) via trypan blue
dye exclusion prior to fusion.
[0300] The M21 cell line, a human melanoma expressing the
.alpha.V.beta.3 and .alpha.V.beta.5 integrins, was expanded and
cryopreserved. The 10-vial research cell bank was received into
Cell Biology Services and stored in liquid nitrogen. The cell bank
was sterile and free of mycoplasma (Bionique Laboratories). The
MDAMB435L2 cell line, a human breast carcinoma, was a gift from Dr.
Janet Price (MD Anderson, Houston Tex.) expresses the integrin
.alpha.V.beta.3. The cell line was cryopreserved in Cell Biology
Services. The cell bank was sterile and free of mycoplasma
(Bionique Laboratories). M21 and MDAMB435L2 cells were thawed,
propagated in appropriate media and maintained in log phase for
several days prior to use in bioassays or allowed to reach
confluency for use in the purification of .alpha.V.beta.3 protein
(M21 cells).
[0301] Cell Fusion
[0302] Fusion was carried out at a 1:1 ratio of murine myeloma
cells (SP2/0) to viable spleen cells. Briefly, spleen cells and
myeloma cells were pelleted together. The pellet was slowly
resuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS solution
(PEG molecular weight 3,000, Sigma) at 37.degree. C. The fusion was
stopped by slowly adding 1 mL of Dulbecco's PBS (JRH) (37.degree.
C.) over 1 minute. An additional 19 mL of PBS was added over the
next 90 seconds. The fused cells were centrifuged for 5 minutes at
750 rpm. The cells were then resuspended in HAT medium (.alpha.MEM
medium containing 20% Fetal Bovine Serum (JRH), 1 mM sodium
pyruvate, 2 mM L-glutamine, 0.1 mM Non-essential amino acids, 10
.mu.g/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 50
.mu.M 2-mercaptoethanol, 100 .mu.M hypoxanthine, 0.4 .mu.M
aminopterin, and 16 .mu.M thymidine) and then plated at 200
.mu.L/well in thirteen 96-well flat bottom tissue culture plates.
The plates were then placed in a humidified 37.degree. C. incubator
containing 5% CO.sub.2 and 95% air for 7-10 days.
[0303] Detection of Human IgG Anti-.alpha.V.beta.3 Antibodies in
Mouse Serum
[0304] Solid phase EIAs were used to screen mouse sera for human
IgG antibodies specific for human .alpha.V.beta.3. Briefly, plates
were coated with .alpha.V.beta.3 at 1 .mu.g/mL in PBS overnight.
After washing in 0.15M saline containing 0.02% (v/v) Tween 20, the
wells were blocked with 1% (w/v) BSA in HBSS with Ca.sup.++ and
Mg.sup.++, 200 .mu.L/well for 1 hour at RT. Plates were used
immediately or frozen at -20.degree. C. for future use. Mouse sera
were incubated in doubling dilutions on the .alpha.V.beta.3 coated
plates at 50 .mu.L/well at RT for 1 hour. The plates were washed
and then probed with 50 .mu.L/well HRP-labeled goat anti-human IgG,
Fc specific (Accurate) diluted 1:30,000 in 1% BSA-PBS for 1 hour at
RT. The plates were again washed and 100 .mu.L/well of the
citrate-phosphate substrate solution (0.1M citric acid and 0.2M
sodium phosphate, 0.01% H.sub.2O.sub.2 and 1 mg/mL OPD) was added
for 15 minutes at RT. Stop solution (4N sulfuric acid) was then
added at 25 .mu.L/well and the OD's were read at 490 nm via an
automated plate spectrophotometer.
[0305] Detection of Totally Human Immunoglobulins in Hybridoma
Supernatants
[0306] Because the GenPharm mouse is capable of generating both
mouse and human immunoglobulin chains, growth positive hybridomas
secreting fully human immunoglobulins were detected using two
separate EIA systems. Plates were coated as described above and
undiluted hybridoma supernatants were incubated on the plates for
one hour at 37.degree. C. The plates were washed and probed with
either HRP labeled goat anti-human kappa (Southern Biotech)
antibody diluted 1:10,000 in 1% BSA-HBSS or HRP labeled goat
anti-human IgG Fc specific antibody diluted to 1:30,000 in 1%
BSA-HBSS for one hour at 37.degree. C. The plates were then
incubated with substrate solution as described above.
[0307] Isotyping
[0308] Isotype determination of the antibodies was accomplished
using an EIA in a format similar to that used to screen the mouse
immune sera for specific titers. .alpha.V.beta.3 was coated on
96-well plates as described above and purified antibody at 2
.mu.g/mL was incubated on the plate for one hour at RT. The plate
was washed and probed with HRP labeled goat anti-human IgG.sub.1
(Binding Site) or HRP labeled goat anti-human IgG.sub.3 diluted at
1:4000 (Zymed) in 1% BSA-HBSS for one hour at RT. The plate was
again washed and incubated with substrate solution as described
above.
[0309] Preparation of Anti-Idiotype Antibodies to CNTO95
[0310] Seventeen monoclonal antibodies were made to the variable
region of CNTO 95. Six of them are non-blocking. The remaining
eleven appear to block the active site of CNTO 95 and inhibit the
binding of CNTO 95 to human integrin aVb3 and do not bind to CNTO
95 prebound to its receptor. The non-blocking Mab, CNTO 1073, can
detect CNTO 95 pre-bound to aVb3. Pooled human serum does not
interfere with the binding of 16 of 17 CNTO 95 anti-variable region
Mabs.
[0311] The anti-idiotype Mabs are useful in pharmacokinetic or
immunohistochemical detection of CNTO 95 in patient and animal
tissue or sera samples as well as in epitope mapping efforts to
define the binding regions of CNTO 95 to its targe
[0312] Binding Characteristics of Human Monoclonal Antibodies to
AlphaV by EIA
[0313] Binding characteristics for the antibodies were assessed
using an .alpha.V.beta.3 capture EIA. Linbro plates were coated
with .alpha.V.beta.3 at 1 .mu.g/mL in TBS with 2 mM calcium
overnight at 4.degree. C. Plates were washed and blocked with
TBS/1% BSA/calcium for at least one hour at room temperature.
Purified antibodies were incubated in doubling dilutions from a
starting concentration of 2 .mu.g/mL. Plates were washed and
conjugated antibodies (HRP-labeled goat anti-human IgG Fc at
1:30,000) were added and incubated on plates for one hour at room
temperature. Plates were washed OPD substrate was added to wells.
Plates were read via an automated plate spectrophotometer.
[0314] Competition of Binding of CNTO95 to M21 Cells by Various
Commercial Anti-Integrin Mabs
[0315] M21 Cells were trypsinized from culture flasks, washed and
resuspended in HBSS/calcium to 2.times.10.sup.6 cell/mL. GenO95 was
prelabeled with FITC-goat anti-human Fc (Jackson) for 30 minutes at
RT. 10.times. concentrations of GenO95 of 200 .mu.g/mL or 20
.mu.g/mL were incubated with FITC-goat anti-human IgG at 250
.mu.g/mL. Aliquots of 100 .mu.L of M21 cells (2.times.10.sup.5
cells) were incubated with 12 .mu.L 10.times. GenO95 at high (20
.mu.g/mL final) and low (2 .mu.g/mL final) concentrations .+-.12
.mu.L of the following murine antibodies: m7E3 IgG,
anti-.alpha.V.beta.3 (clone LM609, Chemicon), anti-.alpha.V.beta.5
(clone P1F6, Gibco), anti-.beta.3 (Chemicon, AMAC), or
anti-.alpha.V (clone VNR139, Gibco) antibodies (at 20 .mu.g/mL) for
45 minutes at 37.degree. C. An aliquot was removed from each tube
(for two-color analysis) and the remainder was fixed with 1%
paraformaldehyde and analyzed on a flow cytometer. For two-color
analysis, an aliquot (50 .mu.L was incubated with PE-goat
anti-mouse IgG for 30 minutes at RT to label murine
anti-.alpha.V.beta.3, anti-.alpha.V.beta.3, anti-.beta.3, or
anti-.alpha.V antibodies for two-color analysis. All tubes were
fixed with 1% paraformaldehyde.
[0316] Inhibition of .alpha.V.beta.3 or .alpha.V.beta.5 Dependent
M21 Cell or MDA MB435L2 Cell Adhesion to Vitronectin Coated Plates
by .alpha.V.beta.3/.alpha.V.beta.5 Specific Mabs
[0317] Linbro plates were coated for 1 hour at room temperature 50
.mu.L/well of vitronectin (Collaborative, Becton Dickinson) at 5
.mu.g/mL in TBS with 2 mM calcium. Plates were washed with
HBSS/calcium and blocked with TBS containing 2 mM calcium and 1%
BSA for 30 minutes at RT. M21 cells were trypsinized, washed once
with media containing FCS and resuspended in 3 mL HBSS without
calcium. All washes were done with 10 minute spins at 1000 rpm in
the Sorvall tabletop centrifuge. To fluorescently label the cells,
calcein (Molecular Probes) (5 mg/mL in DMSO) was added to the cells
to a final concentration of 100 .mu.g/mL in a 50 mL conical tube
(wrapped in foil). Cells were incubated 10 to 15 minutes at
37.degree. C. Calcein labeled cells were washed once with HBSS and
resuspended in HBSS supplemented with 0.1% BSA and 1 mM MgCl.sub.2.
Antibodies were titrated (14-fold dilution series) in HBSS/0.1%
BSA/2 mM calcium at 10.times. final concentration. Cells (300 .mu.L
at 7.5.times.10.sup.6/mL) were preincubated with antibody
titrations (37 .mu.L of 10.times. solution).+-.anti-.alpha.V.beta.5
(P1F6) ascites (Chemicon) (37 .mu.L of 1:600 (10.times.)) for 15
min at 37.degree. C. The cell-antibody mixture was added to the
vitronectin-coated plates at 100 .mu.L/well in triplicate
(approximately 6.times.10.sup.5 cells/well). Plates were incubated
for 45 minutes at 37.degree. C. Unbound cells were removed by two
washes with HB SS/calcium (150 .mu.L/well). 100 .mu.l HBSS/calcium
was added to each well and the plate read on the Fluoroskan at
485-538 nm.
[0318] In a separate assay, MDA-MB435-L2 human breast carcinoma
cells were harvested with versene and suspended in serum free media
at 500,000 cells/mL and incubated with various concentrations of
GenO95. After 10 minutes of incubation tumor cell suspension (100
.mu.L) was added to vitronectin (10 .mu.g/mL) coated Linbro plates
and incubated at 37.degree. C. After 1 hour, wells were washed
three times with serum free media (200 .mu.L/wash) and the MTT
based Cell Titer AQ dye (Promega, Madison, Wis.) was added to each
well. Extent of cell adhesion was determined in an ELISA plate
reader where OD490 nm is directly proportional to cell adhesion.
Cell adhesion to BSA coated wells served as negative control.
[0319] Determination of Ca.sup.++ Dependence for Binding of
Anti-Human alphaVbeta3/alphaVbeta5 Mabs to Their Ligands
[0320] To determine cation dependence in the binding of CNTO 95 and
C372 to .alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5, a
liquid phase EIA was utilized. EIA plates (Corning) were coated
with CNTO 95, C372, c7E3 or LM609 IgG Mabs at 10 :g/mL in carbonate
coating buffer overnight at 4EC. Plates were blocked with 1% BSA
diluted in HBSS in the presence or absence of 2 mM Ca.sup.++ for at
least one hour at 37EC. Doubling dilutions of alphaVbeta3 (log
JG52599) or alphaVbeta5 (Chemicon) starting at 10 :g/mL were
preincubated with 50 mM EDTA (Sigma) in 1% BSA/HBSS without
Ca.sup.-+ or with 1% BSA/HBSS with Ca.sup.-+ for 30 minutes at
37.degree. C. The mixtures were then added to the plates and
incubated for 30 minutes at 37.degree. C. The plates were then
washed and non-competing Mabs were added to the plates as follows:
to the CNTO 95, C372, c7E3 coated plates to detect alphaVbeta3
binding, Mab LM609 was added at 20 microgm/mL in 1% BSA/HBSS
Ca.sup.-+; to the LM609 coated plate to detect alphaVbeta3 binding,
Mab CNTO 95 was added at 20 :g/mL in 1% BSA/HBSS w Ca.sup.++ to the
CNTO 95, C372, c7E3 coated plates to detect alphaVbeta5 binding Mab
VNR139 (Gibco) was added at 10 microg/mL in 1% BSA/HBSS w Ca.sup.++
and incubated for 30 minutes at 37.degree. C. The plates were again
washed and probed with either HRP labeled goat anti-mouse IgG Fc or
HRP labeled goat anti-human IgG Fc in appropriate buffer and
incubated for 30 minutes at 37.degree. C. The plates were washed,
OPD substrate was added and the OD 490 measured as previously
described.
[0321] Results and Discussion
[0322] Generation of Totally Human Anti-Human .alpha.V.beta.3
Integrin Monoclonal Antibodies
[0323] One fusion, named GenO, was performed from a GenPharm mouse
immunized with alphaVbeta3 protein. From this fusion, 129 growth
positive hybrids were screened. Two hybridoma cell lines were
identified that secreted totally human IgG antibodies reactive with
human alphaVbeta3. These two cell lines, CNTO 95.9.12 and
GenO.101.17.22, each secrete immunoglobulins of the human
IgG1.kappa. isotype and both were subcloned twice by limiting
dilution to obtain stable cell lines (>90% homogeneous). CNTO
95.9.12 was assigned C-code #C371A and GenO.101.17.22 was assigned
C-code #C372A. Each of the cell lines was frozen in 12-vial
research cell banks stored in LN2.
[0324] Binding Characteristics of Human Monoclonal Antibodies by
EIA
[0325] ELISA analysis confirmed that purified antibody from the two
hybridomas, C371A (also called Mab CNTO 95) and C372A, bind
alphaVbeta in a concentration-dependent manner. FIG. 1 shows the
results of the relative binding efficiency of the antibodies. Fifty
percent binding is achieved at 0.07 and 0.7 .mu.g/mL for C372A and
CNTO 95 respectively. In the same assay, c7E3 IgG demonstrated
fifty-percent maximal binding at 0.07 .mu.g/mL.
[0326] Competition of Binding of Mab CNTO95 to M21 Cells by
Commercially Available Anti-Integrin Mabs
[0327] By single-color analysis, none of the murine
anti-alphaVbeta3, anti-alphaVbeta5, anti-beta3, or anti-alphaV
antibodies competed with CNTO 95 for binding to M21 cells (Table
1). This experiment also demonstrates that CNTO 95 binds to M21
cells in a dose dependent manner. The two-color analysis
demonstrated that the murine anti-alphaVbeta3, anti-alphaVbeta5,
anti-beta3, or anti-alphaV antibodies were able to bind to M21
cells (data not shown).
