U.S. patent application number 13/641199 was filed with the patent office on 2014-06-12 for anti-vla-4 antibodies.
This patent application is currently assigned to BIOGEN IDEC MA INC.. The applicant listed for this patent is Alexey Lugovskoy, Karen Retta McLachlan, Frederick R. Taylor. Invention is credited to Alexey Lugovskoy, Karen Retta McLachlan, Frederick R. Taylor.
Application Number | 20140161794 13/641199 |
Document ID | / |
Family ID | 44628095 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140161794 |
Kind Code |
A1 |
Lugovskoy; Alexey ; et
al. |
June 12, 2014 |
ANTI-VLA-4 ANTIBODIES
Abstract
This invention relates to alpha-4 binding antibodies, and
fragments thereof.
Inventors: |
Lugovskoy; Alexey; (Woburn,
MA) ; Taylor; Frederick R.; (Milton, MA) ;
McLachlan; Karen Retta; (Encinitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lugovskoy; Alexey
Taylor; Frederick R.
McLachlan; Karen Retta |
Woburn
Milton
Encinitas |
MA
MA
CA |
US
US
US |
|
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
44628095 |
Appl. No.: |
13/641199 |
Filed: |
April 15, 2011 |
PCT Filed: |
April 15, 2011 |
PCT NO: |
PCT/US11/32641 |
371 Date: |
November 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61324944 |
Apr 16, 2010 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/69.6; 530/387.3 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 35/02 20180101; A61K 39/39558 20130101; A61P 1/04 20180101;
C07K 2317/92 20130101; A61P 25/02 20180101; C07K 16/2842 20130101;
A61P 11/14 20180101; A61P 11/06 20180101; A61P 27/02 20180101; A61P
19/02 20180101; A61P 29/00 20180101; A61P 35/00 20180101; C07K
16/3061 20130101; C07K 2317/76 20130101; C07K 2317/567 20130101;
A61P 3/10 20180101; C07K 16/2839 20130101; A61P 43/00 20180101;
C07K 2317/24 20130101; A61P 25/00 20180101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 435/320.1; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/30 20060101
C07K016/30 |
Claims
1. A recombinant antibody molecule, or an .alpha.4-binding fragment
thereof, comprising a variable light chain framework comprising an
acceptor sequence from IGKV4-1 and a variable heavy chain framework
comprising an acceptor sequence from IGHV1-f and having heavy and
light chain CDRs derived from murine antibody HP1/2 or from murine
antibody 21.6.
2. A recombinant antibody molecule, or an .alpha.4-binding fragment
thereof, capable of binding .alpha.4, comprising a variable light
chain framework comprising an acceptor sequence from germline
engineered AAH70335.1 (SEQ ID NO:15) and a variable heavy chain
framework comprising an acceptor sequence from IGHV1-f.
3. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 1, further comprising CDRs derived from murine
antibody HP1/2.
4. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 1, further comprising CDRs derived from murine
antibody 21.6.
5. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 1, wherein the heavy chain variable region
comprises the sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID
NO:5.
6. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 1, wherein the light chain variable region
comprises the sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID
NO:10.
7. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 2, wherein the light chain variable region
comprises the sequence of SEQ ID NO:11.
8. The antibody of claim 1, wherein the antibody binds VLA-4.
9. A vector comprising DNA encoding an antibody heavy chain, or an
.alpha.4-binding fragment thereof, comprising the sequences of SEQ
ID NOs:3, 4, or 5.
10. A vector comprising DNA encoding an antibody light chain, or an
.alpha.4-binding fragment thereof, comprising SEQ ID NOs:8, 9, 10,
or 11.
11. A method of making a recombinant anti-.alpha.4 antibody, or an
.alpha.4-binding fragment thereof, comprising (a) providing a host
cell comprising (i) a DNA sequence encoding an antibody heavy
chain, or an .alpha.4-binding fragment thereof, wherein the DNA
sequence comprises the sequence of SEQ ID NOs:3, 4, or 5, and (ii)
a DNA sequence encoding an antibody light chain, or an
.alpha.4-binding fragment thereof, wherein the DNA sequence
comprises the sequence of SEQ ID NOs:8, 9, 10, or 11, and (b)
culturing the cell to produce the recombinant anti-.alpha.4
antibody molecule or .alpha.4 binding fragment thereof.
12. A method of treating a patient comprising administering to said
patient the composition of claim 1.
13. The method of claim 12, wherein the patient has a cancer.
14. The method of claim 13, wherein the patient has a solid tumor,
a hematological malignancy, a multiple myeloma or acute myelogenous
leukemia (AML).
15. (canceled)
16. (canceled)
17. The method of claim 12, wherein the patient has an inflammatory
disorder, multiple sclerosis, asthma, rheumatoid arthritis,
diabetes, optic neuritis, Crohn's disease, an acute disorder, a
spinal cord injury or traumatic brain injury.
18. (canceled)
19. (canceled)
20. (canceled)
21. The method of claim 12, wherein the composition is administered
as a regimen.
22. The method of claim 12, further comprising administering to the
patient a second therapeutic agent.
23. The method of claim 22, wherein the second therapeutic agent is
a thrombolytic agent, a chemotherapeutic agent, a neuroprotective
agent, an anti-inflammatory agent, a steroid, a cytokine, or a
growth factor.
24. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 2, further comprising CDRs derived from murine
antibody HP1/2.
25. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 2, further comprising CDRs derived from murine
antibody 21.6.
26. The recombinant antibody molecule, or .alpha.4-binding fragment
thereof, of claim 2, wherein the heavy chain variable region
comprises the sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID
NO:5.
27. The antibody of claim 2, wherein the antibody binds VLA-4.
28. A method of treating a patient comprising administering to said
patient the composition of claim 2.
29. The method of claim 28, wherein the patient has a cancer.
30. The method of claim 29, wherein the patient has a solid tumor,
a hematological malignancy, a multiple myeloma or acute myelogenous
leukemia (AML).
31. The method of claim 28, wherein the patient has an inflammatory
disorder, multiple sclerosis, asthma, rheumatoid arthritis,
diabetes, optic neuritis, Crohn's disease, an acute disorder, a
spinal cord injury or traumatic brain injury.
32. The method of claim 28, wherein the composition is administered
as a regimen.
33. The method of claim 28, further comprising administering to the
patient a second therapeutic agent.
34. The method of claim 33, wherein the second therapeutic agent is
a thrombolytic agent, a chemotherapeutic agent, a neuroprotective
agent, an anti-inflammatory agent, a steroid, a cytokine, or a
growth factor.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/324,944, filed Apr. 16, 2010, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to alpha-4 binding antibodies, and
fragments thereof.
BACKGROUND OF INVENTION
[0003] Humanized antibodies can be used as therapeutic agents in
place of murine antibodies to avoid the undesirable immune response
in humans termed the HAMA (Human Anti-Mouse Antibody) response.
Humanized antibodies are generally constructed by replacing the
complementary determining regions (CDRs) of a human antibody with
the CDRs of another species, typically a mouse antibody.
[0004] VLA-4 (also called .alpha.4.beta.1) is a member of the
.beta.1 integrin family of cell surface receptors. VLA-4 contains
an .alpha.4 chain and a .beta.1 chain and is involved in cell-cell
interactions. Its expression is mainly restricted to lymphoid and
myeloid cells. VLA-4 binds the endothelial cell ligand VCAM-1
(Vascular Cell Adhesion Molecule-1), and can mediate T and B
lymphocyte attachment to the heparin II binding fragment of human
plasma fibronectin.
SUMMARY OF INVENTION
[0005] The inventors have discovered that germline variable region
frameworks can be used to optimize CDR-grafted alpha-4 binding
antibodies, such as anti-VLA-4 antibodies. Accordingly, the
invention features anti-VLA-4 variable heavy (VH) and variable
light (VL) chains and antibody molecules including such
frameworks.
[0006] In one aspect, the invention features an anti-.alpha.4
antibody VH chain having CDRs from a donor anti-.alpha.4 antibody,
e.g., an anti-.alpha.4 antibody described herein, and a VH
framework having regions 1, 2, 3, and 4 from the sequence of, or
having no more than 5, 10 or 15 differences from a germline
variable region sequence for the VH chain. In one embodiment,
variable framework region 4 (FR4) is a human consensus sequence. In
one embodiment, the complete VH chain framework regions FR1, FR2,
FR3 and FR4, are present. In another embodiment, the chain is an
antigen-binding fragment of a VH region.
[0007] In one embodiment, the germline sequence is human IGHV1-f
(SEQ ID NO:2), depicted in FIG. 1. In certain embodiments, the VH
framework sequence can differ by at least one, but by no more than
2, 3, 4, 5, 10 or 15 amino acid residues from a germline sequence,
e.g., SEQ ID NO:2. In one embodiment, the VH framework further
includes other than the corresponding human residues. For example,
the VH chain includes non-human residues, at one or more of
framework positions 24, 67, 76, 80, and 94 (Kabat numbering) of SEQ
ID NO:2.
[0008] In one embodiment, at least one or more of the complementary
determining regions (CDRs) of the variable domains are derived from
a donor non-human .alpha.4-binding antibody. In one embodiment, the
antigen binding regions of the CDR-grafted heavy chain variable
domain include the CDRs corresponding to positions 26-34 (CDR1),
50-65 (CDR2) and 95-102 (CDR3) (Kabat numbering; Kabat et al.,
Sequences of Proteins of Immunological Interest, 5.sup.th ed., vol.
4, 1991, U.S. Department of Health and Human Services, NIH,
USA).
[0009] Thus, in one embodiment, the variable heavy chain (VH)
framework has an acceptor sequence derived from human antibody
germline sequence IGHV1-f.
[0010] In another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, or 10 amino acid residues, in the FR1 region of
the VH is other than the corresponding human germline residue. One
or more of such residues can, for example, be identical to the
nonhuman antibody framework region from which the CDR sequences are
derived. In one embodiment, the amino acid residue at Kabat
position 24 is mutated to be identical to the nonhuman antibody
framework region.
[0011] In another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, or 10 amino acid residues, in the FR2 region of
the VH is other than the corresponding human germline residue. One
or more of such residues can, for example, be identical to the
nonhuman antibody framework region from which the CDR sequences are
derived.
[0012] In yet another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, or 10 amino acid residues, in FR3 of the VH chain
is other than the corresponding human germline residue. One or more
of such residues can, for example, be identical to the nonhuman
antibody framework region from which the CDR sequences are derived.
In one embodiment, the amino acid residue at Kabat position 94 is
identical to the nonhuman antibody framework region. In yet another
embodiment, the amino acid residues at Kabat positions 67, 76, 80,
and 94 are identical to the nonhuman antibody framework region.
[0013] In certain embodiments, the VH chain of the antibody has the
sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
[0014] In one aspect, the invention features, an anti-VLA-4 VL
chain having CDRs from a donor anti-VLA-4 antibody, e.g., an
anti-VLA-4 antibody described herein, and a VL framework having
regions 1, 2, 3, and 4 from the sequence of, or having no more than
5, 10 or 15 differences (either per/region or in total) from, a
germline variable region sequence for the VL chain. In one
embodiment, variable framework region 4 (FR4) is a human consensus
sequence. In one embodiment, the complete VL chain framework
regions FR1, FR2, FR3 and FR4, are present. In another embodiment,
the chain is an antigen-binding fragment of a VL region.
[0015] In another embodiment, the germline sequence is IGKV4-1 (SEQ
ID NO:7), depicted in FIG. 2. In yet other embodiments, the VL
framework sequence can differ by at least one, but no more than 2,
3, 4, 5, 10 or 15 amino acid residues from a germline framework
sequence, e.g., SEQ ID NO:7. In another embodiment, the VL further
includes other than the corresponding human amino acid residues.
For example, the VL chain further includes non-human residues at
one or more of framework positions 1, 73, and 87 (Kabat numbering)
of SEQ ID NO:7.
[0016] In one embodiment, the sequence is AAH7035.1 (SEQ ID NO:12)
or its germline engineered version (SEQ ID NO:13), depicted in FIG.
2. In some embodiments, the VL framework sequence can differ by at
least one, but not more than 5, 10, 15, 20, or 25 amino acid
residues from a germline engineered framework sequence, e.g., SEQ
ID NO:13. In one embodiment, the VL chain includes other than the
corresponding human residues. For example, the VL chain includes
non-human residues at one or more of framework positions 1 and 87
(Kabat numbering) of SEQ ID NO:12. In another embodiment, the VL
includes amino acid substitutions in the framework regions to
resemble a different human germline framework sequence, such as
from germline sequence IGKV4-1. In certain embodiments, the VL
framework sequence is altered to be identical to the IGKV4-1
germline sequence at positions 1-3, 5-23, 35-37, 39-42, 45-49, 57,
59-61, 63-64, 70-72, 74-84, 86-88, 99-106 (Kabat numbering) of SEQ
ID NO:12.
[0017] In one embodiment, at least one or more of the complementary
determining regions (CDRs) of the variable domains are derived from
a donor non-human .alpha.4-binding antibody. In another embodiment,
the antigen binding regions of the CDR-grafted heavy chain variable
domain include the CDRs corresponding to positions 24-31 (CDR1),
50-56 (CDR2) and 89-97 (CDR3) (Kabat numbering). Thus, in one
embodiment, the VL framework has an acceptor sequence constructed
from IGKV4-1 germline sequence, from antibody AAH70335.1 or from
germline engineered antibody AAH70335.1.
[0018] In yet another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, 10, or 15 residues, in FR1 of the VL chain is
other than the corresponding human residue. One or more of such
residues can, for example, be identical to the nonhuman antibody
framework region from which the CDR sequences are derived. In one
embodiment, the amino acid residue at the N-terminal position of
FR1 is mutated to be identical to the nonhuman antibody framework
region.
[0019] In another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, 10, or 15 residues, in FR2 of the VL chain is
other than the corresponding human residue. One or more of such
residues can, for example, be identical to the nonhuman antibody
framework region from which the CDR sequences are derived.
[0020] In yet another embodiment, at least one amino acid, and less
than 2, 3, 4, 5, 10, or 15 residues, in FR3 of the VL is other than
the corresponding human residue. One or more of such residues can,
for example, be identical to the nonhuman antibody framework region
from which the CDR sequences are derived. In another embodiment,
the amino acid residue at Kabat position 87 is mutated to be
identical to the nonhuman antibody framework region. In yet another
embodiment, the amino acid residues at Kabat positions 67 and 87
are mutated to be identical to the nonhuman antibody framework
sequence. In yet another embodiment, the amino acid residues at
Kabat positions 67, 73, and 87 of SEQ ID NO:7 are mutated to be
identical to the nonhuman antibody framework sequence.
[0021] In other embodiments, the VL chain of the antibody has the
sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID
NO:11.
[0022] In one embodiment, the CDRs of the VH and VL acceptor
framework sequences are selected to resemble the CDR sequences of a
nonhuman (e.g., murine) antibody sequence, where the nonhuman
antibody binds integrin alpha-4 or a fragment thereof. In another
embodiment, the sequences of the CDRs are selected to resemble the
sequences of the CDRs of a non-human antibody that binds the B1
epitope of the VLA-4 .alpha.4 chain. In one embodiment, the CDRs
are selected to resemble a murine monoclonal antibody, e.g., HP1/2,
HP2/1, HP2/4, L25, P4C2, or 21.6 (Pulido et al., J. Biol. Chem.
266:10241-10245, 1991; U.S. Pat. No. 6,033,665). Modification can
mean, e.g., excision and insertion or alteration, e.g., by directed
mutagenesis.
[0023] In another aspect, the invention features an antibody, or
antigen binding fragment thereof, including: [0024] an anti-VLA-4
VL chain described herein, e.g., an anti-VLA-4 VL chain having
CDR's from a donor anti-VLA-4 antibody, e.g., an anti-VLA-4
antibody described herein, and a VL framework having LC framework
regions 1, 2 and 3 from the sequence of, or having no more than 5,
10, or 15 differences from, a germline variable region sequence for
the VL chain. In one embodiment, variable region 4 is a human
consensus sequence; and [0025] an anti-VLA-4 VH chain described
herein, e.g., an anti-VLA-4 VL chain having CDRs from a donor
anti-VLA-4 antibody, e.g., an anti-VLA-4 antibody described herein,
and a VL framework having LC framework regions 1, 2 and 3 from the
sequence of, or having no more than 5, 10 or 15 differences from, a
germline variable region sequence for the VL chain. In one
embodiment, variable region 4 is a human consensus sequence.
[0026] In one embodiment, the antibody binds one or both of
.alpha.4.beta.1 and .alpha.4.beta.7.
[0027] In another aspect, a VL or VH chain, or antibody, or
fragment thereof, described herein is detectably labeled.