TABLE-US-00001 TABLE 1 Competition of Binding of CNTO95 to M21
Cells by Murine anti-Integrin Mabs FITC-goat anti-human Fc-labeled
CNTO95 2 .mu.g/mL 20 .mu.g/mL Competing Antibody MCF % Positive MCF
% Positive negative (no GenO95) 2.69 2.69 Positive (saline) 4.33
100% 14.33 100% m7E3 IgG 5.73 132% 14.72 103% LM609
(anti-.alpha..sub.v.beta..sub.3) 4.78 110% 13.34 93%
anti-.beta..sub.3 (Chemicon) 5.42 125% 13.10 91% anti-.beta..sub.3
(AMAC) 4.61 106% 13.10 91% P1F6 (anti-.alpha..sub.v.beta..sub.3)
4.87 112% 14.46 101% VNR139 (anti-.alpha..sub.v) 4.61 106% 14.86
104% MCF = Median Channel Fluorescence
[0328] Inhibition of .alpha.V.beta.3 or .alpha.V.beta.5 Dependent
M21 Cell or MDA-MB435-L2 Cell Adhesion to Vitronectin Coated Plates
by .alpha.V.beta.3/.alpha.V.beta.5 Specific Mabs
[0329] M21 cells adhere to vitronectin coated plates in an
.alpha.V.beta.3 and .alpha.V.beta.5 dependent manner. Therefore,
blockade of both .alpha.V.beta.3 and .alpha.V.beta.5 is required to
completely inhibit M21 cell adhesion to vitronectin coated plates.
C372A did not inhibit M21 cell adhesion in the presence or absence
of P1F6, anti-.alpha.V.beta.5 ascites (FIG. 2). GenO95 (CNTO 95)
completely inhibited M21 cell adhesion to vitronectin coated plates
both with and without anti-.alpha..sub.v.beta..sub.5 (P1F6)
ascites, indicating that the antibody blocks both .alpha.V.beta.3
and .alpha.V.beta.5. As a control for the assay parameters, ReoPro
(c7E3 Fab) which blocks .alpha.V.beta.3 (in addition to GPIIb/IIIa)
was included. ReoPro alone only partially inhibited M21 cell
adhesion, ReoPro in the presence of anti-.alpha..sub.v.beta..sub.5
(P1F6) ascites completely inhibited adhesion, which demonstrates
that M21 cells bind to vitronectin through both
.alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5 integrins.
Data were normalized to percent of maximal M21 cell binding in the
absence of antagonist +/- anti-.alpha.V.beta.5 (P1F6) ascites. For
antagonist titration without P1F6, data were normalized to maximal
M21 cell binding in the absence of antagonist or P1F6. For
antagonist titration in the presence of P1F6, data were normalized
to maximal binding in the absence of antagonist but in the presence
of P1F6. Data were graphed as percent of maximal binding (no
antibody) and non-linear regression performed using GraphPad
Prism.
[0330] CNTO95 Mab also demonstrated the ability to completely
inhibit MDAMB435L2 cell adhesion to vitronectin at a minimal
concentration of 1.5 .mu.g/mL (FIG. 3). These data, in combination
with the data indicating inhibition of M21 cell adhesion, confirm
the ability of GenO95 to functionally inhibit the .alpha.V.beta.3
and/or .alpha.V.beta.5 receptor interaction with vitronectin.
[0331] Determination of Ca.sup.++ Dependence for Binding of
Anti-Human alphaVbeta3/alphaVbeta 5 Mabs to Their Ligands
[0332] It is known that the presence of the cation calcium is
necessary for the Mab c7E3 to bind alphaVbeta 3 and is not a
requirement for binding of Mab LM609 to .alpha.V.beta.3 as
demonstrated in FIGS. 4c and 4d respectively. This experiment was
conducted to assess whether calcium dependence also applies to the
binding characteristics of CNTO 95 or C372 for alphaVbeta 3 or
alphaVbeta 5 integrins. An excess concentration of EDTA was
introduced into the assay format to chelate the Ca present within
the binding pocket of the integrin subunits and therefore, binding
was assessed in the absence of the cation. It was found that CNTO
95 and C372 binding to alphaVbeta 3 is not dependent upon the
presence of Ca (FIG. 4a, 4b). The same is true for CNTO 95 binding
to alphaVbeta 5 but not so, however, for C372 binding to alphaVbeta
5 (FIG. 4e, 4f) as binding appears to be increased in the presence
of Ca.
[0333] Conclusion
[0334] The GenO fusion was performed utilizing splenocytes from a
hybrid mouse containing human variable and constant region antibody
transgenes that was immunized with human .alpha.V.beta.3. Two
totally human .alpha.V.beta.3 reactive IgG monoclonal antibodies of
the IgG1.kappa. isotype were generated. These Mabs were further
characterized and it was found that both bind .alpha.V.beta.3 and
.alpha.V.beta.5 integrins. The binding of the two Mabs was
demonstrated to be calcium independent to .alpha.V.beta.3 and
calcium dependent to .alpha.V.beta.5 only for C372 binding.
Moreover, one Mab, GenO95 (CNTO 95), is able to completely inhibit
the binding of .alpha.V.beta.3 and .alpha.V.beta.5 to the ligand
vitronectin in cell based assays. This Mab may prove useful in
anti-angiogenic and other cancer related applications.
[0335] References:
[0336] 1. Taylor et al., International Immunology 6:579-591
(1993).
[0337] 2. Lonberg et al., Nature 368:856-859 (1994).
[0338] 3. Neuberger, Nature Biotechnology 14:826 (1996).
[0339] 4. Fishwild et al.,Nature Biotechnology 14:845-851
(1996).
[0340] 5. Gastl et al., Oncology 54: 177-184 (1997).
[0341] 6. Eliceiri, et al., J. Clin. Invest. 103: 1227-1230
(1999).
[0342] 7. Friedlander et al., Science 270: 1500-1502 (1995).
[0343] 8. Wayner, et al., J. Cell Biol. 113: 919-929 (1991).
[0344] 9.
EXAMPLE 3
Binding Affinities for alpha-V Subunit Antibody
[0345] CNTO 95, (CNTO 95) as described in Example 2, is a human
monoclonal antibody generated by immunizing (CBA/J x C57/BL6/J,
GenPharm International) F2 hybrid mice with
.alpha..sub.v.beta..sub.3 integrin purified from human placenta.
The antibody is composed of human variable and IgG1 kappa constant
regions and found to be reactive to both .alpha..sub.v.beta..sub.3
and .alpha..sub.v.beta..sub.5, suggesting a specificity for the
alpha chain shared by both integrin molecules.
[0346] The purpose of this study is to characterize the binding
affinity of GenO.05 for .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 purified integrins and for beta integrin
expressing cell lines. For further characterization, the binding
values will be compared between CNTO 95 and ReoPro.
[0347] Abbreviations
[0348] K.sub.D, equilibrium dissociation constant, expressed in
M
[0349] Bmax=maximal number of binding sites
[0350] Materials and Methods
[0351] Cell Lines
[0352] A37552 cells, a human melanoma cell line expressing
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins,
were cultured in Dulbelcco's minimal media (DMEM) containing 10%
fetal bovine serum (FBS, Cell Culture Labs), 1 mM sodium pyruvate,
0.1 mM nonessential amino acids, and 2 mM L-glutamine (all from JRH
Biosciences).
[0353] HT29 cells, a human colon carcinoma cell line expressing
.alpha..sub.v.beta..sub.5 and minimal .alpha..sub.v.beta..sub.3 (NB
4546, p207) were cultured in DMEM containing 10% FBS, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, and 2 mM
L-glutamine.
[0354] M21 cells, a human melanoma expressing
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins,
obtained from Dr. J. Jakubowski (Eli Lilly, Inc.), were cultured in
RPMI media (JRH Biosciences) containing 10% FBS, 1 mM sodium
puruvate, 0.1 mM nonessential amino acids, and 2 mM
L-glutamine.
[0355] Integrins
[0356] .alpha..sub.v.beta..sub.3 lot JG22499 was purified at
Centocor from human placenta. Another .alpha..sub.v.beta..sub.33
integrin lot (octyl formulation, lot 19100991) was purchased from
Chemicon. .alpha..sub.v.beta..sub.5 (Triton formulation, lot
20030055, lot 1910990 and octyl formulation, lot 19060747) was
purchased from Chemicon.
[0357] Antibodies
[0358] CNTO 95 was purified from cell culture supernatant by
Protein A chromatography. ReoPro was manufactured at Centocor, Inc.
LM609, a murine anti-human .alpha..sub.v.beta..sub.3 antibody,
(1976ZK, lot 20020559 and lot 1910329) and P1F6, a murine
anti-human .alpha..sub.v.beta..sub.5 antibody (1961 P-K, lot
17110560) were purchased from Chemicon.
[0359] Radiolabeling
[0360] Antibodies were radiolabeled with 125-I Na (Amersham, Ill.)
using Iodobeads (Pierce Chemicals, IL) to a specific activity of
1-2 .mu.Ci/.mu.g. Antibody concentration (mg/ml) was determined by
dividing the adsorption (OD/ml) at 280 nm by 1.4. Specific activity
of the iodinated antibody was determined by diluting the antibody
and counting an aliquot in the gamma counter or Topcounter
(Packard).
Specific activity (cpm/ug)=cpm /volume (ml).times.dilution
factor
[0361] concentration (.mu.g/ml determined by OD.sub.280
reading)
[0362] Integrin-coated plate binding assay
[0363] .alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5
integrin was diluted to 1 .mu.g/ml in Tris-buffered saline (TBS, 10
mM Tris, 100 mM NaCl, pH 7.5) containing 2 mM calcium chloride
(TBS/Ca.sup.++) and coated at 50 .mu.l per well onto 96 well
polystyrene Linbro plates (Flow/ICN) overnight at 4.degree. C.
Plates were washed with TBS/Ca.sup.++ and blocked with 1% bovine
serum albumin (BSA) in TBS/Ca.sup.++ for 1 h at room temperature.
Fifty microliters of diluted antibody was added in triplicate to
coated wells and incubated for 2 h at 37.degree. C. After three
washes with TBS-Tween buffer (TBS+0.1% Tween 20), peroxidase
conjugated goat anti-human IgG F(ab').sub.2 (H+L, Jackson lot
16869), at 1:40:000 dilution in 1% BSA-TBS was added and incubated
for 1 h at room temperature. Plates were washed three times, and
developed with o-phenylenediamine dihydrochloride substrate
solution (OPD, Sigma) consisting of 0.1 M citric acid, 0.2M sodium
phosphate, 0.01% H.sub.2O.sub.2 and 1 mg/ml OPD. Color development
was stopped after 15 min at room temperature with 0.3 N
H.sub.2SO.sub.4, and plates were read at OD.sub.490 nm in the
Molecular Dynamics plate reader.
[0364] Binding curves were generated with GraphPad PRISM (version
3, GraphPad Software). Results were expressed as % maximal binding
of the saturation value. K.sub.D, the equilibrium dissociation
binding constant (expressed as M), was determined from a non-linear
regression fit of the data using PRISM.
[0365] Cell Binding Assay
[0366] Fifty microliters of diluted radiolabeled antibody in 2%
RPMI media containing 2% bovine serum albumin (JRH Biosciences)
were added in triplicate to confluent cells cultured in 96 well
tissue culture plates (Packard). Cells were incubated for 1.5 h at
37.degree. C.; gently washed three times with Hanks buffered saline
containing calcium and magnesium (HBSS++, JRH Biosciences) and then
aspirated. One hundred microliters of Mycosinct 20 (Packard) was
added per well, and cell-bound radioactivity was quantified in the
TopCounter (Packard).
[0367] To determine nonspecific binding, experiments were performed
with a similar set of dilutions in the presence of 100-fold excess
of unlabeled antibody.
[0368] To determine the number of cells plated in each well, cells
from several wells were removed with trypsin, pooled and counted
under the microscope. The receptor number per cell was calculated
as follows:
Receptor number/cell=specific bound
cpms.times.6.023.times.10.sup.23 molecules/mole specific activity
(cpm/g).times.mol.wt. (g/mole).times.cell number
[0369] Bmax, the maximal binding sites per cell, and the K.sub.D
were determined from a nonlinear regression fit of the data using
PRISM.
Results and Discussion
[0370] Determination of the binding affinity values was performed
by measuring the binding of various concentrations of CNTO 95 (and
ReoPro) to purified .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 integrins and to cell surface receptors
at equilibrium. The saturation binding curves were rectangular
hyperbolas, suggesting a single receptor binding site for CNTO 95
and ReoPro (FIGS. 5-6; Motulsky H, 1999). Analysis of these
saturation binding data (sometimes called Scatchard experiments)
were performed using a one-site hyperbola nonlinear regression fit
in PRISM to obtain an affinity, K.sub.D, and receptor number, Bmax
(Motulsky H, 1999).
[0371] Several lots of CNTO 95, ReoPro and purified integrins were
used to ensure an accurate determination of binding affinity
values. The saturation binding curve of CNTO 95 on an
.alpha..sub.v.beta..sub.3 coated plate (FIG. 5A) and the binding
curve of ReoPro on an .alpha..sub.v.beta..sub.3coated plate (FIG.
5B) represent the mean and standard deviation of six separate
experiments. Results obtained with Triton formulation of
.alpha..sub.v.beta..sub.3 were found to be more reproducible than
those obtained from the octyl formulation. On .alpha..sub.v.beta.
coated plates, the CNTO 95 mean K.sub.D was
2.1.+-.1.33.times.10.sup.-10 M; and the mean ReoPro Kd was
2.5.+-.1.46.times.10.sup.-10 M.
[0372] The saturation binding curve of CNTO 95 on an
.alpha..sub.v.beta..sub.5 coated plate (FIG. 6A) and the binding
curve of ReoPro on an .alpha..sub.v.beta..sub.5 coated plate (FIG.
6B) are shown as the mean and standard deviation of six separate
experiments. Results obtained with the octyl formulation were more
consistent than those obtained with the Triton formulation. The
CNTO 95 mean K.sub.D on .alpha..sub.v.beta..sub.5 was
2.5+1.04.times.10.sup.-11 M. ReoPro showed no binding and no
dose-response on .alpha..sub.v.beta..sub.5 coated plates.
[0373] The binding affinity values for purified integrins were
compared to binding to receptors expressed on various cell lines.
FIG. 7A-C shows the binding of 125-I CNTO 95 with A375S2 cells
which express .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 (FIG. 7A). Mean affinity values on A375S2
cells were: Kd=5.2.+-.2.04.times.10.sup.-9 M; and 120,000.+-.37,000
receptors/cell. HT-29 cells express .alpha..sub.v.beta..sub.5.
Affinity values for 125-I CNTO 95 binding to HT-29 cells were:
Kd=1.3.+-.3.76.times.10.sup.-10 M; and 81,000.+-.24,000
receptors/cell (FIG. 7B). M21 cells express
.alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.5 integrins.