[0028] In yet another aspect, the invention features a vector
containing DNA encoding an antibody heavy chain, or an .alpha.4
binding fragment thereof, described herein. In some embodiments,
the DNA of the vector encodes a VH having the sequence of SEQ ID
NO:3, SEQ ID NO:4, or SEQ ID NO:5.
[0029] In yet another aspect, the invention features a vector
containing DNA encoding an antibody light chain, or an .alpha.4
binding fragment thereof, described herein. In some embodiments,
the DNA of the vector encodes a VL chain having the sequence of SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
[0030] In yet another aspect, the invention features a vector
containing DNA encoding an antibody heavy chain, or an .alpha.4
binding fragment thereof, described herein and an antibody light
chain, or an .alpha.4 binding fragment thereof, described
herein.
[0031] In another aspect, the invention features a host cell
containing a vector described herein, e.g., one capable of
expressing a heavy and/or light chain antibody or antibody fragment
described herein.
[0032] In one aspect, the invention features a method of making a
recombinant anti-.alpha.4 antibody, or an .alpha.4-binding fragment
thereof, by providing a host cell transfected with (a) a DNA
sequence encoding an antibody heavy chain described herein, or an
.alpha.4-binding fragment thereof, and (b) a DNA sequence encoding
an antibody light chain, or an .alpha.4-binding fragment thereof,
and culturing the transfected cell to produce the recombinant
anti-.alpha.4 antibody molecule or .alpha.4 binding fragment
thereof. The DNA encoding the antibody heavy and light chains can
be produced on the same vector or on different vectors.
[0033] In one aspect, the invention features a method of making a
recombinant anti-.alpha.4 antibody, or an .alpha.4-binding fragment
thereof, by providing a host cell transfected with (a) a DNA
sequence encoding an antibody heavy chain, or an .alpha.4-binding
fragment thereof, e.g., where the DNA sequence has the sequence of
SEQ ID NOs:3, 4, or 5, and (b) a DNA sequence encoding an antibody
light chain, or an .alpha.4-binding fragment thereof, e.g., wherein
the DNA sequence has the sequence of SEQ ID NOs: 8, 9, 10, or 11,
and culturing the transfected cell line to produce the recombinant
anti-.alpha.4 antibody molecule or .alpha.4 binding fragment
thereof. The DNA encoding the antibody heavy and light chains can
be produced on the same vector or on different vectors.
[0034] In another aspect, the invention features a method of
treating a disease or disorder mediated by an .alpha.4 integrin,
e.g., an .alpha.4.beta.1 (VLA-4) or .alpha.4.beta.7 integrin, by
administering an .alpha.4 antibody or antibody fragment described
herein, or a pharmaceutical composition containing the antibody or
fragment, to a subject in need of such treatment. The subject can
have or be at risk for developing, for example, inflammatory,
immune, or autoimmune disorders (e.g., inflammation of the central
nervous system, such as multiple sclerosis, meningitis,
neuromyelitis optica, neurosarcoidosis, CNS vasculitis,
encephalitis, and transverse myelitis), tissue or organ graft
rejection or graft-versus-host disease, acute CNS injury, such as
stroke, traumatic brain injury (TBI), or spinal cord injury (SCI);
chronic renal disease; allergy, e.g., allergic asthma; type 1
diabetes mellitus; inflammatory bowel disorders, such as Crohn's
disease, ulcerative colitis; myasthenia gravis; fibromyalgia;
arthritic disorders, such as rheumatoid arthritis, psoriatic
arthritis; inflammatory/immune skin disorders, such as psoriasis,
vitiligo, dermatitis, lichen planus; systemic lupus erythematosus;
Sjogren's Syndrome; hematological cancers, such as multiple
myeloma, leukemia, lymphoma; solid cancers, such as sarcomas or
carcinomas, e.g., of the lung, breast, prostate, brain; and
fibrotic disorders, such as pulmonary fibrosis, myelofibrosis,
liver cirrhosis, mesangial proliferative glomerulonephritis,
crescentic glomerulonephritis, diabetic nephropathy, and renal
interstitial fibrosis.
[0035] In another aspect, the invention features a method of
treating a patient by administering to the patient an
.alpha.4-binding antibody or antibody fragment. In one embodiment,
the patient has a cancer, such as a solid tumor or a hematological
malignancy. For example, a patient treated with an .alpha.4-binding
antibody or antibody fragment can have acute myelogenous leukemia
(AML) or multiple myeloma (MM).
[0036] In another embodiment, the patient has an inflammatory
disorder, such as multiple sclerosis, asthma (e.g., moderate to
severe asthma), rheumatoid arthritis, diabetes, or Crohn's disease.
In another embodiment, the composition is administered as a
regimen. In yet another embodiment, the method further includes
selecting a patient suitable for treatment with the composition. A
patient suitable for treatment, for example, has demonstrated a
sign or symptom indicative of disease onset, such as a sign or
symptom indicative of MS.
[0037] In yet another embodiment, the method further includes
administering to the patient a second therapeutic agent, such as, a
chemotherapeutic agent, a thrombolytic agent, a neuroprotective
agent, an anti-inflammatory agent, a steroid, a cytokine, or a
growth factor.
[0038] In one embodiment, the patient is administered a humanized
anti-VLA-4 antibody, or fragment thereof, described herein, such as
HuHP1/2, H1L1, H1L2 or H1L3.
[0039] In one embodiment, the composition containing an
.alpha.4-binding antibody is administered as a regimen, such at
regular intervals. For example, the composition can be administered
once daily, weekly or monthly; once per week, twice per week, three
times per week, four times per week or more; or once every two
weeks, once every three weeks, once every four weeks or more.
[0040] In one embodiment, dosing can be adjusted according to a
patient's rate of clearance of a prior administration of
anti-.alpha.4 antibody. For example, in one embodiment, a patient
will not be administered a second or follow-on dose before the
level of anti-.alpha.4 antibodies in the patient's system has
dropped below a pre-determined level. In one embodiment, a sample
from a patient (e.g., plasma, serum, blood or urine sample) is
assayed for the presence of anti-.alpha.4 antibodies, and if the
level of anti-.alpha.4 antibodies is above a pre-determined level,
the patient will not be administered a second or follow-on dose. If
the level of anti-.alpha.4 antibodies in the patient's system is
below a pre-determined level, then the patient is administered a
second or follow-on dose.
[0041] In one embodiment, the composition is administered
continuously, e.g., over a period of more than 30 minutes but less
than 1, 2, 4, or 12 hours. The composition containing the antibody
and the second agent can be administered by any appropriate method,
e.g., subcutaneously, intramuscularly, or intravenously.
[0042] In some embodiments, each of the antibody and the second
agent is administered at the same dose as each is prescribed for
monotherapy. In other embodiments, the antibody is administered at
a dosage that is equal to or less than an amount required for
efficacy if administered alone. Likewise, the second agent can be
administered at a dosage that is equal to or less than an amount
required for efficacy if administered alone.
[0043] Another aspect featured in the disclosure is a method of
evaluating a patient by determining if the patient meets a
preselected criterion, and if the patient meets the preselected
criterion approving, providing, prescribing, or administering a
VLA-4 binding antibody formulation described herein to the patient.
In one embodiment, the preselected criterion is the failure of the
patient to adequately respond to a prior alternate therapeutic
treatment or regimen, e.g., for treatment of MS. In another
embodiment, the preselected criterion is the absence of any signs
or symptoms of progressive multifocal leukoencephalopathy (PML), or
the absence of any diagnosis of PML. In some cases, the selection
is based on the absence of a risk factor for PML, for example, the
subject does not test positive for JC virus DNA or does not test
positive for JC virus antibodies. In another embodiment, the
criterion is as described in PCT/US07/75577 (published as
WO2008/021954), hereby incorporated by reference, which describes
methods and systems for drug distribution and for providing drugs
to patients.
[0044] In another aspect, a method of distributing a composition
described herein is provided. The composition contains an alpha-4
binding antibody. The method includes providing a recipient (e.g.,
an end user, patient, physician, retail or wholesale pharmacy,
distributor, or pharmacy department at a hospital, nursing home
clinic or HMO) with a package containing sufficient unit dosages of
the drug to treat a patient for at least 6, 12, 24, 36, or 48
months. In another aspect, the invention features a method of
evaluating the quality of a package or lot of packages (e.g., to
determine if it has expired) of a composition described herein
containing an alpha-4 binding antibody. The method includes
evaluating whether the package has expired. The expiration date is
at least 6, 12, 24, 36, or 48 months, e.g., greater than 24 or 36
months, from a preselected event, such as manufacturing, assaying,
or packaging. In some embodiments, a decision or step is taken as a
result of the analysis. For example, depending on the right
analysis, the antibody in the package is used or discarded,
classified, selected, released or withheld, shipped, moved to a new
location, released into commerce, sold, or offered for sale,
withdrawn from commerce or no longer offered for sale, depending on
whether the product has expired.