125-I CNTO 95 binding to M21 cellls were:
Kd=8.5.+-.3.03.times.10.sup.-9 M; and 200,000.+-.80,000
receptors/cell (FIG. 7C).
[0374] Similar cell binding studies were performed with 125-I
ReoPro on various cell lines. FIG. 8A-C shows the binding of 125-I
ReoPro with A375S2 cells and the mean values obtained were:
Kd=22.+-.3.7.times.10.sup.-9 M; and 370,000.+-.190,000
receptors/cell (FIG. 8A). On HT-29 cells, 125-I ReoPro showed
minimal binding (FIG. 8B). 125-I ReoPro binding to M21 cells
showed: Kd=10.+-.2.00.times.10.sup.-9 M and 660,000.+-.120,000
receptors/cell (FIG. 8C). The binding values of 125-I ReoPro on M21
cells are consistent with values previously published (Tam et al,
1998).
[0375] A summary of binding results is shown in Tables 2-3.
TABLE-US-00002 TABLE 2 Summary of CNTO 95 and abciximab affinities
to purified integrins mAb alphaVbeta 3 coated alphaVbeta 5 coated
plate (n = 6) plate (n = 6) Kd (M) Kd (M) CNTO 95 2.1 + 1.33
.times. 10.sup.-10 2.5 + 1.04 .times. 10.sup.-11 abciximab 2.5 +
1.46 .times. 10.sup.-10 Negligible
TABLE-US-00003 TABLE 3 Summary of CNTO 95 and abciximab affinities
to cells A375S2 cells HT-29 cells M21 cells A375S2 cells Receptors
HT-29 cells Receptors M21 cells Receptors Kd (M) per cell Kd(M) per
cell Kd (M) Per cell CNTO 95 5.2 .+-. 2.04 .times. 10.sup.-9
120,000 .+-. 37,000 1.3 .+-. 0.38 .times. 10.sup.-9 81,000 .+-.
24,000 8.5 .+-. 3.03 .times. 10.sup.-9 200,000 .+-. 80,000 (n = 5)
(n = 7) (n = 5) (n = 7) (n = 4) (n = 8) abciximab 22 .+-. 3.7
.times. 10.sup.-9 370,000 .+-. 190,000 Negligible Negligible 10
.+-. 2.00 .times. 10.sup.-9 660,000 .+-. 120,000 (n = 3) (n = 6) (n
= 4) (n = 4) (n = 3) (n = 7) anti .alpha..sub.v.beta..sub.3 nd
300,000 nd nd nd nd LM609 (n = 2) anti-.alpha..sub.v.beta..sub.5 nd
70,000 .+-. 50,000 nd 73,000 nd 44,000 P1F6 (n = 4) (n = 1) (n =
2)
[0376] Several observations were notable in the binding
characterizations. Affinity values (Kd) of CNTO 95 on
.alpha..sub.v.beta..sub.5 were lower than on
.alpha..sub.v.beta..sub.3. Lower Kd values indicate a higher
affinity; thus the affinity for CNTO 95 binding to
.alpha..sub.v.beta..sub.5 purified integrin was about 8-fold higher
than binding to .alpha..sub.v.beta..sub.3 purified integrin.
However, when both integrin receptors are present on the same
cells, the overall affinity value more closely approximates the
value corresponding to the integrin in greater abundance. Thus, on
A375 S2 and M21 cells where there is more .alpha..sub.v.beta..sub.3
than .alpha..sub.v.beta..sub.5, the affinity of CNTO 95 binding to
these cells was similar to the affinity on
.alpha..sub.v.beta..sub.3, .about.7.times.10.sup.-9 M. In contrast,
on HT-29 cells which express .alpha..sub.v.beta..sub.5, the CNTO 95
affinity was slightly higher, 1.times.10.sup.-9 M. The
approximately 2-fold discrepancy in receptor sites per cell between
CNTO 95 and ReoPro binding may be explained by the difference in
antibody valency. CNTO 95 (IgG) is bivalent and likely binds two
adjacent receptors, whereas ReoPro (Fab) is monovalent and can only
bind to one receptor (BRD930001).
References
[0377] Fraker D J, Speck J C. Protein and cell membrane iodination
with a sparingly soluble chloramide
1,3,4,5-tetrachloro-3a-diphenyl-glycoluril. Biochem Biophys Res
Commun. 80:849, 1978. Motulsky H. Analyzing Data with GraphPad
Prism. GraphPad Software, Inc. San Diego, Calif. 1999. [0378] Tam S
H, Sassoli P M, RJordan, M T Nakada. Circulation, 1999.
EXAMPLE 4
Effect of alpha-V Subunit Antibody on Angiogenesis Modulation
[0379] GenO95 is a human IgG1.kappa. monoclonal antibody that
recognizes integrins .alpha.v.beta.3 and .alpha.v5 as well as
.alpha..sub.v.beta..sub.1 and .alpha..sub.v.beta..sub.6. These
integrins participate in endothelial cell adhesion, migration,
survival and proliferation, processes that are important for
angiogenesis. Endothelial cell sprouting mimics angiogenesis in
vitro because it involves cell adhesion, migration, proliferation
and survival. We utilized the sprouting assay to determine whether
GenO95 could inhibit .alpha.v.beta.3 and .alpha.v.beta.5 function.
This example describes that GenO95 is an inhibitor of sprouting of
endothelial cells that are cultured in three dimensional fibrin
matrix, thereby demonstrating that this antibody may have potential
anti-angiogenic properties.
[0380] There is now considerable evidence that progressive tumor
growth is dependent upon angiogenesis, the formation of new blood
vessels. These blood vessels provide tumors with nutrients and
oxygen, carry away waste products and act as conduits for the
metastasis of tumor cells to distant sites (1). Recent studies have
further defined various roles of integrins in the angiogenic
process. Integrins are subunitic transmembrane proteins that play
an important role in mediating cell adhesion, migration, survival,
and proliferation (2). Expression of integrin .alpha.v.beta.3 is
minimal on resting or normal blood vessels but is significantly
up-regulated on angiogenic vascular cells (1-3). The closely
related but distinct integrin .alpha.v.beta.5 has also been shown
to mediate the angiogenic process. An antibody generated against
.alpha.v.beta.3 blocked basic fibroblast growth factor (bFGF)
induced angiogenesis, whereas an antibody specific to
.alpha.v.beta.5 inhibited vascular endothelial growth factor (VEGF)
induced angiogenesis (1-5).
[0381] Angiogenesis can be mimicked in vitro by an endothelial
sprouting assay. This system involves endothelial cell migration
and proliferation. GenO95 is a human monoclonal antibody that
recognizes integrins .alpha.v.beta.3 and .alpha.v.beta.5, and these
integrins regulate endothelial cell migration and proliferation.
Therefore, we determined whether GenO95 could inhibit sprouting of
endothelial cells. This example describes experiments that
demonstrate that GenO95 inhibits sprouting of human endothelial
cells growing in a fibrin matrix.
[0382] Materials
[0383] Human basic fibroblast growth factor (bFGF) and human
vascular endothelial growth factor 165 (VEGF.sub.165) were obtained
from R&D Systems (Minneapolis, Minn.). MAB 1976Z (LM609), a
monoclonal antibody against integrin .alpha.v.beta.3 and MAB1961
(PIF6), a monoclonal antibody against integrin .alpha.v.beta.5 were
purchased from Chemicon (Temecula, Calif.). ReoPro and GenO95 were
obtained from Centocor's Clinical Pharmacology and Antibody
Technology Department. Human fibrinogen (plasminogen free, >95%
clottable protein) and bovine skin gelatin were purchased from
Sigma (Saint Louis, Mich.).
[0384] Cell Lines
[0385] Huvecs, Human umbilical vein endothelial cells, were
purchased from Clonetics (Walkersville, Mass.). Huvecs were
cultured in endothelial basal media (EBM) kit (Clonetics)
containing 10% FBS, long R insulin-like growth factor-1, ascorbic
acid, hydrocortisone, human epidermal growth factor, human vascular
endothelial growth factor, hFGF-b, gentamicin sulfate, and
amphotericin-B. Cells were incubated at 37.degree. C. and 5%
CO.sub.2 and media was changed every 2 to 3 days. Only passages 3
to 8 were used in all experiments.
[0386] Fibrin Microcarrier-Based Sprouting Assay
[0387] A modification of the methods of Nehls and Drenckhahn (6)
was used to measure capillary tube formation in three-dimensional
fibrin-based matrix. Gelatin-coated cytodex-3 microcarriers (MCs,
Sigma) were prepared according to recommendations of the supplier.
Freshly autoclaved MCs were suspended in EBM-2+20% FBS and
endothelial cells were added to a final concentration of 40
cells/MC. The cells were allowed to attach to the MCs during a
4-hour incubation at 37.degree. C. The MCs were then suspended in a
large volume of medium and cultured for 2 to 4 days at 37.degree.
C. in 5% CO.sub.2 atmosphere. MCs were occasionally agitated to
prevent aggregation of cell coated beads. MCs were embedded in a
fibrin gel that was prepared as follows: human fibrinogen (2 mg/ml)
was dissolved in plain, bFGF or serum containing EBM-2 media. This
solution also contained various antibodies. To prevent excess
fibrinolysis by fibrin-embedded cells, aprotinin was added to the
fibrinogen solution and to growth media at 200 U/ml. Cell-coated
microcarriers were added to the fibrinogen solution at a density of
100 to 200 MCs/ml (50-100 beads/ per well-48 well plate) and
clotting was induced by addition of thrombin (0.5 U/ml). After
clotting was complete, 0.5 ml solution (containing all components
described above except fibrinogen and thrombin) was added to the
fibrin matrices. The plates were incubated at 37.degree. C. and 5%
CO.sub.2 for 1 to 3 days. After 1-3 days, gels were fixed with 3%
paraformaldehyde dissolved in PBS, and the number of capillary
sprouts with length exceeding the diameter of the MC bead (150
.mu.m) was quantified.
[0388] Results and Discussion
[0389] Huvecs can form capillary-like sprouts when cultured in a
fibrin gel (FIG. 9).
[0390] Endothelial cells migrate outwards from the gelatin coated
beads and extend into long filopodia. The long sprouts consist of
several cells forming a lumen. This process resembles
microcapillary formation in vivo, because it involves endothelial
cell migration, invasion and cell proliferation. Quantification of
sprout formation revealed that GenO95 inhibited endothelial cell
sprout formation in bFGF or complete media (FIG. 10). Combination
of LM609 and P1F6 routinely inhibited sprouting more effectively
than GenO95 (FIG. 11).
[0391] Conclusion
[0392] Formation of new blood vessels from existing blood vessels
is a hallmark of angiogenesis. This process can be mimicked in
vitro by the endothelial sprouting assay. These sprouts represent
microcapillaries that are formed in response to angiogenic stimuli
such as bFGF or a variety of stimuli that are present in serum.
GenO95 dose dependently inhibited bFGF- and complete
media-stimulated endothelial cell sprouting, suggesting that this
antibody can effectively inhibit .alpha.v.beta.3 and
.alpha.v.beta.5 function. Why GenO95 was not as effective as the
combination of LM609 and P1F6 is unknown, but it is possible that
GenO95 recognizes .alpha.v.beta.3 and .alpha.v.beta.5 with lower
affinity when compared to LM609 and P1F6, respectively.
Collectively, these data demonstrate that GenO95 can inhibit the
complex process of microcapillary formation in vitro.
[0393] References [0394] 1. Gastl G, Hermann T, Steurer M, Zmija J,
Gunsilius E, Unger C, and Kraft A. 1997. Angiogenesis as a Target
for Tumor Treatment. Oncology 54:177-184. [0395] 2. Eliceiri B P,
and Cheresh D A. 1999. The role of .alpha.V integrins during
angiogenesis: insights into potential mechanisms of action and
clinical development. The Journal of Clinical Investigation
103:1227-1230. [0396] 3. Brooks P C, Montgomery A M, Rosenfeld M,
Reisfeld R A, 1994. Integrin .alpha.v.beta.3 antagonists promote
tumor regression by inducing apoptosis of angiogenic blood vessels.
Cell 79: 1157-1164. [0397] 4. Enenstein J, Walweh N S, and Kramer R
H. 1992. Basic FGF and TGF-.beta. differentially modulate integrin
expression of human microvascular endothelial cells. Exp. Cell Res.
203 :499-503. [0398] 5. Friedlander M, Brooks P C, Shaffer R W,
Kincaid C M, Varner J A, and Cheresh D A. 1995. Definition of two
angiogenic pathways by distinct .alpha.V integrins. Science
270:1500-1502. [0399] 6. Nehls, V and Drenckhahn, D. 1995. A novel,
microcarrier-based in vitro assay for rapid and reliable
quantification of three-dimensional cell migration and
angiogenesis. Microvascular Res. 50:311-322.
EXAMPLE 5
Effect of alpha-V Subunit Antibody on Endothelial and Tumor Cell
Ashesion, Migration and Invasion
[0400] (CBA/J x C57/BL6/J) F.sub.2 hybrid mice (1-4) containing
human variable and constant region antibody transgenes for both
heavy and light chains were immunized with human placental
.alpha.V.beta.3. One fusion yielded a totally human .alpha.V.beta.3
reactive IgG1.kappa. monoclonal antibody named GenO95. The totally
human antibody was found to be reactive to the .alpha.V.beta.3 and
.alpha.V.beta.5 integrins (5). These integrins participate in
endothelial and tumor cell adhesion, migration, and invasion.
Therefore, we characterized the effect of CNTO 95 on integrin
mediated cell motility. CNTO 95 inhibits human umbilical vein
endothelial (HUVEC) and human melanoma cell binding to vitronectin,
denatured collagen, fibrinogen and fibrin, but it does not block
cell adhesion to fibronectin and type I collagen. GenO95 also
inhibits migration of endothelial cells that have been stimulated
with basic fibroblast growth factor and low-dose serum. GenO95
inhibits invasion of tumor cells through a fibrin gel. In
conclusion, GenO95 functionally blocks .alpha.V.beta.3 and
.alpha.V.beta.5 in a variety of cell-based assays in vitro.
[0401] Abbreviations
[0402] BSA--bovine serum albumin
[0403] CO.sub.2--carbon dioxide
[0404] DMSO--dimethyl sulfoxide
[0405] FBS--fetal bovine serum
[0406] Ig--immunoglobulin
[0407] Mab--monoclonal antibody
[0408] OD--optical density
[0409] RT--room temperature
[0410] HUVECS--human umbilical vein endothelial cells
[0411] bFGF--bovine basic fibroblast growth factor
[0412] Introduction
[0413] There is now considerable evidence that progressive tumor
growth is dependent upon angiogenesis. The formation of new blood
vessels provide tumors with nutrients and oxygen, carry away waste
products and act as conduits for the spread of tumor cells to
distant sites. Several studies have defined the role of integrins
in the angiogenic process. Integrins are subunitic trans-membrane
proteins that play a critical role in cell adhesion to the
extracellular matrix (ECM) and mediate cell survival, proliferation
and migration (6). During the angiogenic process, .alpha.v.beta.3
and .alpha.v.beta.5 are upregulated on the surface of activated
endothelial cells, which in turn helps these cells to migrate and
proliferate (6). An antibody generated against .alpha.V.beta.3
blocks basic fibroblast growth factor (bFGF) induced angiogenesis,
whereas an antibody specific to .alpha.V.beta.5 inhibits vascular
endothelial growth factor (VEGF) induced angiogenesis (6,7). In
addition to regulating angiogenesis, .alpha.V.beta.5 and
.alpha.V.beta.3 regulate tumor cell adhesion, migration and
invasion, processes required for tumor cell metastases. Previous
studies indicated that CNTO 95 binds to purified .alpha.V.beta.5
and .alpha.V.beta.3 integrins, therefore, we determined whether
this antibody could functionally block .alpha.V.beta.3- and
.alpha.V.beta.5-mediated endothelial and tumor cell adhesion,
migration and invasion.
[0414] Materials and Methods
[0415] Materials
[0416] Bovine fibroblast growth factor (bFGF) and human vascular
endothelial growth factor 165 (VEGF.sub.165) were obtained from
R&D Systems (Minneapolis, Minn.). MAB 1976Z (LM609), a
monoclonal antibody against integrin .alpha.v.beta.3 and MAB1961
(PIF6), a monoclonal antibody against integrin .alpha.v.beta.5 were
purchased from Chemicon (Temecula, Calif.). ReoPro (lot: 94A04ZE)
and CNTO 95 (lot: JG100899) were obtained from Centocor. BIOCOAT
cell culture inserts (pore size: 8 .mu.m) were purchased from
Becton Dickinson (Bedford, Mass.). Vybrant.TM. cell adhesion assay
kit (V-13181) was purchased from Molecular Probes (Eugene, Oreg.).
Human plasminogen free fibrinogen (VWF/Fn depleted) was purchased
from Enzyme Research Labs (South Bend, Ind.). Bovine skin gelatin
was purchased from Sigma (Saint Louis, Mo.). Human vitronectin was
purchased from Promega (Madison, Wis.), and type I collagen was
purchased from GIBCO BRL (Gaithersburg, Md.).
[0417] Cell Lines
[0418] Human umbilical vein endothelial cells (HUVECS), were
purchased from Clonetics (Walkersville, Mass.), and they were
cultured in EBM medium kit (Clonetics) containing 10% FBS, long R
insulin-like growth factor-1, ascorbic acid, hydrocortisone, human
epidermal growth factor, human vascular endothelial growth factor,
gentamicin sulfate and amphotericin-B. Cells were grown at
37.degree. C. and 5% CO.sub.2 and media was changed every 2 to 3
days. Cells were passaged when they reached 80% confluence.
Passages 3 to 8 were used in all experiments.
[0419] The A375S.2 human melanoma cell line expressing the
.alpha.V.beta.3 and .alpha.V.beta.5 integrins was obtained from
Centocor Cell Bank where the cell line was deemed free of
mycoplasma and bacterial contaminants. The cells were cultured in
DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM
sodium pyruvate, and 0.1 mM non-essential amino acids.
[0420] Human colon carcinoma HT29 cells were obtained from Centocor
Cell Biology
[0421] Service Department, where the cell line was deemed free of
mycoplasma and bacterial contaminant. The cells were cultured in
a-MEM medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM
sodium pyruvate, and 0.1 mM nonessential amino acids.
[0422] Flow Cytometry
[0423] For the detection of surface integrins, cells were
harvested, rinsed, suspended in unsupplemented RPMI media, and
sequentially incubated for 60 minutes on ice with anti-integrin mAb
(10 .mu.g/ml) and FITC-labeled goat anti-mouse antibody (1:100) or
FITC-labeled anti-integrin antibody (10 .mu.g/ml). Absence of
primary antibody or substitution of primary antibody with isotype
matched antibody served as negative controls. Cells were
immediately analyzed with a FACS Scan II flow cytometer (Becton
Dickinson, Mountain View, Calif.).
[0424] Adhesion Assay
[0425] Microtiter plates (Linbro-Titertek, ICN Biomedicals, Inc)
were coated at 4.degree. C. overnight with vitronectin (1
.mu.g/ml), gelatin (0.1%), fibrinogen (100 .mu.g/ml), type I
collagen (10 .mu.g/ml), or fibronectin (10 .mu.g/ml). Immediately
before use plates were rinsed with PBS and blocked for 1 hour with
1% BSA/PBS (pH 7.4). Fibrin-coated Microtiter wells were formed by
thrombin treatment (1 U/ml) of fibrinogen. Adherent cells (HUVECS
HT29 and A375S.2) were labeled with Calcein AM fluorescent dye
(Molecular Probes, Eugene, Oreg.) according to the manufacturer's
instructions, harvested, washed twice, and suspended in 0.1% BSA in
DMEM medium. After cell density was adjusted to
5.times.10.sup.5/ml, cells were incubated with various
concentrations of antibodies for 15 min at 37.degree. C. The
cell-antibody mixture was added to wells (100 .mu.l per well) and
incubated for 1 h at 37.degree. C. Plates were rinsed twice with
PBS to remove unbound cells and adhesion was measured in a
fluorescence plate reader (Fluoroskan) at 485-538 nm. Cell adhesion
to BSA-coated wells served as a negative control. Isotype matched
antibodies served as a negative control.
[0426] Chemotactic Migration Assay
[0427] Cell migration assays were performed in 24-Transwell
chambers with a polystyrene membrane (6.5 mm diameter, 10 .mu.m
thickness, and a pore size of 8 .mu.m). Sub-confluent 24-hr cell
cultures (HUVECS or A375S.2) were harvested with trypsin-EDTA,
washed twice, and resuspended in their respective serum free medium
containing 0.1% BSA. Cells (100,000/500 .mu.l) were added to the
upper chamber in the presence or absence of antibodies. To
facilitate chemotactic cell migration, 750 .mu.l of medium
containing 0.1% BSA and vitronectin (2 .mu.g/ml) or serum (2% for
HUVECS and 10% for A375S2 cells) was added to the bottom chambers
and the plate was placed in a tissue culture incubator. Migration
was terminated after 4 to 8 hrs by removing the cells on the top
with a cotton swab and then the filters were fixed with 3%
paraformaldehyde and stained with Crystal Violet. The extent of
cell migration was determined by light microscopy and images were
analyzed using the Phase 3 image analysis software (Glen Mills,
Pa.). The software analyzes the total area occupied by the stained
cells on the bottom side of the filter and this is directly
proportional to the extent of cell migration.
[0428] Haptotactic Migration Assay
[0429] Cell migration assays were performed using the transwell
chambers as described above with slight modifications. Briefly, the
underside of the membrane was coated with vitronectin (2 .mu.g/ml)
for 60 minutes at room temperature, and then blocked with a
solution of 1% BSA/PBS at room temperature for 60 min. Next,
membranes were washed with PBS and air dried. Serum free medium
(750 .mu.l) containing 0.1% BSA and bFGF (20 ng/ml) was added to
the lower chambers. Sub-confluent 24 h cultures were harvested with
trypsin-EDTA, washed twice, and resuspended in serum free medium.
Cells (100,000/500 .mu.l) were added to the upper chambers in the
presence or absence of antibodies. The chambers were placed in a
tissue culture incubator and migration was allowed to proceed for 6
h. Extent of cell migration was determined as described above.
[0430] Invasion Assay
[0431] Fibrinogen (Plasminogen-free, 100 .mu.l of 10 mg/ml) and 100
.mu.l of 1 U/ml thrombin was mixed and immediately added to the top
chamber of 24 well transwell plates (6.5 mm diameter, 10 .mu.m
thickness and a pore size of 8.0 .mu.m, Costar). The plates were
incubated at 37.degree. C. for 30 minutes to form a fibrin gel.
Confluent tumor cells (A375S.2) were trypsinized, centrifuged,
resuspended in basal medium supplemented with 0.1% BSA and 10
.mu.g/ml plasminogen
[0432] (Enzyme Research Labs, South Bend, Ind.) with various
concentrations of antibodies, and incubated for 15 minutes at room
temperature. Cells (100,000/500 .mu.l) were added to the upper
chamber in the presence or absence of antibodies. The lower
compartment of the invasion chamber was filled with 0.75 ml of 10%
FBS-DMEM, which served as a chemoattractant and the plate was
transferred to a tissue culture incubator. After 24 hours, invasion
was terminated by removing the cells on the top with a cotton swab,
and the filters were fixed with 3% paraformaldehyde and stained
with Crystal Violet. The extent of cell migration was analyzed
using the Phase 3 image analysis software as described above.
[0433] Results and Discussion
[0434] CNTO 95 Inhibits .alpha.v.beta.3- and
.alpha.v.beta.5-mediated Cell Adhesion
[0435] Since CNTO 95 binds to .alpha.V.beta.3 and .alpha.V.beta.5
integrins, we determined whether our tumor cells (A375S.2 and HT29)
and endothelial cells express these integrins. Flow cytometry
indicated that A375S.2 and HUVEC cells express both .alpha.V.beta.3
and .alpha.V.beta.5 integrins, but HT29 cells express
.alpha.V.beta.5, but not .alpha.V.beta.3 integrin (FIG. 12A-I).
[0436] HT29 cells (12A, B and C) express .alpha.v.beta.5, but not
.alpha.v.beta.3 integrin on their surface. HUVEC (12D, E and F) and
A375S.2 (12G, H and I) cells express .alpha.v.beta.5 and
.alpha.v.beta.3 integrin on their surface. Tumor cells and
endothelial cells were stained by immunofluorescence and analyzed
by flow cytometry. The histogram on the left represents background
fluorescence in the presence of isotype matched antibody. The
histogram on the right indicates positive staining. A, D, G, LM609
(mAb directed to .alpha.v.beta.3, 10 .mu.g/ml); B, E, H, PIF6 (mAb
directed to .alpha.v.beta.5, 10 .mu.g/ml); and C, F, I, GenO95 (10
.mu.g/ml).
[0437] The effect of CNTO 95 on adhesion of HUVEC, A375S.2 and HT
29 cells to various matrix proteins was determined in detail.
GenO95 completely inhibited adhesion of HUVEC and A375S.2 cells to
vitronectin, and partially to fibrinogen, gelatin and fibrin coated
plates, indicating that the antibody can block .alpha.V.beta.3 and
.alpha.V.beta.5 (FIGS. 13 and 14, Table 1 and 2). GenO95 completely
inhibited HT-29 cell adhesion to vitronectin coated plates,
indicating that the antibody blocks .alpha.V.beta.5 (FIG. 15).
GenO95 completely inhibited adhesion of HUVEC and A375S.2 cells to
vitronectin coated plates, indicating that the antibody blocks
.alpha.V.beta.3 and .alpha.V.beta.5 (FIGS. 13 and 14). Data were
graphed as percent of maximal binding (no antibody) and non-linear
regression performed using GraphPad Prism.
[0438] Adhesion of HUVECS to matrix protein-coated plates. Adhesion
assay was performed as described in Methods. Plate was read on a
fluorometer at 485-538 nm. Cell adhesion to BSA coated wells served
as a negative control. In FIG. 13, the extent of cell adhesion in
the presence of various concentrations of antibody was plotted as a
percent of cell adhesion in the absence of antibody that was
considered as 100%. Each data point is the mean of triplicate
determinations (+/-SD).
[0439] Adhesion of human melanoma cells to matrix protein-coated
plates. Adhesion assay was performed as described in Methods. Cell
adhesion to BSA coated wells served as a negative control. In FIG.
14 the extent of cell adhesion in the presence of various
concentrations of antibody was plotted as a percent of cell
adhesion in the absence of antibody that was considered as 100%.
Each data point is the mean of triplicate determinations
(+/-SD).
[0440] Table 4 shows the extent of HUVECs adhesion to vitronectin,
gelatin, fibrinogen, fibrin, fibronectin and type I collagen in the
presence of various concentration of antibody was plotted as a
percent of cell adhesion in the absence of antibody that was
considered as 100%. Each data point is the mean of triplicate
determinations (+/-SD). The concentration of antibodies used was 10
.mu.g/ml.
TABLE-US-00004 TABLE 4 Type I Vitronectin Gelatin Fibrinogen Fibrin
Fibronectin collagen Human IgG 96.3 .+-. 11.4 109.0 .+-. 8.8 108.0
.+-. 6.3 99.7 .+-. 4.5 96.8 .+-. 4.7 99.3 .+-. 4.1 LM609 26.3 .+-.
3.7 36.5 .+-. 4.7 14.3 .+-. 2.5 48.1 .+-. 1.5 102.8 .+-. 7.2 108.8
.+-. 12.7 PIF6 39.8 .+-. 5.9 94.4 .+-. 15.1 94.5 .+-. 4.2 96.7 .+-.
4.5 103.2 .+-. 3.8 115.7 .+-. 8.1 LM609-PIF6 3.7 .+-. 0.4 32.2 .+-.
5.2 10.7 .+-. 1.1 30.7 .+-. 8.9 99.6 .+-. 4.7 116.2 .+-. 4.1 CNTO
95 3.3 .+-. 0.6 54.8 .+-. 4.0 34.5 .+-. 1.7 45.1 .+-. 2.4 101.6
.+-. 6.1 97.7 .+-. 3.9 ReoPro 54.9 .+-. 0.9 2.5 .+-. 2.3 8.7 .+-.
2.9 35.8 .+-. 3.0 96.3 .+-. 2.8 99.6 .+-. 6.0
[0441] Table 5 shows the extent of A375S.2 cells adhesion to
vitronectin, gelatin, fibrinogen, fibrin, fibronectin and type I
collagen in the presence of various concentration of antibody. The
data is expressed as a percent of cell adhesion in the absence of
antibody that was considered as 100%. Each data point is the mean
of triplicate determinations (+/-SD). The concentration of
antibodies used is 10 .mu.g/ml.
TABLE-US-00005 TABLE 5 Type I Vitronectin Gelatin Fibrinogen Fibrin
Fibronectin collagen Human IgG 104.0 .+-. 5.3 94.6 .+-. 12.4 102.5
.+-. 5.9 99.5 .+-. 4.0 100.0 .+-. 5.5 99.1 .+-. 3.3 LM609 42.1 .+-.
6.1 25.2 .+-. 7.1 14.0 .+-. 1.8 50.0 .+-. 1.9 104.0 .+-. 8.1 100.0
.+-. 1.5 PIF6 28.5 .+-. 3.8 87.4 .+-. 7.8 99.4 .+-. 3.6 92.9 .+-.