[0045] In another aspect, the invention features a package
containing at least two unit doses of an aqueous composition
containing an .alpha.4 binding antibody. In one embodiment, all of
the unit doses contain the same amount of antibody, and in other
embodiments there are unit dosages of two or more strengths, or two
or more different formulations, e.g., having different strengths or
release properties.
[0046] In another aspect, the invention includes a method of
instructing a recipient on the administration of a formulation
containing .alpha.4 binding antibody. The method includes
instructing the recipient (e.g., an end user, patient, physician,
retail or wholesale pharmacy, distributor, or pharmacy department
at a hospital, nursing home clinic or HMO) that the antibody should
be administered to a patient according to a regimen described
herein. The method can also include instructing the recipient that
the antibody should be administered prior to the expiration date.
The expiration date is at least 6, 12, 24, 36, or 48 months, e.g.,
greater than 24 or 36 months, from a preselected event, such as
manufacturing, assaying, or packaging. In one embodiment, the
recipient also receives a supply of the antibody, e.g., a supply of
unit dosages of the antibody.
[0047] In another aspect, the invention features a method of making
an antibody which includes CDRs from a donor antibody, such as a
non-human, e.g., a murine antibody, and one or both heavy and light
chain variable region frameworks derived from human germline
variable region framework region or regions. The method includes
one or both of 1 and 2, where 1 and 2 are as follows:
[0048] 1. identifying or selecting a stable human acceptor heavy
chain variable framework which has the same residues as the
non-human donor heavy chain at one or more of the residues in one
or more of a), b) and c):
[0049] a) VH Kabat #2, 4, 24, 26, 27, 29, 36, 38, 46, 47, 48, 49,
66, 67, 69, 71, 78, 93, and 94, which, without being bound by
theory, are believed to be important for maintaining CDR
conformations;
[0050] b) VH Kabat #1, 2, 27, 28, 30, 43, 66, 68, 70, 72, 73, 74,
and 75 which, without being bound by theory, are believed to be
able to interact with antigen; and
[0051] c) VH Kabat #37, 39, 44, 45, 47, 91, 93 and 103, which,
without being bound by theory, are believed to be important for
VH/VL interface integrity; and
[0052] 2. identifying or selecting a stable acceptor light chain
variable framework which has the same residues as the donor light
chain at one or more of the residues in one or more of a), b) and
c):
[0053] a) VL Kabat #2, 4, 38, 43, 44, 48, 58, 64, 71, and 73, which
without being bound by theory, are believed to be important for
maintaining CDR conformations;
[0054] b) VL Kabat #1, 2, 49, 57, 60, 63, 65, 66, 67, 68, 69, and
70 which without being bound by theory, are believed to potentially
be able to interact with antigen; and
[0055] c) VL Kabat #36, 38, 43, 44, 46, 49, 87, and 98, which
without being bound by theory, are believed to be important for
VH/VL interface integrity;
[0056] 3. providing a variable region having donor CDRs and the
selected germline framework having matched residues identified in 1
or 2, such as by selecting a germline sequence and further
backmutating additional residues identified in 1 or 2 of the
germline to murine sequence so as to further maximize matching at
the residues identified in 1 and 2; and
[0057] 4. evaluating each matched position, such as by 3D
structural analysis or modeling, and if a position meets a
predetermined standard for risk of, for example, interfering with
CDR conformations, antigen interactions or VH/VL interface
integrity, then reintroducing an equivalent murine residue, or a
common human antibody residue, compatible with antibody
structure.
[0058] In one embodiment, at least 3, 4 or 5 of the residues
identified in (1.a) are matched. For example, in one embodiment,
residues 24, 29, or 94 are matched.
[0059] In one embodiment, at least 3, 4 or 5 of the residues
identified in (1.b) are matched. For example, in one embodiment,
residues 1, 73, or 75 are matched.
[0060] In one embodiment, at least 3, 4 or 5 of the residues
identified in (1.c) are matched. For example, in one embodiment,
residues 37, 93, or 103 are matched.
[0061] In one embodiment, at least 3, 4 or 5 of the residues
identified in (2.a) are matched. For example, in one embodiment,
residues 2, 71 and 73 are matched.
[0062] In one embodiment, at least 3, 4 or 5 of the residues
identified in (2.b) are matched. For example, in one embodiment,
residues 1, 68, or 70 are matched.
[0063] In one embodiment, at least 3, 4 or 5 of the residues
identified in (2.c) are matched. For example, in one embodiment,
residues 46, 87, or 98 are matched.
[0064] In one embodiment, residue 6 in (1.a), residue 2 in (1.b),
and residue 4 in (1.c) are matched.
[0065] In another embodiment, residue 4 in (2.a), residue 2 in
(2.b), and residue 4 in (2.c) are matched.
[0066] In one embodiment, the heavy chain germline sequence is of
VH3, VH1 and VH5 germline class. In another embodiment, the light
chain germline sequence is a Vkappa or Vlambda sequence.
[0067] The term "treating" refers to administering a therapy in an
amount, manner, and/or mode effective to improve a condition,
symptom, or parameter associated with a disorder or to prevent
progression of a disorder, to either a statistically significant
degree or to a degree detectable to one skilled in the art. An
effective amount, manner, or mode can vary depending on the subject
and may be tailored to the subject.
[0068] An ".alpha.4 binding antibody" refers to an antibody that
binds to the .alpha.4 subunit of the VLA-4 (.alpha.4.beta.1)
integrin, and at least partially inhibits an activity of VLA-4,
particularly a binding activity of a VLA-4 integrin or a signaling
activity, e.g., ability to transduce a VLA-4 mediated signal. For
example, a VLA-4 binding antibody may inhibit binding of VLA-4 to a
cognate ligand of VLA-4, e.g., a cell surface protein such as
VCAM-1 (Vascular Cell Adhesion Molecule-1), or to an extracellular
matrix component, such as fibronectin or osteopontin. An alpha-4
binding antibody may bind to both .alpha.4.beta.1 or
.alpha.4.beta.7. Typically, the antibody binds to the B1 epitope of
.alpha.4. An .alpha.4 binding antibody may bind to VLA-4 with a
K.sub.d of less than about 10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9, 10.sup.-10, or 10.sup.-11 M.
[0069] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable region, e.g., an
amino acid sequence that provides an immunoglobulin variable domain
or immunoglobulin variable domain sequence. For example, an
antibody can include a heavy (H) chain variable region (abbreviated
herein as VH), and a light (L) chain variable region (abbreviated
herein as VL). In another example, an antibody includes two heavy
(H) chain variable regions and two light (L) chain variable
regions. The light chains of the immunoglobulin may be of types
kappa or lambda. In one embodiment, the antibody is glycosylated.
An antibody can be functional for antibody dependent cytotoxicity
and/or complement-mediated cytotoxicity, or may be non-functional
for one or both of these activities.
[0070] The VH and VL regions can be further subdivided into regions
of hypervariability, termed "complementarity determining regions"
("CDR"), interspersed with regions that are more conserved, termed
"framework regions" (FR). The extent of the FRs and CDRs has been
precisely defined (see, Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242; and
Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabat
definitions are used herein. Each VH and VL is typically composed
of three CDRs and four FRs, arranged from amino-terminus to
carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4.
[0071] An "immunoglobulin domain" refers to a domain from the
variable or constant domain of immunoglobulin molecules.
Immunoglobulin domains typically contain two .beta.-sheets formed
of about seven .beta.-strands, and a conserved disulphide bond
(see, e.g., A. F. Williams and A. N. Barclay (1988) Ann Rev.
Immunol. 6:381-405).
[0072] As used herein, an "immunoglobulin variable domain sequence"
refers to an amino acid sequence that can form the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
omit one, two or more N- or C-terminal amino acids, internal amino
acids, may include one or more insertions or additional terminal
amino acids, or may include other alterations. In one embodiment, a
polypeptide that includes an immunoglobulin variable domain
sequence can associate with another immunoglobulin variable domain
sequence to form a target binding structure (or "antigen binding
site"), e.g., a structure that interacts with VLA-4.