4.7 101.0 .+-. 5.7 101.0 .+-. 7.3 LM609-PIF6 0.9 .+-. 0.3 1.1 .+-.
1.5 10.3 .+-. 2.6 47.6 .+-. 3.2 109.0 .+-. 4.1 102.0 .+-. 4.6 CNTO
95 1.4 .+-. 0.4 23.2 .+-. 7.2 11.4 .+-. 2.8 43.3 .+-. 3.5 103.0
.+-. 4.5 104.0 .+-. 5.9 ReoPro 38.1 .+-. 0.7 6.0 .+-. 1.0 6.5 .+-.
2.1 12.9 .+-. 3.8 104.0 .+-. 5.6 93.1 .+-. 3.1
[0442] The adhesion human colon carcinoma HT29 cells to vitronectin
in the presence of antibody was performed as described above. Cell
adhesion to BSA coated wells served as a negative control. The data
shown in FIG. 15 are plotted as percent of maximum binding (absence
of antibody), and are the mean of triplicate determinations
(+/-SD).
[0443] CNTO95 Blocks Human Melanoma and Endothelial Cell
Migration
[0444] Integrins .alpha.V.beta.3 and .alpha.V.beta.5 participate in
cell migration, therefore we determined whether CNTO 95 could block
vitronectin-stimulated cell migration. Vitronectin-stimulated cell
migration involves .alpha.V.beta.3 and .alpha.V.beta.5. CNTO 95
dose dependently inhibited endothelial cell migration when
vitronectin was used as a chemoattractant (FIG. 17). Interestingly,
CNTO 95 also inhibited migration of both HUVECS and A375S.2 cells
to serum (FIGS. 18 and 19). These findings could be potentially
important for angiogenic and tumor therapy because they suggest
that the targets for CNTO 95, .alpha.V.beta.3 and .alpha.V.beta.5,
are central receptors that are activated by a variety of migratory
factors that are present in serum.
[0445] FIG. 16 shows the migration of HUVECS toward 2 .mu.g/ml
vitronectin. The assay was performed as described in Methods and
cells were allowed to migrate for 6 h. Photomicrographs are
representative fields (10.times. objective lens) of cell migration
in FIG. 16A, absence of antibody, (16B), CNTO 95 (5 .mu.g/ml),
(16C), CNTO 95 (40 .mu.g/ml). FIG. 16D is graphical representation
of cell migration in the presence of varying concentrations of
GenO95. The data were normalized to percent of control (no
antibody) which was considered as 100%, and each point is the mean
of three transwell filters (+/-SD).
[0446] FIG. 17 shows the migration of HUVECS toward 2 .mu.g/ml
vitronectin in the presence of antibodies to .alpha.v.beta.3 and
.alpha.v.beta.5. The migration assay was performed as described in
Methods, and cells were allowed to migrate for 6 hours. LM609 and
P1F6 are mAbs directed to .alpha.v.beta.3 and .alpha.v.beta.5,
respectively. The data shown in FIG. 17 were normalized to percent
of control (no antibody) which was considered as 100%, and each bar
is the mean of three transwell filters (+/-SD). BSA, mouse IgG and
human IgG served as negative controls. LM609-PIF6 represents
combinations of both antibodies. The antibodies and BSA were used
at a concentration of 10 .mu.g/ml.
[0447] FIG. 18 shows the migration of HUVECS towards 2% FBS.
Migration assay was allowed to proceed for 4 h and the data was
captured as described in Methods. FIG. 18(A) is a graphical
representation of cell migration in the presence of LM609, P1F6,
combination of LM609+P1F6, isotype matched control antibodies
(human and mouse). The antibodies and proteins were used at a
concentration of 10 .mu.g/ml. FIG. 18(B) is a graphical
representation of cell migration in the presence of ReoPro and
GenO95. Photomicrographs are representative fields (10.times.
objective lens) of cell migration in FIG. 18(C), the absence of
antibody, FIG. 18(D), GenO95 (5 .mu.g/ml), and FIG. 18(E), GenO95
(20 .mu.g/ml). The data were normalized to percent of control (no
antibody) which was considered as 100%, and each point is the mean
of three transwell filters (+/-SD).
[0448] FIG. 19 shows the migration of A375S.2 cells toward 10% FBS.
Migration assay was allowed to proceed for 4 h and the data was
captured as described in Methods. Antibodies were used at a
concentration of 10 .mu.g/ml. FIG. 19(A) is a graphical
representation of cell migration in the presence of varying
concentrations of GenO95. FIG. 19(B) is a graphical representation
of cell migration in the presence of LM609, P1F6, combination of
LM609+P1F6, isotype matched control antibodies (human and mouse).
The data were normalized to percent of control, which was
considered as 100%, and each point is the mean of three transwell
filters (+/-SD). Photomicro-graphs are representative fields
(10.times. objective lens) of cell migration in FIG. 19(C), absence
of antibody, FIG. 19(D), GenO95 (5 .mu.g/ml), and FIG. 19(E),
GenO95 (20 .mu.g/ml).
[0449] Results described above indicate that CNTO 95 blocks tumor
and endothelial migration to vitronectin and serum. Next, we
determined whether this antibody could inhibit bFGF-stimulated cell
migration. As shown in FIG. 20, bFGF stimulated HUVEC cell
migration towards vitronectin, and CNTO 95 significantly blocked
this stimulated cell migration.
[0450] FIG. 20 shows the migration of HUVECS towards vitronectin in
the presence of bFGF. The undersides of migration chamber filters
were coated with 2 .mu.g/ml vitronectin, and the assay was
performed as described in Methods. Cells were allowed to migrate
for 6 h. In FIG. 20A-E, each data point is the mean of 3 transwell
filters (+/-SD). FIG. 20(A), bFGF; FIG. 20(B), CNTO 95 (5
.mu.g/ml); FIG. 20 (C), CNTO 95 (40 .mu.g/ml); FIG. 20(D), no-bFGF.
FIG. 20(E), Inhibition of cell migration in the presence of various
antibodies is shown graphically.
GenO95 Blocks Human Melanoma Cell Invasion
[0451] Results described above indicate that CNTO 95 can inhibit
cell adhesion and migration. Therefore, we questioned whether this
antibody could block tumor cell invasion, a multistep process that
involves cell adhesion, degradation of the matrix, and migration of
cells through the degraded matrix. We chose fibrin as a matrix for
tumor cells because CNTO 95 was able to block tumor cell adhesion
to fibrin (FIG. 3). As shown in FIG. 10, invasion of A375S.2 cells
could be inhibited by LM609, suggesting the involvement of at least
.alpha.v.beta.3 in this process. CNTO 95 dose dependently inhibited
tumor cell invasion through fibrin. Irrelevant
[0452] IgG and a mAb directed to platelet GPIIb/IIIa (10E5) served
as negative controls. Collectively, these data suggest that CNTO 95
can effectively block invasion of human melanoma cells.
[0453] Invasion of A375S.2 cells through a fibrin gel (5 mg/ml).
Invasion assay was allowed to proceed for 24 h and data was
captured as decribed in Methods. Photomicrographs are
representative fields (4.times. objective lens) of cell invasion in
FIG. 21(A) the absence of antibodies, FIG. 21(B) CNTO 95 (10
.mu.g/ml), FIGS. 21(C) and (D) are graphical representation of cell
invasion in presence of CNTO 95, 10E5 F(ab').sub.2, LM609, P1F6,
LM-PIF6 (LM609+P1F6), human and mouse IgGs (H-IgG and M-IgG). Graph
FIG. 21(D): The concentration of all antibodies and proteins is 10
.mu.g/ml. The data were normalized to percent of control (no
antibody) which was considered as 100%, and each point is the mean
of three transwell filters (+/-SD).
[0454] Conclusion
[0455] Cell adhesion, migration and invasion requires integrins
such as .alpha.v.beta.3 and .alpha.v.beta.5. CNTO 95 is able to
functionally block .alpha.v.beta.3 and .alpha.v.beta.5 integrins
that are expressed by endothelial and tumor cells. CNTO 95 was able
to block migration and invasion of cells that were stimulated by
bFGF or serum. These results suggest that the CNTO 95 is a potent
inhibitor of tumor and endothelial cell expressed .alpha.v.beta.3
and .alpha.v.beta.5 integrins.
[0456] References [0457] 1. Taylor, L. D., C. E. Carmack, D.
Huszar, K. M. Higgins, R. Mashayekh, G. Sequar, S. R. Schramm, C-C.
Kuo, S. L. O'Donnell, R. M. Kay, C. S. Woodhouse, and N. Lonberg.
1993. Human immunoglobulin transgenes undergo rearrangement,
somatic mutation and class switching in mice that lack endogenous
IgM. International Immunology 6:579-591. [0458] 2. Lonberg, N., L.
D. Taylor, F. A. Harding, M. Trounstine, K. M. Higgins, S. R.
Schramm, C-C. Kuo. R. Mashayekh, K. Wymore, J. G. McCabe, D.
Munoz-O'Regan, S. L. O'Donnell, E. S. G. Lapachet, T. Bengoechea,
D. M. Fishwild, C. E. Carmack, R. M. Kay, and D. Huszar. 1994.
Antigen-specific human antibodies from mice comprising four
distinct genetic modifications. Nature 368:856-859. [0459] 3.
Neuberger, M. 1996. Generating high-avidity human Mabs in mice.
Nature Biotechnology 14:826. [0460] 4. Fishwild, D. M., S. L.
O'Donnell, T. Bengoechea, D. V. Hudson, F. Harding, S. L. Bernhard,
D. Jones, R. M. Kay, K. M. Higgins, S. R. Schramm, and N. Lonberg.
1996. High-avidity human IgG monoclonal antibodies from a novel
strain of minilocus transgenic mice. Nature Biotechnology
14:845-851. [0461] 5. Gastl, G., T. Hermann, M. Steurer, J. Zmija,
E. Gunsilius, C. Unger, and A. Kraft. 1997. Angiogenesis as a
Target for Tumor Treatment. Oncology 54: 177-184. [0462] 6.
Eliceiri, B. P., and D. A. Cheresh. 1999. The role of .alpha.V
integrins during angiogenesis: insights into potential mechanisms
of action and clinical development. The Journal of Clinical
Investigation 103: 1227-1230. [0463] 7. Friedlander M., P. C.
Brooks, R. W. Shaffer, C. M. Kincaid, J. A. Varner, and D. A.
Cheresh. 1995. Definition of two angiogenic pathways by distinct
.alpha.V integrins. Science 270: 1500-1502.
EXAMPLE 6
Production and Characterization of Antibodies to the Variable
Region of CNTO 95
[0464] Anti-anti-bodies were prepared by immunization of Balb/c
mice with CNTO 95. Initial titers from mice immunized with CNTO 95
ranged from a high of >1:40,000, to a low of 1:20,000.
[0465] For fusions C371idD, C371idH, C371idI and C371idJ, mice #2,
11, 12 and 14 respectively, were IV boosted with 50 mg of CNTO 95
diluted to 100 mL in phosphate buffered saline (PBS). For fusions
C371idK and C371idL, mice #16 and 17 respectively, were boosted
using 50 mg CNTO 95-mouse albumin conjugate as above. Three days
after the IV injection, the mice were sacrificed by cervical
dislocation and the spleen was removed aseptically, spleenocytes
were isolated, and fused to the non-secreting mouse myeloma fusion
partner, P3.times.63 Ag 8.653.
[0466] From six fusions utilizing CNTO 95 immunized Balb/c mice,
seventeen anti-variable region antibody-producing hybridomas were
identified based on their specific reactivity with the variable
region of the human aVb3 and aVb5 antibody, CNTO 95 (IgG1k), and
for their nonrecognition of isotypic antigenic determinants.
TABLE-US-00006 TABLE 2 Characterization of CNTO 95 Anti-variable
region Mabs Anti-ID Mab Anti-ID Mab Inhibition of Inhibition of
Inhibition of binding to inhibition of ID binding ID binding ID
binding CNTO 95 CNTO 95 in the in the in the Murine Prebound to
binding to presence of presence of presence of Isotype
.alpha.V.beta.3 .alpha.V.beta.3 0.5% NHS 5% NHS 50% NHS C508 IgG2b
.kappa. - + - - - C577 IgG1 .kappa. - + - - inhibition C580 IgG1
.kappa. - - - - - C581 IgG1 .kappa. - + - - - C582 IgG1 .kappa. - +
- - - C583 IgG1 .kappa. - + - - - C571 IgG1 .kappa. - - - - - C578
IgG2b .kappa. + - - - - C585 IgG2b .kappa. - + - - - C572 IgG1
.kappa. - - - - - C573 IgG1 .kappa. - - - - - C574 IgG1 .kappa. - +
- - - C575 IgG1 .kappa. - - - - - C576 IgG1 .kappa. - + - - - C579
IgG1 .kappa. - + - - - C584 IgG1 .kappa. - + - - - C586 IgG1
.kappa. - + - - - (NHS = Normal Human Serum)
[0467] Seventeen monoclonal antibodies were made to the variable
region of CNTO 95. Six of them (C571-3, C575, C578 and C580) were
demonstrated not to block binding of the antibody to alpha-V-beta3.
The remaining eleven (C508, C576-7, C581-6,C574 and C579) appear to
block the active site of CNTO 95 and inhibit the binding of CNTO 95
to human integrin .alpha.Vb3 and do not bind to CNTO 95 prebound to
its receptor. The non-blocking Mab, C578, can detect CNTO 95
pre-bound to aV.beta.3. Pooled human serum does not interfere with
the binding of 16 of 17 CNTO 95 anti-variable region Mabs.
[0468] These seventeen Mabs could prove useful in pharmacokinetic
or immunohistochemical detection of CNTO 95 in patient and animal
tissue or sera samples.
EXAMPLE 7
Demonstration of Anti-alpha-V Subunit Antibody Binding to alpha-V
beta-6 on the Surfaces of Cells
[0469] CNTO 95 is capable of recognizing the alpha-V (or alpha5)
subunit as it is presented on cell surfaces when complexed with the
beta subunits designated beta-III (beta-3) and beta-5. It is
important to ascertain that the epitope for binding the alpha-V
subunit is still available when alpha-V is present in other
heterodimeric forms of integrins such as alpha-V, beta-1 or
alpha-V, beta-6.
[0470] The following study confirms that CNTO 95 has the capability
to bind the heterodimeric receptor alpha-5, beta-6 as it occurs on
the surface of a cell.
[0471] Materials and Methods
[0472] CNTO 95 antibody was from Centocor (Malvern, Pa. 19355). All
other antibody reagents listed below were purchased from Chemicon,
International, Inc. (Temecula Calif.). The control and comparator
antibodies included: H IgG 557276, MAB 1959 to beta1, MAB 2076Z to
beta6, MAB 1953Z to alpha-V, MAB 1976Z to alpha-V-beta-3, MAB 1961Z
to alpha-V-beta-5, MAB 2077Z to alpha-V-beta6, MAB 2075Z to beta6,
and MAB 2074Z to alpha-V-beta6.
[0473] HEK-293 cells (Human embryonal kidney cells, ATCC CRC-1573)
were transfected with cDNA constructs to overexpress either human
av, b6, or avb6 integrins.
[0474] Cell Staining and Flow Cytometric Analysis:
[0475] Cell suspensions were prepared by trypsinizing adherent cell
cultures, washing and resuspending the cells in serum free media
(SFM). Thereafter, the cells were reacted with primary antibody,
washed and reacted with a second antibody carrying a fluorescent
marker.
[0476] Primary antibody reaction: cells (1.times.10.sup.6 cells/ml)
were resuspended in 200 microL SFM, and 2 .mu.L of antibody was
added to give a final antibody concentration 10 mcirogm/ml in each
tube. Cells were incubated from 45 minutes to 1 hour on ice,
keeping tubes in the dark. To wash away extra antibody, 3 ml of
DPBS was added, and tubes were centrifuged at 1300 RPM for 3
minutes at 4.degree. C.
[0477] Secondary antibody: Phycoerythrin-conjugated secondary
antibodies were added as described above. After 1 hour, tubes were
centrifuged at 1300 RPM for 3 minutes at 4.degree. C., and cells
were resuspended in 0.5 ml of FACS buffer.
[0478] Flow cytometry: Samples were mixed thoroughly before
analysis. Flow cytometric analysis was performed on a
Becton-Dickinson FACSCalibur, using both green (FITC) and red
(phycoerythrin) channels.
[0479] Results
[0480] As seen in the upper row of FIG. 22A, mock transfected HEK
293 cells demonstrated immunoreactivity for integrin alpha-V, and
some immunoreactivity for beta-6 and avb6 (weak), as shown by
comparing the position of the open curve (control stain) against
the shaded curve (test antibody). Transfection of HEK 293 cells
with either b6 or avb6 cDNA caused a high level of immunoreactivity
for avb6 integrin as demonstrated by comparing the shaded curves in
the third panels of rows 3 and 4 against the shaded curve in the
third panel of row 1. A stronger shift toward the right indicates
stronger immunoreactivity and higher protein expression.
[0481] Transfection with avb6 did not cause a change in expression
of avb3 (column 1), avb5 (column 2), or b1 (column 3) integrins
compared to mock transfected cells (FIG. 22B). The positions of the
shaded curves in each of the panels in the lower row (avb6
transfected) are nearly identical to the positions of the
corresponding shaded curves in the upper row (mock transfected).
Therefore, any change seen in CNTO 95 binding would not be due to
changes in avb3 or avb5 expression.
[0482] As shown in FIG. 22C, overexpression of human integrin
subunits aV (panel 2) or b6 (panel 3) alone caused a small increase
in CNTO 95 immunoreactivity, which is indicated by a shift toward
the right of the shaded curves compared to panel 1. Transfection
with the heterodimeric human avb6 integrin (panel 4) caused a
dramatic increase in CNTO 95 staining as demonstrated by a large
proportion of the shaded curve to the right of the vertical line.
As a further confirmation, cells were double-stained for both avb6
and CNTO 95 (FIG. 22D). As seen previously, mock transfected cells
showed a small amount of immunoreactivity for avb6 (panel A) and
substantial immunoreactivity for CNTO 95 (FIG. 22D panel B).
Plotting immunoreactivity of avb6 (vertical axis) against CNTO 95
(horizontal axis), a large proportion of mock transfected cells
fell into the lower-right quadrant (FIG. 22D, panel C), indicating
immunoreactivity to CNTO 95 alone. Analysis of avb6 transfected
cells showed a large increase in immunoreactivity for avb6 (FIG.
22D panel D), as well as for CNTO 95 (FIG. 22D panel E). Plotting
immunoreactivity of avb6 (vertical axis) against CNTO 95
(horizontal axis) revealed a strong shift in the population of
cells toward the upper-right quadrant (FIG. 22D panel F),
indicating that cells which stained intensely for avb6 also stained
intensely for CNTO 95. Taken together, the results indicate that
CNTO 95 binds to alphavbeta6 integrin.
EXAMPLE 8
Characterization of CNTO 95 Ligands in Human Placental Tissue
[0483] Human placenta is a source of a spectrum of adhesion
molecules including known integrins. Ligands binding CNTO 95 from a
human placental extract were identified using commercially
available Mabs with known specificity.
[0484] Human placenta was donated with consent. Approximately 300 g
tissue was washed with ice cold buffered saline followed by
addition of 600 ml of extraction buffer (TBS, pH7.5, 1 mM
CaCl.sub.2, 1 mM MnCl.sub.2, 100 mM Octylglucoside (OTG), and
EDTA-free protease inhibitor tablets from Roche Applied Sciences).
The tissue was minced with scissors and then homogenized using a
blender. After homogenation, 17.52 g OTG was added to the
homogenate and the mixture was rotated at 4.degree. C. overnight.
The extract was the supernatant taken after centrifugation in a
SORVALL RC5CPLUS centrifuge in a SLA 3000 rotor at 10,000 rpm for 1
hour at 4.degree. C. Extracts were stored at 4.degree. C.
[0485] CNTO 95 (Centocor, Malvern, Pa.) and MAB1978 (anti-integrin
alphaV purchased from Chemicon, Temecula, Calif. as ascites fluid
and purified using an immobilized protein A (Pierce Chemicals) were
coupled to CNBr-activated sepharose 4 Fast Flow (Amersham)
according to standard procedures.
[0486] Placenta extracts were incubated with CNTO 95- or
MAB1978-conjugated resin overnight at 4.degree. C. Resins were then
loaded to empty columns. Columns were washed with 10 ml of column
wash buffer I followed by 10 ml of column wash buffer II. Integrin
fractions were eluted with 10 ml of elution buffer from the
columns. The eluted materials were concentrated and stored at
4.degree. C. for further analysis.
[0487] Western blot. The integrin fraction were separated by
electrophoresis on 4-12% SDS polyacrylamide gels and then
transferred to nitrocellulose filters. The filters were blocked
with 5% nonfat dry milk in TBS containing 0.05% Tween 20 (wash
buffer) at room temperature for 1 hour and then incubated with
anti-integrin antibodies (anti-a5 (P-19) Santa Cruz, goat
polyclonal IgG 1:500 dilution; anti-aIIb, Chemicon, mouse
monoclonal IgG1, 1:1000 dilution; anti-aV (Q-20), Santa Cruz, goat
polyclonal IgG, 1:500 dilution; anti-b1 (4B7R), Santa Cruz, mouse
monoclonal IgG1, 1:500 dilution; anti-b1 (N-20), Santa Cruz, goat
polyclonal IgG, 1:500 dilution; anti-.beta.3 (H-96), Santa Cruz,
rabbit polyclonal IgG, 1:500 dilution; anti-.beta.5 (H-96), Santa
Cruz, rabbit polyclonal IgG, 1:500 dilution; anti-.beta.6 (H-110),
Santa Cruz, rabbit polyclonal IgG, 1:250 dilution). After thorough
washing, filters were incubated with appropriate
peroxidase-conjugated secondary antibodies (1:20000 dilution). The
antigen-antibody complexes were visualized using SuperSignal West
Pico Chemiluminescent Substrate kit (Pierce).
[0488] To identify which integrins eluted fraction from CNTO 95
affinity column Western blot analysis was performed on strips of
the blot incubated with individual antibody preparations. The
strips labeled positively with anti-integrin-aV, -b1, -b3, -b5, and
-b6, demonstrating that the above integrin subunits are components
of integrin complexes bound by CNTO 95. Since integrins are
subunits of alpha subunits and beta subunits, the results indicate
that CNTO 95 binds aVb1, aVb3, aVb5, and aVb6 subunit integrins. To
further define the integrin specificity of CNTO 95, integrin
fractions purified from CNTO 95 affinity column were examined by
Western blots with antibodies against integrin a5 and integrin
aIIb, it should be noted that placenta contains abundant integrin
a5b1 and integrin aIIbb3. Purified integrin a5b1 and crude placenta
extract, were stained by anti-integrin a5 or anti-aIIb antibody.
However, there were no detectable signals for a5 and alIb in lanes
loaded with proteins purified by CNTO 95 affinity
chromatography.
[0489] Therefore, CNTO 95 binds all subunit of the integrins
containing alphaV tested and not a5 or aIIb containing
heterodimers.
EXAMPLE 9
In Vitro Angiogenesis: Microvessel Sprouting
[0490] Rat aortic ring assay. An early event during tumor-induced
angiogenesis is the formation of microvessel sprouts from
established blood vessels that migrate towards the tumor. A
microvessel sprouting assay was used to test the in vitro
anti-angiogenic activity of CNTO 95. In this assay, freshly excised
rat aortas were cultured in 3-dimensional fibrin or collagen gels.
In the presence of various angiogenic factors such as bFGF, the
aorta sprouts microcapillaries after a few days. This process is a
representation of angiogenesis, as it involves endothelial cell
adhesion, migration, invasion, and proliferation.
[0491] The rat aortic ring assay was performed as described by
Nicosia et al. (Lab Invest. 63: 115-122, 1990) with slight
modifications (Sassoli, et al. Thromb Haemost, 85: 896-902, 2001).
10 mm diameter agarose wells (1.5%) prepared in 100.times.15 mm
tissue culture dishes were filled with M199 media containing rat
tail type 1 collagen (2.8 mg/ml, Becton Dickinson), NaHCO3 (28 mM)
and either CNTO 95 (10 ug/ml), nonspecific control murine IgG (20
mg/ml) or BSA (20 mg/ml). Rat aortic ring sections (1 mm) were
placed on top of collagen gels within the agarose rings; wells were
filled with collagen solution and incubated at 37.degree. C. After
the collagen had gelled, the collagen-aortic ring sandwiches were
transferred to 12 well plates containing 1 ml EBM-2 medium, BSA
(0.1%), bFGF (10 ng/ml), penicillin (100 U/ml), streptomycin (100
mg/ml), amphotericin B (0.25 mg/ml) (all from Clonetics), and
either CNTO 95 (increasing concentrations), non-specific control
mIgG (20 ug/ml), or BSA (20 mg/ml). Plates were maintained in a
37.degree. C. tissue culture incubator with media changes every
other day. After 10 days, the number and length of microvessel
sprouts originating from each aortic ring was quantified
microscopically using the Phase 3 Image Analysis System.
[0492] As shown in FIG. 23, CNTO 95 inhibited sprouting of
microvessels from aortas excised from rats in a dose-dependent
manner These results demonstrate that CNTO 95 is an inhibitor of
angiogenesis in vitro.
EXAMPLE 10
Anti-alpha V Antibody Blocks Angiogensis In Vivo
[0493] Rat and Monkey Matrigel Assays
[0494] In order to determine whether CNTO 95 could functionally
block aVb3 and aVb5 integrins, a non-human primate model and a nude
rat model of growth factor-induced angiogenesis and in a nude rat
model of tumor cell-induced angiogenesis were used. The monkey
study was performed at Charles River Laboratories (Worcester,
Mass.) on young adult female Cynomolgus monkeys (species macaca
fascicularis). Nude female rats (5-7 weeks old) were obtained from
Harlan (Indianapolis, Ind.). Recombinant human bFGF was obtained
from R&D Systems. Matrigel, prepared from the
Engelbreth-Holm-Swarm tumor, was obtained from Becton
Dickinson.
[0495] Liquid Matrigel was maintained at 4.degree. C. The
angiogenesis assays were performed as described (Trikha, et al.
Cancer Res., 62: 2824-2833, 2002). For growth factor induced
angiogenesis, human bFGF (5 mg/ml) was added to the Matrigel
solution and allowed to mix thoroughly overnight. The Matrigel was
then mixed with antibodies or control solutions and kept on ice.
For tumor-cell induced angiogenesis, human melanoma M21 cells were
harvested with trypsin-EDTA, centrifuged, washed twice with DMEM,
and resuspended in ice-cold DMEM. M21 cells were gently added to
the Matrigel solution at a final concentration of
0.5.times.10.sup.6/ml. The tumor cell-Matrigel solution was gently
mixed and stored on ice until it was injected into nude rats.
[0496] Monkeys were injected at each site subcutaneously with 2 ml
of Matrigel solution, while rats were injected with 1 ml of
Matrigel each. In the tumor cell-induced study, rats were injected
at two sites with 1 ml of the ice-cold tumor cell-Matrigel
solution. Gel formation was confirmed after injection. Animals
received test article via intravenous or intraperitoneal bolus
injection. At the end of each study, animals were euthanized and
Matrigel harvested from the injection sites. Matrigel implants were
weighed, photographed and graded for angiogenesis using the Phase 3
Image Analysis System. To measure the total area of neovessels,
photomicrographs were taken from both the top surface and the
bottom surface of each Matrigel plug at 2.times. magnification on
the inverted phase contrast microscope. The vessel length and
number of vessels per field were calculated using the tracing
function within the Phase 3 Image System. The mean value from all
2.times. fields was calculated for each Matrigel plug, and the mean
vessel number and vessel length for each test group was
calculated.
[0497] As a third method to measure angiogenesis,
immunohistochemistry was performed on 10 mm serial cryostat
sections cut from frozen Matrigel plugs. Sections were immediately
fixed in cold acetone (5 min) and air-dried. The sections were
washed 3 times in PBS to remove frozen mounting media, blocked for
1 hour with 5% mouse serum and 5% goat serum in PBS and rinsed in
PBS. The sections were then blocked with Avidin-Biotin solution
(X0590, DAKO Corporation, Carpinteria, Calif.) for 10 min. After
washing, endogenous peroxidase was quenched by incubation in 3%
hydrogen peroxide for 10 min. Then, the sections were incubated for
60 min with primary antibody (mouse anti-human PECAM, BD
PharMingen, Bedford, Mass.; 10 mg/ml) diluted with DAKO antibody
diluent solution (S3022, DAKO). Immunoreactive sites were detected
using a DAKO Kit, and sections were counterstained with
hematoxylin. An irrelevant mouse IgG1 was used as a negative
control in all cases. Photomicrographs were taken from all slides
at 20.times. magnification; each entire section was photographed.
The vessel density per field was calculated using the Phase 3 Image
System software. Vessel density was quantitated by measuring the
percentage of cross-sectional area of each Matrigel section
occupied by stained microvessels. The mean value for each slide was
calculated, and the mean vessel density for each group was
determined
[0498] Results
[0499] Inclusion of human bFGF in Matrigel implants in monkeys and
rats resulted in increased angiogenesis as measured by vessel
length, number, and vessel density (FIGS. 24-26). Systemic
treatment of rats with CNTO 95 significantly inhibited
bFGF-stimulated increases in vessel length and total vessel number
within Matrigel implants as measured by visual inspection.