[0073] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region, to thereby form
a heavy or light immunoglobulin chain, respectively. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains. The heavy and light
immunoglobulin chains can be connected by disulfide bonds. The
heavy chain constant region typically includes three constant
domains, CH1, CH2 and CH3. The light chain constant region
typically includes a CL domain. The variable region of the heavy
and light chains contains a binding domain that interacts with an
antigen. The constant regions of the antibodies typically mediate
the binding of the antibody to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system.
[0074] The term "immunoglobulin" comprises various broad classes of
polypeptides that can be distinguished biochemically. Those skilled
in the art will appreciate that heavy chains are classified as
gamma, mu, alpha, delta, or epsilon (.gamma., .mu., .alpha.,
.delta., .epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgD, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1,
IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known
to confer functional specialization. Modified versions of each of
these classes and isotypes are readily discernable to the skilled
artisan in view of the instant disclosure and, accordingly, are
within the scope of the instant invention. All immunoglobulin
classes are clearly within the scope of the present invention.
Light chains are classified as either kappa or lambda (.kappa.,
.lamda.). Each heavy chain class may be bound with either a kappa
or lambda light chain.
[0075] The term "antigen-binding fragment" of a full length
antibody refers to one or more fragments of a full-length antibody
that retain the ability to specifically bind to a target of
interest, e.g., VLA-4. Examples of binding fragments encompassed
within the term "antigen-binding fragment" of a full length
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment including two Fab fragments linked by
a disulfide bridge at the hinge region; (iii) an Fd fragment
consisting of the VH and CH1 domains; (iv) an 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) that retains functionality. 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. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883.
[0076] In some embodiments, the above-described antibodies are
pegylated.
[0077] In some embodiments, the above-described antibodies or
fragments thereof are multispecific. In further embodiments, the
above-described antibodies or fragments thereof are monovalent or
bispecific.
[0078] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF DRAWINGS
[0079] FIG. 1 displays the three sequence variants of HP1/2 heavy
chain to a human heavy germline IGHV1-f. The lower case letters
above the sequence represent insertions according to the Kabat
numbering scheme.
[0080] FIG. 2 displays the four sequence variants of HP1/2 light
chain to a germline IGKV4-1 antibody sequence (Design L0, L1, and
L2) or human kappa germline engineered AAH7033.1 antibody sequence
(Design L3). The lower case letters above the sequence represent
insertions according to the Kabat numbering scheme.
[0081] FIG. 3 is a graph depicting the results of ELISA assays.
[0082] FIG. 4 is a graph depicting the results of ELISA assays.
[0083] FIG. 5 is the amino acid sequence of an IgG4 Fc
(hinge+CH2+CH3 domain). The hinge region is depicted in bold, and
the CH3 domain is underlined. The boxed "S" is Ser228. The circled
"N" is Asn297.
[0084] FIG. 6 is a graph depicting flow cytometry data from binding
of HuHP1/2 to various tumor cell lines. "HP1/2" refers to humanized
HP1/2.
[0085] FIGS. 7A-7C is a panel of graphs depicting inhibition of
binding of AML cell lines to fibronectin or VCAM1-Ig coated wells
by HuHP1/2. FIG. 7A depicts inhibition of binding of HL60 and KG1
cells to FN-coated wells. FIG. 7B depicts inhibition of binding of
KG1 cells to VCAM1-Ig-coated wells. FIG. 7C depicts inhibition of
binding of HL60 cells to FN- and VCAM1-Ig-coated wells when
incubated with 20 .mu.g/mL HuHP1/2 (Solid bars). Clear bars
indicate percent cell adhesion in the presence of an isotype
control. "HP1/2" refers to humanized HP1/2.
[0086] FIGS. 8A-8C make up a panel of graphs depicting inhibition
of binding of MM cell lines to fibronectin or VCAM1-Ig coated wells
by HuHP1/2. FIG. 8A depicts inhibition of binding of U266 and H929
cells to FN-coated wells. FIG. 8B depicts inhibition of binding of
U266 and H929 cells to VCAM1-Ig-coated wells. FIG. 8C depicts
inhibition of binding of U266 cells to FN- and VCAM1-Ig-coated
wells when incubated with 20 .mu.g/mL HuHP1/2 (Solid bars). Clear
bars indicate percent cell adhesion in the presence of an isotype
control. "HP1/2" refers to humanized HP1/2.
[0087] FIGS. 9A-9C makes up a panel of graphs depicting inhibition
of binding of CLL cell lines to fibronectin or VCAM1-Ig coated
wells by HuHP1/2. FIG. 9A depicts inhibition of binding of Mec1 and
JM1 cells to FN-coated wells. FIG. 9B depicts inhibition of binding
of Mec1 and JM1 cells to VCAM1-Ig-coated wells. FIG. 9C depicts
inhibition of binding of Mec1 cells to FN- and VCAM1-Ig-coated
wells when incubated with 20 .mu.g/mL HuHP1/2 (Solid bars). Clear
bars indicate percent cell adhesion in the presence of an isotype
control. "HP1/2" refers to humanized HP1/2.
DETAILED DESCRIPTION
[0088] Antibodies against VLA-4 have been demonstrated to be useful
in treating disease. For example, natalizumab (Tysabri.RTM.), an
anti-VLA-4 antibody is used for treating relapsing multiple
sclerosis and Crohn's disease. However, for treatment of certain
conditions, for example acute conditions such as spinal cord injury
(SCI) or traumatic brain injury (TBI), or treatments that are
administered in a finite number such as treatment of cancer, it may
be advantageous to treat with an anti-VLA-4 antibody that binds
with an affinity different than natalizumab, e.g., a higher
affinity. In addition, treatment with anti-VLA-4 antibodies is
associated with a rare but sometimes fatal disorder, progressive
multifocal leukoencephalopathy (PML), for which a part of the
treatment requires removal of antibody from the treated subject,
for example using plasma exchange or immunoabsorption. Because of
the need to remove antibody, it is also desirable to balance the
advantages of an antibody that has increased affinity for VLA-4
with the disadvantage of an antibody that binds so tightly as to
make removal difficult or to create a risk associated with a slow
turnover rate. Such antibodies may also be useful for treating
conditions such as multiple sclerosis in that less frequent
treatment may be required or administration by means other than
infusion may be more efficient. Enabling treatment with lower doses
may also lower the risk of adverse events such as PML. Accordingly,
the present invention provides antibodies having such desirable
properties.
[0089] The invention is based at least in part on the unexpected
characteristics of newly designed humanized .alpha.4-binding
antibodies that have a binding affinity for .alpha.4 that is
10-fold higher than that of the anti-.alpha.4 antibody
natalizumab.
[0090] Alpha-4 binding antibodies, and fragments thereof, are
provided where the variable light chain (VL) and variable heavy
chain (VH) frameworks have acceptor sequences constructed from
germline or germline engineered antibody sequences, such as IGKV4-1
or geAAH70335.1 or IGHV1-f antibodies. The CDR sequences are
derived from nonhuman anti-.alpha.4 binding antibodies such as the
anti-VLA-4 antibody HP1/2. Antibodies described herein can have an
increase of at least 1.5, 2.0, 2.5, 3.0 fold in affinity, e.g.,
relative to its murine parent. In one embodiment, the increase in
affinity is at least 1.5, 2.0, 2.5, 3.0 fold but is respectively,
less than 25, 20, or 15 fold.
[0091] Pharmaceutical Compositions
[0092] An .alpha.4 binding agent, such as a VLA-4 binding antibody,
can be formulated as a pharmaceutical composition. Typically, a
pharmaceutical composition includes a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically
compatible.
[0093] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0094] The antibody compositions described herein can be formulated
according to methods known in the art. Pharmaceutical formulation
is a well-established art, and is further described in Gennaro
(ed.), Remington: The Science and Practice of Pharmacy, 20.sup.th
ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472);
Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery
Systems, 7.sup.th Ed., Lippincott Williams & Wilkins Publishers
(1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of
Pharmaceutical Excipients American Pharmaceutical Association,
3.sup.rd ed. (2000) (ISBN: 091733096X).
[0095] In one embodiment, the .alpha.4 antibody can be formulated
with excipient materials, such as sodium chloride, sodium dibasic
phosphate heptahydrate, sodium monobasic phosphate, and polysorbate
80. In another embodiment, the .alpha.4 antibody can be formulated
in a citrate buffer, e.g., at pH 5, 5.5, 6, 6.5, 7, or 7.5. In yet
another embodiment, the .alpha.4 antibody can be formulated in a
solution including 2, 4, 5, 6, 8, 10, 12, 14, or 15% sucrose. It
can be provided, for example, in a buffered solution at a
concentration of about 20 mg/ml and can be stored at 2-8.degree.