Inhibition of angiogenesis by CNTO 95 was dose-dependent, with a
dose of 1 mg/kg being active in this model (FIG. 24). Results of
the immunostaining of vessels with anti-CD31 were consistent with
those obtained by direct visual counting of microvessels.
[0500] Subcutaneous injection of Matrigel containing human bFGF in
cynomolgus monkeys resulted in increased angiogenesis as measured
by vessel length, number, and vessel density (FIG. 25A-C). Systemic
treatment of monkeys with CNTO 95 significantly inhibited
bFGF-stimulated increases in vessel length (FIG. 25A) and total
vessel number (FIG. 25B) within Matrigel implants as measured by
image analysis quantification. Systemic treatment of monkeys with
CNTO 95 also reduced the bFGF-stimulated increase in microvessel
density within Matrigel implants as measured by immunostaining for
CD31 expression and image analysis (FIG. 25C).
[0501] In the tumor cell-induced angiogenesis model, a single dose
of 10 mg/kg of CNTO 95 inhibited tumor cell-induced angiogenesis.
No difference in inhibitory activity was observed when CNTO 95 was
administered as a single intravenous dose or when it was mixed with
the Matrigel-tumor cell suspension prior to injection into the rats
Inhibition of angiogenesis by CNTO 95 was demonstrated by decrease
in the length and number of microvessels (FIG. 26) and decrease in
hemoglobin content in the Matrigel plugs (data not shown).
[0502] Collectively, these results indicated that CNTO 95 inhibited
angiogenesis stimulated by either bFGF in both nude rats and
non-human primates. CNTO 95 also prevents human tumor cell induced
angiongenesis in an immunosuppressed animal.
EXAMPLE 11
Anti-Tumor Effect of Anti-alpha V Antibody in Nude Mice and
Rats
[0503] For studies performed in nude mice, female nude mice, aged
4-5 weeks, were purchased from Charles River Laboratories
(Wilmington, Mass.), and were maintained according to the NIH
standards established in the `Guidelines for the Care and Use of
Experimental Animals`. Twenty mice were inoculated subcutaneously
with A375.S2 cells (3.times.106) in the flank region (day 0). On
day 3, the mice were randomly divided into two groups. One group
was injected i.p. with CNTO 95 (10 mg/kg in PBS), while the other
group received vehicle. Dosing was continued three times a week
thereafter until day 26. Tumors were measured by calipers twice a
week, and tumor volumes were calculated by the formula
(length.times.width2/2). Body weights were also recorded
weekly.
[0504] For studies performed in nude rats, female nude rats, aged
6-7 weeks, were purchased from Harlan (Indianapolis, Ind.), and
were maintained according to the NIH standards established in the
`Guidelines for the Care and Use of Experimental Animals`. Twenty
rats were inoculated subcutaneously with A375.S2 cells
(3.times.106) in the flank region (day 1). On day 4, the rats were
randomly assigned to two groups. One group was injected i.v. with
CNTO 95 (10 mg/kg in PBS), while the other group received an
isotype-matched control IgG (10 mg/kg). Dosing was continued weekly
thereafter until day 46 (total of 6 doses). Tumors were measured by
calipers twice a week, and tumor volumes were calculated by the
formula (length.times.width2/2). Body weights were also recorded
weekly. Statistical comparison of group mean tumor volumes was
performed using Student's t-test, 2-tailed analysis.
[0505] Results
[0506] To determine the anti-tumor efficacy of CNTO 95 in vivo, a
human A375.S2 melanoma xenograft tumor model was established in
nude mice. Mice were treated with CNTO 95 (10 mg/kg) 3 times per
week by i.p. injection, starting 3 days after tumor inoculation. As
shown in FIG. 27, dosing with CNTO 95 inhibited growth of human
melanoma tumors in nude mice. At day 26 CNTO 95 inhibited tumor
growth by .about.80% compared to tumors from control-treated
animals. In this model CNTO 95 does not interact with host
angiogenic vessels, since CNTO 95 does not bind mouse integrins,
suggesting that blockade of human tumor-expressed integrins alone
can inhibit tumor growth in mice independent of anti-angiogenic
effects.
[0507] To determine the anti-tumor efficacy of CNTO 95 in another
xenograft animal model, an A375.S2 human melanoma model was
developed in female nude rats. In this model CNTO 95 is capable of
blocking both rat angiogenic integrins and human tumor cell
expressed integrins. Weekly treatment of tumor-bearing nude rats
with CNTO 95 at 10 mg/kg reduced tumor growth compared to the
isotype-matched human IgG control mAb (FIG. 28). By day 46,
treatment with CNTO 95 resulted in a significant reduction in final
tumor size compared to control-treated nude rats (P=0.0007).
[0508] In addition to blocking integrins on angiogenic endothelium,
CNTO 95 has the ability to inhibit integrin function on tumor cells
themselves. AlphaV integrins have been suggested to play critical
roles in tumor cell biology. Therefore, the use of CNTO 95 has
applicability to multiple tumor types with different integrin
expression patterns.
[0509] In the nude mouse xenograft model, CNTO 95 does not
cross-react with host integrins, however, treatment with CNTO 95
significantly inhibited the growth of the
.alpha.v.beta.3/.beta.5positive melanoma tumors. In a previous
report (Trikha et al. supra), it was demonstrated that antibody
against .alpha.v.beta.3 on the surface of human melanoma cells
partially inhibited tumor growth in nude mice. In that study, m7E3
F(ab')2, a murine antibody which binds and blocks human
.alpha.v.beta.3 and .alpha.II.beta.3, directly reduced growth of a
human melanoma xenograft without blocking host cell integrins. CNTO
95 differs from m7E3 F(ab')2 in that it is a full-length human IgG
that recognizes .alpha.v.beta.3 and .alpha.v.beta.5, but not the
.alpha.IIb.beta.3 that is predominantly expressed on platelets.
[0510] Together these data suggest that through combined blockade
of .alpha.v.beta.3 and .alpha.v.beta.5 integrins on tumor and
endothelial cells, CNTO 95 may have multiple mechanisms of action
that contribute to its observed antitumor efficacy in animal
models.
[0511] It is becoming increasingly clear that although targeted
therapy holds great promise, combination drug regimens will likely
be necessary for optimal efficacy. CNTO 95 by itself targets
multiple crucial receptors involved in tumor growth, angiogenesis
and metastasis. An additional advantage of CNTO 95 is its
fully-human nature, which will allow long-term and repeated use
with anticipated safety due to lack of the HAMA reactions seen with
murine antibodies.
[0512] It will be clear that the invention can be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0513] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
Sequence CWU 1
1
1715PRTHomo sapiens 1Arg Tyr Thr Met His1 5217PRTHomo sapiens 2Val
Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Lys Gly1 5
10 15310PRTHomo sapiens 3Glu Ala Arg Gly Ser Tyr Ala Phe Asp Ile1 5
10411PRTHomo sapiens 4Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala1
5 1057PRTHomo sapiens 5Asp Ala Ser Asn Arg Ala Thr1 568PRTHomo
sapiens 6Gln Gln Arg Ser Asn Trp Pro Pro1 57119PRTHomo sapiens 7Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Val Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn
Thr Leu Tyr65 70 75 80Leu Gln Val Asn Ile Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ala Arg Gly Ser Tyr Ala Phe
Asp Ile Trp Gly Gln Gly 100 105 110Thr Met Val Thr Val Ser Ser
1158108PRTHomo sapiens 8Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Phe Thr Phe Gly
Pro Gly Thr Lys Val Asp Ile Lys 100 10591048PRTHomo sapiens 9Met
Ala Phe Pro Pro Arg Arg Arg Leu Arg Leu Gly Pro Arg Gly Leu1 5 10
15Pro Leu Leu Leu Ser Gly Leu Leu Leu Pro Leu Cys Arg Ala Phe Asn
20 25 30Leu Asp Val Asp Ser Pro Ala Glu Tyr Ser Gly Pro Glu Gly Ser
Tyr 35 40 45Phe Gly Phe Ala Val Asp Phe Phe Val Pro Ser Ala Ser Ser
Arg Met 50 55 60Phe Leu Leu Val Gly Ala Pro Lys Ala Asn Thr Thr Gln
Pro Gly Ile65 70 75 80Val Glu Gly Gly Gln Val Leu Lys Cys Asp Trp
Ser Ser Thr Arg Arg 85 90 95Cys Gln Pro Ile Glu Phe Asp Ala Thr Gly
Asn Arg Asp Tyr Ala Lys 100 105 110Asp Asp Pro Leu Glu Phe Lys Ser
His Gln Trp Phe Gly Ala Ser Val 115 120 125Arg Ser Lys Gln Asp Lys
Ile Leu Ala Cys Ala Pro Leu Tyr His Trp 130 135 140Arg Thr Glu Met
Lys Gln Glu Arg Glu Pro Val Gly Thr Cys Phe Leu145 150 155 160Gln
Asp Gly Thr Lys Thr Val Glu Tyr Ala Pro Cys Arg Ser Gln Asp 165 170
175Ile Asp Ala Asp Gly Gln Gly Phe Cys Gln Gly Gly Phe Ser Ile Asp
180 185 190Phe Thr Lys Ala Asp Arg Val Leu Leu Gly Gly Pro Gly Ser
Phe Tyr 195 200 205Trp Gln Gly Gln Leu Ile Ser Asp Gln Val Ala Glu
Ile Val Ser Lys 210 215 220Tyr Asp Pro Asn Val Tyr Ser Ile Lys Tyr
Asn Asn Gln Leu Ala Thr225 230 235 240Arg Thr Ala Gln Ala Ile Phe
Asp Asp Ser Tyr Leu Gly Tyr Ser Val 245 250 255Ala Val Gly Asp Phe
Asn Gly Asp Gly Ile Asp Asp Phe Val Ser Gly 260 265 270Val Pro Arg
Ala Ala Arg Thr Leu Gly Met Val Tyr Ile Tyr Asp Gly 275 280 285Lys
Asn Met Ser Ser Leu Tyr Asn Phe Thr Gly Glu Gln Met Ala Ala 290 295
300Tyr Phe Gly Phe Ser Val Ala Ala Thr Asp Ile Asn Gly Asp Asp
Tyr305 310 315 320Ala Asp Val Phe Ile Gly Ala Pro Leu Phe Met Asp
Arg Gly Ser Asp 325 330 335Gly Lys Leu Gln Glu Val Gly Gln Val Ser
Val Ser Leu Gln Arg Ala 340 345 350Ser Gly Asp Phe Gln Thr Thr Lys
Leu Asn Gly Phe Glu Val Phe Ala 355 360 365Arg Phe Gly Ser Ala Ile
Ala Pro Leu Gly Asp Leu Asp Gln Asp Gly 370 375 380Phe Asn Asp Ile
Ala Ile Ala Ala Pro Tyr Gly Gly Glu Asp Lys Lys385 390 395 400Gly
Ile Val Tyr Ile Phe Asn Gly Arg Ser Thr Gly Leu Asn Ala Val 405 410
415Pro Ser Gln Ile Leu Glu Gly Gln Trp Ala Ala Arg Ser Met Pro Pro
420 425 430Ser Phe Gly Tyr Ser Met Lys Gly Ala Thr Asp Ile Asp Lys
Asn Gly 435 440 445Tyr Pro Asp Leu Ile Val Gly Ala Phe Gly Val Asp
Arg Ala Ile Leu 450 455 460Tyr Arg Ala Arg Pro Val Ile Thr Val Asn
Ala Gly Leu Glu Val Tyr465 470 475 480Pro Ser Ile Leu Asn Gln Asp
Asn Lys Thr Cys Ser Leu Pro Gly Thr 485 490 495Ala Leu Lys Val Ser
Cys Phe Asn Val Arg Phe Cys Leu Lys Ala Asp 500 505 510Gly Lys Gly
Val Leu Pro Arg Lys Leu Asn Phe Gln Val Glu Leu Leu 515 520 525Leu
Asp Lys Leu Lys Gln Lys Gly Ala Ile Arg Arg Ala Leu Phe Leu 530 535
540Tyr Ser Arg Ser Pro Ser His Ser Lys Asn Met Thr Ile Ser Arg
Gly545 550 555 560Gly Leu Met Gln Cys Glu Glu Leu Ile Ala Tyr Leu
Arg Asp Glu Ser 565 570 575Glu Phe Arg Asp Lys Leu Thr Pro Ile Thr
Ile Phe Met Glu Tyr Arg 580 585 590Leu Asp Tyr Arg Thr Ala Ala Asp
Thr Thr Gly Leu Gln Pro Ile Leu 595 600 605Asn Gln Phe Thr Pro Ala
Asn Ile Ser Arg Gln Ala His Ile Leu Leu 610 615 620Asp Cys Gly Glu
Asp Asn Val Cys Lys Pro Lys Leu Glu Val Ser Val625 630 635 640Asp
Ser Asp Gln Lys Lys Ile Tyr Ile Gly Asp Asp Asn Pro Leu Thr 645 650
655Leu Ile Val Lys Ala Gln Asn Gln Gly Glu Gly Ala Tyr Glu Ala Glu
660 665 670Leu Ile Val Ser Ile Pro Leu Gln Ala Asp Phe Ile Gly Val
Val Arg 675 680 685Asn Asn Glu Ala Leu Ala Arg Leu Ser Cys Ala Phe
Lys Thr Glu Asn 690 695 700Gln Thr Arg Gln Val Val Cys Asp Leu Gly
Asn Pro Met Lys Ala Gly705 710 715 720Thr Gln Leu Leu Ala Gly Leu
Arg Phe Ser Val His Gln Gln Ser Glu 725 730 735Met Asp Thr Ser Val
Lys Phe Asp Leu Gln Ile Gln Ser Ser Asn Leu 740 745 750Phe Asp Lys
Val Ser Pro Val Val Ser His Lys Val Asp Leu Ala Val 755 760 765Leu
Ala Ala Val Glu Ile Arg Gly Val Ser Ser Pro Asp His Ile Phe 770 775
780Leu Pro Ile Pro Asn Trp Glu His Lys Glu Asn Pro Glu Thr Glu
Glu785 790 795 800Asp Val Gly Pro Val Val Gln His Ile Tyr Glu Leu
Arg Asn Asn Gly 805 810 815Pro Ser Ser Phe Ser Lys Ala Met Leu His
Leu Gln Trp Pro Tyr Lys 820 825 830Tyr Asn Asn Asn Thr Leu Leu Tyr
Ile Leu His Tyr Asp Ile Asp Gly 835 840 845Pro Met Asn Cys Thr Ser
Asp Met Glu Ile Asn Pro Leu Arg Ile Lys 850 855 860Ile Ser Ser Leu
Gln Thr Thr Glu Lys Asn Asp Thr Val Ala Gly Gln865 870 875 880Gly
Glu Arg Asp His Leu Ile Thr Lys Arg Asp Leu Ala Leu Ser Glu 885 890
895Gly Asp Ile His Thr Leu Gly Cys Gly Val Ala Gln Cys Leu Lys Ile
900 905 910Val Cys Gln Val Gly Arg Leu Asp Arg Gly Lys Ser Ala Ile
Leu Tyr 915 920 925Val Lys Ser Leu Leu Trp Thr Glu Thr Phe Met Asn
Lys Glu Asn Gln 930 935 940Asn His Ser Tyr Ser Leu Lys Ser Ser Ala
Ser Phe Asn Val Ile Glu945 950 955 960Phe Pro Tyr Lys Asn Leu Pro
Ile Glu Asp Ile Thr Asn Ser Thr Leu 965 970 975Val Thr Thr Asn Val
Thr Trp Gly Ile Gln Pro Ala Pro Met Pro Val 980 985 990Pro Val Trp
Val Ile Ile Leu Ala Val Leu Ala Gly Leu Leu Leu Leu 995 1000
1005Ala Val Leu Val Phe Val Met Tyr Arg Met Gly Phe Phe Lys Arg Val
1010 1015 1020Arg Pro Pro Gln Glu Glu Gln Glu Arg Glu Gln Leu Gln
Pro His Glu1025 1030 1035 1040Asn Gly Glu Gly Asn Ser Glu Thr
10451015DNAHomo sapiens 10agatatacta tgcac 151151DNAHomo sapiens
11gttatatcat ttgatggaag caataaatac tacgtagact ccgtgaaggg c
511230DNAHomo sapiens 12gaggcccggg gatcgtatgc ttttgatatc
301342DNAHomo sapiens 13ctctcctgca gggccagtca gagtgttagc agctacttag
cc 421418DNAHomo sapiens 14gatgcatcca acagggcc 181521DNAHomo
sapiens 15cagcagcgta gcaactggcc t 2116788PRTHomo sapiens 16Met Arg
Ala Arg Pro Arg Pro Arg Pro Leu Trp Ala Thr Val Leu Ala1 5 10 15Leu
Gly Ala Leu Ala Gly Val Gly Val Gly Gly Pro Asn Ile Cys Thr 20 25
30Thr Arg Gly Val Ser Ser Cys Gln Gln Cys Leu Ala Val Ser Pro Met
35 40 45Cys Ala Trp Cys Ser Asp Glu Ala Leu Pro Leu Gly Ser Pro Arg
Cys 50 55 60Asp Leu Lys Glu Asn Leu Leu Lys Asp Asn Cys Ala Pro Glu
Ser Ile65 70 75 80Glu Phe Pro Val Ser Glu Ala Arg Val Leu Glu Asp
Arg Pro Leu Ser 85 90 95Asp Lys Gly Ser Gly Asp Ser Ser Gln Val Thr
Gln Val Ser Pro Gln 100 105 110Arg Ile Ala Leu Arg Leu Arg Pro Asp
Asp Ser Lys Asn Phe Ser Ile 115 120 125Gln Val Arg Gln Val Glu Asp
Tyr Pro Val Asp Ile Tyr Tyr Leu Met 130 135 140Asp Leu Ser Tyr Ser
Met Lys Asp Asp Leu Trp Ser Ile Gln Asn Leu145 150 155 160Gly Thr
Lys Leu Ala Thr Gln Met Arg Lys Leu Thr Ser Asn Leu Arg 165 170
175Ile Gly Phe Gly Ala Phe Val Asp Lys Pro Val Ser Pro Tyr Met Tyr
180 185 190Ile Ser Pro Pro Glu Ala Leu Glu Asn Pro Cys Tyr Asp Met
Lys Thr 195 200 205Thr Cys Leu Pro Met Phe Gly Tyr Lys His Val Leu
Thr Leu Thr Asp 210 215 220Gln Val Thr Arg Phe Asn Glu Glu Val Lys
Lys Gln Ser Val Ser Arg225 230 235 240Asn Arg Asp Ala Pro Glu Gly
Gly Phe Asp Ala Ile Met Gln Ala Thr 245 250 255Val Cys Asp Glu Lys
Ile Gly Trp Arg Asn Asp Ala Ser His Leu Leu 260 265 270Val Phe Thr
Thr Asp Ala Lys Thr His Ile Ala Leu Asp Gly Arg Leu 275 280 285Ala
Gly Ile Val Gln Pro Asn Asp Gly Gln Cys His Val Gly Ser Asp 290 295
300Asn His Tyr Ser Ala Ser Thr Thr Met Asp Tyr Pro Ser Leu Gly
Leu305 310 315 320Met Thr Glu Lys Leu Ser Gln Lys Asn Ile Asn Leu
Ile Phe Ala Val 325 330 335Thr Glu Asn Val Val Asn Leu Tyr Gln Asn
Tyr Ser Glu Leu Ile Pro 340 345 350Gly Thr Thr Val Gly Val Leu Ser
Met Asp Ser Ser Asn Val Leu Gln 355 360 365Leu Ile Val Asp Ala Tyr
Gly Lys Ile Arg Ser Lys Val Glu Leu Glu 370 375 380Val Arg Asp Leu
Pro Glu Glu Leu Ser Leu Ser Phe Asn Ala Thr Cys385 390 395 400Leu
Asn Asn Glu Val Ile Pro Gly Leu Lys Ser Cys Met Gly Leu Lys 405 410
415Ile Gly Asp Thr Val Ser Phe Ser Ile Glu Ala Lys Val Arg Gly Cys
420 425 430Pro Gln Glu Lys Glu Lys Ser Phe Thr Ile Lys Pro Val Gly
Phe Lys 435 440 445Asp Ser Leu Ile Val Gln Val Thr Phe Asp Cys Asp
Cys Ala Cys Gln 450 455 460Ala Gln Ala Glu Pro Asn Ser His Arg Cys
Asn Asn Gly Asn Gly Thr465 470 475 480Phe Glu Cys Gly Val Cys Arg
Cys Gly Pro Gly Trp Leu Gly Ser Gln 485 490 495Cys Glu Cys Ser Glu
Glu Asp Tyr Arg Pro Ser Gln Gln Asp Glu Cys 500 505 510Ser Pro Arg
Glu Gly Gln Pro Val Cys Ser Gln Arg Gly Glu Cys Leu 515 520 525Cys
Gly Gln Cys Val Cys His Ser Ser Asp Phe Gly Lys Ile Thr Gly 530 535
540Lys Tyr Cys Glu Cys Asp Asp Phe Ser Cys Val Arg Tyr Lys Gly
Glu545 550 555 560Met Cys Ser Gly His Gly Gln Cys Ser Cys Gly Asp
Cys Leu Cys Asp 565 570 575Ser Asp Trp Thr Gly Tyr Tyr Cys Asn Cys
Thr Thr Arg Thr Asp Thr 580 585 590Cys Met Ser Ser Asn Gly Leu Leu
Cys Ser Gly Arg Gly Lys Cys Glu 595 600 605Cys Gly Ser Cys Val Cys
Ile Gln Pro Gly Ser Tyr Gly Asp Thr Cys 610 615 620Glu Lys Cys Pro
Thr Cys Pro Asp Ala Cys Thr Phe Lys Lys Glu Cys625 630 635 640Val
Glu Cys Lys Lys Phe Asp Arg Glu Pro Tyr Met Thr Glu Asn Thr 645 650
655Cys Asn Arg Tyr Cys Arg Asp Glu Ile Glu Ser Val Lys Glu Leu Lys
660 665 670Asp Thr Gly Lys Asp Ala Val Asn Cys Thr Tyr Lys Asn Glu
Asp Asp 675 680 685Cys Val Val Arg Phe Gln Tyr Tyr Glu Asp Ser Ser
Gly Lys Ser Ile 690 695 700Leu Tyr Val Val Glu Glu Pro Glu Cys Pro
Lys Gly Pro Asp Ile Leu705 710 715 720Val Val Leu Leu Ser Val Met
Gly Ala Ile Leu Leu Ile Gly Leu Ala 725 730 735Ala Leu Leu Ile Trp
Lys Leu Leu Ile Thr Ile His Asp Arg Lys Glu 740 745 750Phe Ala Lys
Phe Glu Glu Glu Arg Ala Arg Ala Lys Trp Asp Thr Ala 755 760 765Asn
Asn Pro Leu Tyr Lys Glu Ala Thr Ser Thr Phe Thr Asn Ile Thr 770 775
780Tyr Arg Gly Thr78517799PRTHomo sapiens 17Met Pro Arg Ala Pro Ala
Pro Leu Tyr Ala Cys Leu Leu Gly Leu Cys1 5 10 15Ala Leu Leu Pro Arg
Leu Ala Gly Leu Asn Ile Cys Thr Ser Gly Ser 20 25 30Ala Thr Ser Cys
Glu Glu Cys Leu Leu Ile His Pro Lys Cys Ala Trp 35 40 45Cys Ser Lys
Glu Asp Phe Gly Ser Pro Arg Ser Ile Thr Ser Arg Cys 50 55 60Asp Leu
Arg Ala Asn Leu Val Lys Asn Gly Cys Gly Gly Glu Ile Glu65 70 75
80Ser Pro Ala Ser Ser Phe His Val Leu Arg Ser Leu Pro Leu Ser Ser
85 90 95Lys Gly Ser Gly Ser Ala Gly Trp Asp Val Ile Gln Met Thr Pro
Gln 100 105 110Glu Ile Ala Val Asn Leu Arg Pro Gly Asp Lys Thr Thr
Phe Gln Leu 115 120 125Gln Val Arg Gln Val Glu Asp Tyr Pro Val Asp
Leu Tyr Tyr Leu Met 130 135 140Asp Leu Ser Leu Ser Met Lys Asp Asp
Leu Asp Asn Ile Arg Ser Leu145 150 155 160Gly Thr Lys Leu Ala Glu
Glu Met Arg Lys Leu Thr Ser Asn Phe Arg 165 170 175Leu Gly Phe Gly
Ser Phe Val Asp Lys Asp Ile Ser Pro Phe Ser Tyr 180 185 190Thr Ala
Pro Arg Tyr Gln Thr Asn Pro Cys Ile Gly Tyr Lys Leu Phe 195 200
205Pro Asn Cys Val Pro Ser Phe Gly Phe Arg His Leu Leu Pro Leu Thr
210 215 220Asp Arg Val Asp Ser Phe Asn Glu Glu Val Arg Lys Gln Arg
Val Ser225 230 235 240Arg Asn Arg Asp Ala Pro Glu Gly Gly Phe Asp
Ala Val Leu Gln Ala 245
250 255Ala Val Cys Lys Glu Lys Ile Gly Trp Arg Lys Asp Ala Leu His
Leu 260 265 270Leu Val Phe Thr Thr Asp Asp Val Pro His Ile Ala Leu
Asp Gly Lys 275 280 285Leu Gly Gly Leu Val Gln Pro His Asp Gly Gln
Cys His Leu Asn Glu 290 295 300Ala Asn Glu Tyr Thr Ala Ser Asn Gln
Met Asp Tyr Pro Ser Leu Ala305 310 315 320Leu Leu Gly Glu Lys Leu
Ala Glu Asn Asn Ile Asn Leu Ile Phe Ala 325 330 335Val Thr Lys Asn
His Tyr Met Leu Tyr Lys Asn Phe Thr Ala Leu Ile 340 345 350Pro Gly
Thr Thr Val Glu Ile Leu Asp Gly Asp Ser Lys Asn Ile Ile 355 360
365Gln Leu Ile Ile Asn Ala Tyr Asn Ser Ile Arg Ser Lys Val Glu Leu
370 375 380Ser Val Trp Asp Gln Pro Glu Asp Leu Asn Leu Phe Phe Thr
Ala Thr385 390 395 400Cys Gln Asp Gly Val Ser Tyr Pro Gly Gln Arg
Lys Cys Glu Gly Leu 405 410 415Lys Ile Gly Asp Thr Ala Ser Phe Glu
Val Ser Leu Glu Ala Arg Ser 420 425 430Cys Pro Ser Arg His Thr Glu
His Val Phe Ala Leu Arg Pro Val Gly 435 440 445Phe Arg Asp Ser Leu
Glu Val Gly Val Thr Tyr Asn Cys Thr Cys Gly 450 455 460Cys Ser Val
Gly Leu Glu Pro Asn Ser Ala Arg Cys Asn Gly Ser Gly465 470 475
480Thr Tyr Val Cys Gly Leu Cys Glu Cys Ser Pro Gly Tyr Leu Gly Thr
485 490 495Arg Cys Glu Cys Gln Asp Gly Glu Asn Gln Ser Val Tyr Gln
Asn Leu 500 505 510Cys Arg Glu Ala Glu Gly Lys Pro Leu Cys Ser Gly
Arg Gly Asp Cys 515 520 525Ser Cys Asn Gln Cys Ser Cys Phe Glu Ser
Glu Phe Gly Lys Ile Tyr 530 535 540Gly Pro Phe Cys Glu Cys Asp Asn
Phe Ser Cys Ala Arg Asn Lys Gly545 550 555 560Val Leu Cys Ser Gly
His Gly Glu Cys His Cys Gly Glu Cys Lys Cys 565 570 575His Ala Gly
Tyr Ile Gly Asp Asn Cys Asn Cys Ser Thr Asp Ile Ser 580 585 590Thr
Cys Arg Gly Arg Asp Gly Gln Ile Cys Ser Glu Arg Gly His Cys 595 600
605Leu Cys Gly Gln Cys Gln Cys Thr Glu Pro Gly Ala Phe Gly Glu Met
610 615 620Cys Glu Lys Cys Pro Thr Cys Pro Asp Ala Cys Ser Thr Lys
Arg Asp625 630 635 640Cys Val Glu Cys Pro Leu Leu His Ser Gly Lys
Pro Asp Asn Gln Thr 645 650 655Cys His Ser Leu Cys Arg Asp Glu Val
Ile Thr Trp Val Asp Thr Ile 660 665 670Val Lys Asp Asp Gln Glu Ala
Val Leu Cys Phe Tyr Lys Thr Ala Lys 675 680 685Asp Cys Val Met Met
Phe Thr Tyr Val Glu Leu Pro Ser Gly Lys Ser 690 695 700Asn Leu Thr
Val Leu Arg Glu Pro Glu Cys Gly Asn Thr Pro Asn Ala705 710 715
720Met Thr Ile Leu Leu Ala Val Val Gly Ser Ile Leu Leu Val Gly Leu
725 730 735Ala Leu Leu Ala Ile Trp Lys Leu Leu Val Thr Ile His Asp
Arg Arg 740 745 750Glu Phe Ala Lys Phe Gln Ser Glu Arg Ser Arg Ala
Arg Tyr Glu Met 755 760 765Ala Ser Asn Pro Leu Tyr Arg Lys Pro Ile
Ser Thr His Thr Val Asp 770 775 780Phe Thr Phe Asn Lys Phe Asn Lys
Ser Tyr Asn Gly Thr Val Asp785 790 795
* * * * *
References