C.
[0096] Pharmaceutical compositions may also be in a variety of
other forms. These include, for example, liquid, semi-solid and
solid dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The form can depend on the
intended mode of administration and therapeutic application.
Typically, compositions for the agents described herein are in the
form of injectable or infusible solutions.
[0097] Such compositions can be administered by a parenteral mode
(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular
injection). The phrases "parenteral administration" and
"administered parenterally" as used herein mean modes of
administration other than enteral and topical administration,
usually by injection, and include, without limitation, intravenous,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion.
[0098] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. A
pharmaceutical composition can also be tested to insure it meets
regulatory and industry standards for administration.
[0099] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration. Sterile injectable solutions
can be prepared by incorporating an agent described herein in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
an agent described herein into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, typical methods of
preparation are vacuum drying and freeze-drying that yields a
powder of an agent described herein plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0100] Administration
[0101] An .alpha.4 binding antibody can be administered to a
subject, e.g., a human subject, by a variety of methods. For many
applications, the route of administration is one of: intravenous
injection or infusion, subcutaneous injection, or intramuscular
injection. An .alpha.4 binding antibody can be administered as a
fixed dose, or in a mg/kg dose. The antibody can be administered
intravenously (IV) or subcutaneously (SC). For example, the
antibody can be administered at a fixed unit dose of between about
50-600 mg IV, e.g., every 4 weeks, or between about 50-100 mg SC
(e.g., 75 mg), e.g., at least once a week (e.g., twice a week). In
one embodiment, the antibody is administered IV at a fixed unit
dose of 50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 130 mg, 140 mg, 150
mg, 160 mg, 180 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg or
more. Administration of the IV dose can be once or twice or three
times or more per week, or once every two, three, four, or five
weeks, or less frequently.
[0102] In one embodiment, the antibody is administered SC at a
fixed unit dose of 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 100 mg, or
120 mg or more. Administration of the SC dose can be once or twice
or three times or more per week, or once every two, three, four, or
five weeks, or less frequently.
[0103] An anti-.alpha.4 antibody can also be administered in a
bolus at a dose of between about 1 and 10 mg/kg, e.g., about 6.0
mg/kg, 4.0 mg/kg, 3.0 mg/kg, 2.0 mg/kg, 1.0 mg/kg. Modified dose
ranges include a dose that is less than about 600 mg/subject, about
400 mg/subject, about 300 mg/subject, about 250 mg/subject, about
200 mg/subject, or about 150 mg/subject, typically for
administration every fourth week or once a month. The .alpha.4
binding antibody can be administered, for example, every three to
five weeks, e.g., every fourth week, or monthly.
[0104] Dosing can be adjusted according to a patient's rate of
clearance of a prior administration of anti-.alpha.4 antibody. For
example, a patient may not be administered a second or follow-on
dose before the level of anti-.alpha.4 antibodies in the patient's
system has dropped below a pre-determined level. In one embodiment,
a sample from a patient (e.g., plasma, serum, blood, urine, or
cerebrospinal fluid (CSF)) is assayed for the presence of
anti-.alpha.4 antibodies, and if the level of anti-.alpha.4
antibodies is above a pre-determined level, the patient will not be
administered a second or follow-on dose. If the level of
anti-.alpha.4 antibodies in the patient's system is below a
pre-determined level, then the patient is administered a second or
follow-on dose. A patient whose anti-.alpha.4 levels are determined
to be too high (above the pre-determined level) can be tested again
after one or two or three days, or a week, and if the level of
anti-.alpha.4-antibody in the patient samples has dropped below the
pre-determined level, the patient may be administered a second or
follow-on dose of antibody.
[0105] The dose can also be chosen to reduce or avoid production of
antibodies against the .alpha.4 binding antibody, to achieve
greater than 40, 50, 70, 75, or 80% saturation of the .alpha.4
subunit, to achieve less than 80, 70, 60, 50, or 40% saturation of
the .alpha.4 subunit, or to prevent an increase in the level of
circulating white blood cells
[0106] In certain embodiments, the active agent may be prepared
with a carrier that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known. See, e.g., Controlled
Drug Delivery (Drugs and the Pharmaceutical Sciences), Second
Edition, J. Robinson and V. H. L. Lee, eds., Marcel Dekker, Inc.,
New York, 1987.
[0107] Pharmaceutical compositions can be administered with a
medical device. For example, pharmaceutical compositions can be
administered with a needleless hypodermic injection device, such as
the devices disclosed in U.S. Pat. No. 5,399,163; 5,383,851;
5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples
of well-known implants and modules are discussed in, e.g., U.S.
Pat. No. 4,487,603, which discloses an implantable micro-infusion
pump for dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicaments through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system. Of
course, many other such implants, delivery systems, and modules are
also known.
[0108] This disclosure also features a device for administering a
first and second agent.
[0109] The device can include, for example, one or more housings
for storing pharmaceutical preparations, and can be configured to
deliver unit doses of the first and second agent. The first and
second agents can be stored in the same or separate compartments.
For example, the device can combine the agents prior to
administration. It is also possible to use different devices to
administer the first and second agent.
[0110] Dosage regimens are adjusted to provide the desired
response, such as a therapeutic response or a combinatorial
therapeutic effect. Generally, any combination of doses (either
separate or co-formulated) of the VLA-4 binding agent and the
second agent can be used in order to provide a subject with both
agents in bioavailable quantities.
[0111] Dosage unit form or "fixed dose" as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier and
optionally in association with the other agent.
[0112] A pharmaceutical composition may include a "therapeutically
effective amount" of an agent described herein. Such effective
amounts can be determined based on the combinatorial effect of the
administered first and second agent. A therapeutically effective
amount of an agent may also vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the compound to elicit a desired response in the
individual, such as amelioration of at least one disorder
parameter, e.g., a multiple sclerosis parameter, or amelioration of
at least one symptom of the disorder, e.g., a symptom of multiple
sclerosis, such as muscle atrophy, ataxia, and tremors. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the composition are outweighed by the
therapeutically beneficial effects.
[0113] Devices and Kits
[0114] Formulations containing an antibody described herein can be
administered with a medical device. The device can be designed with
features such as portability, room temperature storage, and ease of
use so that it can be used in emergency situations, such as by an
untrained subject or by emergency personnel in the field, removed
to medical facilities and other medical equipment. The device can
include, for example, one or more housings for storing
pharmaceutical preparations that include an .alpha.4-binding
antibody, and can be configured to deliver one or more unit doses
of the agent.
[0115] For example, the pharmaceutical composition can be
administered with a transcutaneous delivery device, such as a
syringe, including a hypodermic or multichamber syringe. Other
suitable delivery devices include stents, catheters, microneedles,
and implantable controlled release devices. The composition can be
administered intravenously with standard IV equipment, including,
e.g., IV tubings, with or without in-line filters. In certain
embodiments, the device will be a syringe for use in SC or IM
administration.
[0116] Pharmaceutical compositions can be administered with medical
devices. For example, pharmaceutical compositions can be
administered with a needleless hypodermic injection device, such as
the devices disclosed in U.S. Pat. No. 5,399,163; 5,383,851;
5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples
of well-known implants and modules are described in, e.g., U.S.
Pat. No. 4,487,603, which discloses an implantable micro-infusion
pump for dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system. The
therapeutic composition can also be in the form of a biodegradable
or nonbiodegradable sustained release formulation for subcutaneous
or intramuscular administration. Methods for such compositions are
known in the art. Continuous administration can also be achieved
using an implantable or external pump. The administration can also
be conducted intermittently, such as by single daily injection, or
continuously at a low dose, such as in a sustained release
formulation. The delivery device can be modified to be optimally
suited for administration of an .alpha.4-binding antibody. For
example, a syringe can be siliconized to an extent that is optimal
for storage and delivery of the antibody. Of course, many other
such implants, delivery systems, and modules are also known.
[0117] This disclosure also features a device for administering a
first and second agent (e.g., an antibody and a second agent). The
device can include, for example, one or more housings for storing
pharmaceutical preparations, and can be configured to deliver unit
doses of the first and second agent. The first and second agents
can be stored in the same or separate compartments. In one
embodiment, the device combines the agents prior to administration.
In some embodiments, the first and second agents are administered
by different devices.
[0118] An .alpha.4-binding antibody can be provided in a kit. In
one embodiment, the kit includes (a) a container that contains a
composition that includes a high concentration of VLA-4-binding
antibody, optionally (b) a container that contains a composition
that includes a second agent, and optionally (c) informational
material. The informational material can be descriptive,
instructional, marketing or other material that relates to the
methods described herein and/or the use of the agents for
therapeutic benefit. In one embodiment, the kit also includes a
second agent. For example, the kit includes a first container that
contains a composition that includes the .alpha.4-binding antibody,
and a second container that includes the second agent.
[0119] The informational material of the kits is not limited in its
form. In one embodiment, the informational material can include
information about production of the antibody, concentration, date
of expiration, batch or production site information, and so forth.
In one embodiment, the informational material relates to methods of
administering the .alpha.4-binding antibody, e.g., in a suitable
dose, dosage form, or mode of administration (e.g., a dose, dosage
form, or mode of administration described herein), to treat a
subject who has an acute disorder such as a spinal cord injury or
traumatic brain injury, or an inflammatory disease (e.g., MS), or
who is at risk for experiencing an episode associated with an
inflammatory disease. The information can be provided in a variety
of formats, including printed text, computer readable material,
video recording, or audio recording, or information that provides a
link or address to substantive material.
[0120] In addition to the agent, the composition in the kit can
include other ingredients, such as a solvent or buffer, a
stabilizer, or a preservative. The agent can be provided in any
form, e.g., liquid, dried or lyophilized form, and substantially
pure and/or sterile. When the agents are provided in a liquid
solution, the liquid solution typically is an aqueous solution.
When the agents are provided as a dried form, reconstitution
generally is by the addition of a suitable solvent. The solvent,
e.g., sterile water or buffer, can optionally be provided in the
kit.
[0121] The kit can include one or more containers for the
composition or compositions containing the agents. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle, vial, or
syringe, and the informational material can be contained in a
plastic sleeve or packet. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of the agents. The containers can include a
combination unit dosage, e.g., a unit that includes both the
.alpha.4 binding antibody and the second agent, such as in a
desired ratio. For example, the kit can include a plurality of
syringes, ampoules, foil packets, blister packs, or medical devices
each containing, for example, a single combination unit dose. The
containers of the kits can be air tight, waterproof (e.g.,
impermeable to changes in moisture or evaporation), and/or
light-tight.
[0122] The kit optionally includes a device suitable for
administering the composition, e.g., a syringe or other suitable
delivery device. The device can be provided pre-loaded with one or
both of the agents or can be empty but suitable for loading.
[0123] Oncology
[0124] The .alpha.4-binding antibodies and methods described herein
can be used to treat cancer, including solid cancers and
hematological malignancies. Exemplary solid cancers include
sarcomas and carcinomas, such as of the lung, breast, pancreas,
colon, prostate, bladder and brain. Hemotological malignancies
include cancers such as multiple myeloma, leukemia, and
lymphoma.
[0125] Methods are provided for treating a patient having a
hematological malignancy with a composition containing an
.alpha.4-binding antibody, such as anti-VLA-4 antibody described
herein. Hematological malignancies are cancers of the body's
blood-forming and immune systems. Cancers of this type affect the
blood, bone marrow, and/or lymph nodes. Hematological malignancies
include leukemias, such as acute lymphoblastic leukemia (ALL),
acute myelogenous leukemia (AML), chronic myelogenous leukemia
(CML), chronic lymphocytic leukemia (CLL), acute promyelocytic
leukemia, acute erythroleukemia, and hairy cell leukemia (HCL);
lymphomas, such as Hodgkin's disease and Non-Hodgkin's lymphoma;
and multiple myeloma; Waldenstrom's macroglobulinemia;
myelodysplastic syndrome (MDS) (which can culminate in AML); a
myeloproliferative disease, such as polycythemia vera (also called
PV, PCV or polycythemia rubra vera (PRV)), Essential thrombocytosis
(ET), myelofibrosis, heavy chain disease; and amyloid due to
light-chain disease.
[0126] Patients having a hematological malignancy may be identified
by analysis of blood count and blood film by, for example, light
microscopy, which is useful for identifying malignant cells. A
biopsy, such as from bone marrow, can also be used to identify
malignant cells, and a biopsy from a lymph node can be useful for
identifying a lymphadenopathy.
[0127] An .alpha.4-binding antibody (e.g., a humanized anti-VLA-4
antibody, such as HuHP1/2, H1L0, H1L1, H1L2 or H1L3) is useful for
the treatment of a leukemia, such as AML. Leukemias are cancers
that originate in the bone marrow, where the malignant cells are
white blood cells (leukocytes). AML (also called acute myelocytic
leukemia, acute myeloblastic leukemia, acute granulocytic leukemia,
and acute nonlymphocytic leukemia) is a malignancy that arises in
either granulocytes or monocytes. AML is characterized by the
uncontrolled, exaggerated growth and accumulation of cells called
leukemic blasts, which fail to function as normal blood cells, and
the blockade of the production of normal marrow cells, leading to a
deficiency of red cells (anemia), and platelets (thrombocytopenia)
and normal white cells (especially neutrophils, i.e., neutropenia)
in the blood.
[0128] All subtypes of AML are suitable for treatment with a VLA-4
binding antibody. The subtypes of AML are classified based on the
stage of development myeloblasts have reached at the time of
diagnosis. The categories and subsets allow the physician to decide
what treatment works best for the cell type and how quickly the
disease may develop. The subsets are: M0, myeloblastic, on special
analysis; M1, Myeloblastic, without maturation; M2, Myeloblastic,
with maturation; M3, Promyelocytic; M4, Myelomonocytic; M5,
Monocytic; M6, Erythroleukemia; and M7, Megakaryocytic. A VLA-4
antibody can be administered with a secondary agent that is
particularly suited to the subtype of AML. For example, acute
promyelocytic leukemia (APL) and acute monocytic leukemia are
subtypes of AML that need different treatment than other subtypes
of AML. A second agent for treatment of APL can include all-trans
retinoic acid (ATRA) or an antimetabolite, such as cytarabine. A
second agent for treatment of acute monocytic leukemia can include
a deoxyadenosine analog, such as 2-chloro-2'-deoxyadenosine
(2-CDA).
[0129] Risk factors of AML include the presence of certain genetic
disorders, such as Down syndrome, Fanconi anemia, Shwachman-Diamond
syndrome and others. A patient having AML and a genetic disorder
can be administered a VLA-4 binding antibody and a second agent to
treat a symptom of the genetic disorder. For example, a patient
with AML and Fanconi anemia can be administered a VLA-4 binding
antibody and an antibiotic.
[0130] Other risk factors for AML include chemotherapy or
radiotherapy for treatment of a different cancer, tobacco smoke,
and exposure to large amounts of benzene.
[0131] Other cancers suitable for treatment with an
.alpha.4-binding antibody include, solid tumors such as sarcomas
and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, ovarian cancer, squamous cell carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinomas,
systadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, uterine cancer, testicular cancer, small cell lung
carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma,
meningioma, melanoma, neuroblastoma, and retinoblastoma).
[0132] Other Disorders
[0133] The formulations and methods described herein can also be
used to treat other inflammatory, immune, or autoimmune disorders,
e.g., inflammation of the central nervous system (e.g., in addition
to multiple sclerosis, meningitis, neuromyelitis optica,
neurosarcoidosis, CNS vasculitis, encephalitis, and transverse
myelitis); tissue or organ graft rejection or graft-versus-host
disease; acute CNS injury, e.g., stroke or spinal cord injury
(SCI); chronic renal disease; allergy, e.g., allergic asthma,
moderate to severe allergic rhinitis, ocular allergy; type 1
diabetes mellitus; inflammatory bowel disorders, e.g., Crohn's
disease, ulcerative colitis (e.g., for treatment or maintenance of
remission); eosinophilic gastroenteritis; myasthenia gravis;
fibromyalgia; disorders associated with rheumatology/immunology,
such as arthritic disorders, e.g., rheumatoid arthritis, psoriatic
arthritis; dermatological disorders, such as inflammatory/immune
skin disorders, e.g., psoriasis, vitiligo, dermatitis (e.g., atopic
dermatitis), lichen planus, moderate to severe chronic urticaria;
systemic lupus erythematosus (SLE; e.g., lupus nephritis);
scleroderma (e.g., Progressive Systemic Sclerosis (PSS), such as
PSS of the lung); acute or chronic primary eosinophilic pneumonia;
Sjogren's Syndrome; acute coronary syndrome (ACS); acute myocardial
infarction; atherosclerosis; and fibrotic disorders, e.g.,
pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), lung
fibrosis (e.g., XRT induced), myelofibrosis, liver cirrhosis,
mesangial proliferative glomerulonephritis, crescentic
glomerulonephritis, diabetic nephropathy, and renal interstitial
fibrosis.
[0134] The formulations and methods described herein can also be
used to treat neurological disorders, such as cerebral ischemia,
including prevention in patients with transient ischemic attacks
and/or arterial stenosis. Other exemplary neurological disorders
include chronic inflammatory demyelinating polyneuropathy (CIDP);
Guillian-Barre Syndrome (GBS); ocular diseases, such as macular
degeneration (e.g., wet macular degeneration), and anteriorischemic
optic neuropathy; neuropathic pain (e.g., symptomatic neuropathic
pain); Alzheimer's Disease; Amyotrophic Lateral Sclerosis (ALS)
(e.g., disease modifying ALS)' and Parkinson's Disease.
[0135] The formulations and methods described herein can also be
used to treat patients who have undergone transplantation, such as
renal, heart, or bone marrow transplantation.
[0136] Multiple Sclerosis
[0137] Formulations containing an alpha-4 binding antibody
described herein are useful for the treatment of inflammatory
diseases, such as multiple sclerosis (MS). Multiple sclerosis is a
central nervous system disease that is characterized by
inflammation and loss of myelin sheaths.
[0138] Patients having MS may be identified by criteria
establishing a diagnosis of clinically definite MS as defined by
the workshop on the diagnosis of MS (Poser et al., Ann. Neurol.
13:227, 1983). For example, an individual with clinically definite
MS has had two attacks and clinical evidence of either two lesions
or clinical evidence of one lesion and paraclinical evidence of
another, separate lesion. Definite MS may also be diagnosed by
evidence of two attacks and oligoclonal bands of IgG in
cerebrospinal fluid or by combination of an attack, clinical
evidence of two lesions and oligoclonal band of IgG in
cerebrospinal fluid. The McDonald criteria can also be used to
diagnose MS. (McDonald et al., 2001, "Recommended diagnostic
criteria for multiple sclerosis: guidelines from the International
Panel on the Diagnosis of Multiple Sclerosis," Ann. Neurol.
50:121-127). The McDonald criteria include the use of MRI evidence
of CNS impairment over time to be used in diagnosis of MS, in the
absence of multiple clinical attacks. Effective treatment of
multiple sclerosis may be evaluated in several different ways. The
following parameters can be used to gauge effectiveness of
treatment. Two exemplary criteria include: EDSS (extended
disability status scale), and appearance of exacerbations on MRI
(magnetic resonance imaging). The EDSS is a method for grading
clinical impairment due to MS (Kurtzke, Neurology 33:1444, 1983).
Eight functional systems are evaluated for the type and severity of
neurologic impairment. Briefly, prior to treatment, patients are
evaluated for impairment in the following systems: pyramidal,
cerebella, brainstem, sensory, bowel and bladder, visual, cerebral,
and other. Follow-ups are conducted at defined intervals. The scale
ranges from 0 (normal) to 10 (death due to MS). A decrease of one
full step indicates an effective treatment (Kurtzke, Ann. Neurol.
36:573-79, 1994). Patients may also be diagnosed using other
criteria used by those in the art.
[0139] Exacerbations are defined as the appearance of a new symptom
that is attributable to MS and accompanied by an appropriate new
neurologic abnormality (IFNB MS Study Group, supra). In addition,
the exacerbation must last at least 24 hours and be preceded by
stability or improvement for at least 30 days. Briefly, patients
are given a standard neurological examination by clinicians.
Exacerbations are either mild, moderate, or severe according to
changes in a Neurological Rating Scale (Sipe et al., Neurology
34:1368, 1984). An annual exacerbation rate and proportion of
exacerbation-free patients are determined.
[0140] Therapy can be deemed to be effective if there is a
statistically significant difference in the rate or proportion of
exacerbation-free or relapse-free patients between the treated
group and the placebo group for either of these measurements. In
addition, time to first exacerbation and exacerbation duration and
severity may also be measured. A measure of effectiveness as
therapy in this regard is a statistically significant difference in
the time to first exacerbation or duration and severity in the
treated group compared to control group. An exacerbation-free or
relapse-free period of greater than one year, 18 months, or 20
months is particularly noteworthy. Efficacy may also be assessed
using any method used in the art, for example to assess symptoms of
MS, including mobility improvement using a timed walk test used
alone or in combination with other criteria,
[0141] Efficacy of administering a first agent and, optionally, a
second agent, can also be evaluated based on one or more of the
following criteria: frequency of MBP reactive T cells determined by
limiting dilution, proliferation response of MBP reactive T cell
lines and clones, cytokine profiles of T cell lines and clones to
MBP established from patients. Efficacy is indicated by decrease in
frequency of reactive cells, a reduction in thymidine incorporation
with altered peptide compared to native, and a reduction in TNF and
IFN-.alpha..
[0142] Clinical measurements include the relapse rate in one and
two-year intervals, and a change in EDSS, including time to
progression from baseline of 1.0 unit on the EDSS that persists for
six months. On a Kaplan-Meier curve, a delay in sustained
progression of disability shows efficacy. Other criteria include a
change in area and volume of T2 images on MRI, and the number and
volume of lesions determined by gadolinium enhanced images.
[0143] MRI can be used to measure active lesions using
gadolinium-DTPA-enhanced imaging (McDonald et al. Ann. Neurol.
36:14, 1994) or the location and extent of lesions using
T.sub.2-weighted techniques. Briefly, baseline MRIs are obtained.
The same imaging plane and patient position are used for each
subsequent study. Positioning and imaging sequences can be chosen
to maximize lesion detection and facilitate lesion tracing. The
same positioning and imaging sequences can be used on subsequent
studies. The presence, location and extent of MS lesions can be
determined by radiologists. Areas of lesions can be outlined and
summed slice by slice for total lesion area. Three analyses may be
done: evidence of new lesions, rate of appearance of active
lesions, percentage change in lesion area (Paty et al., Neurology
43:665, 1993). Improvement due to therapy can be established by a
statistically significant improvement in an individual patient
compared to baseline or in a treated group versus a placebo
group.
[0144] Exemplary symptoms associated with multiple sclerosis, which
can be treated with the methods described herein, include: optic
neuritis, diplopia, nystagmus, ocular dysmetria, internuclear
ophthalmoplegia, movement and sound phosphenes, afferent pupillary
defect, paresis, monoparesis, paraparesis, hemiparesis,
quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia,
quadraplegia, spasticity, dysarthria, muscle atrophy, spasms,
cramps, hypotonia, clonus, myoclonus, myokymia, restless leg
syndrome, footdrop, dysfunctional reflexes, paraesthesia,
anaesthesia, neuralgia, neuropathic and neurogenic pain,
l'hermitte's, proprioceptive dysfunction, trigeminal neuralgia,
ataxia, intention tremor, dysmetria, vestibular ataxia, vertigo,
speech ataxia, dystonia, dysdiadochokinesia, frequent micturation,
bladder spasticity, flaccid bladder, detrusor-sphincter
dyssynergia, erectile dysfunction, anorgasmy, frigidity,
constipation, fecal urgency, fecal incontinence, depression,
cognitive dysfunction, dementia, mood swings, emotional lability,
euphoria, bipolar syndrome, anxiety, aphasia, dysphasia, fatigue,
uhthoffs symptom, gastroesophageal reflux, and sleeping
disorders.
[0145] Each case of MS displays one of several patterns of
presentation and subsequent course. Most commonly, MS first
manifests itself as a series of attacks followed by complete or
partial remissions as symptoms mysteriously lessen, only to return
later after a period of stability. This is called
relapsing-remitting (RR) MS. Primary-progressive (PP) MS is
characterized by a gradual clinical decline with no distinct
remissions, although there may be temporary plateaus or minor
relief from symptoms. Secondary-progressive (SP) MS begins with a
relapsing-remitting course followed by a later primary-progressive
course. Rarely, patients may have a progressive-relapsing (PR)
course in which the disease takes a progressive path punctuated by
acute attacks. PP, SP, and PR are sometimes lumped together and
called chronic progressive MS.
[0146] A few patients experience malignant MS, defined as a swift
and relentless decline resulting in significant disability or even
death shortly after disease onset. This decline may be arrested or
decelerated by administration of a combination therapy described
herein.
[0147] Administration of an anti-.alpha.4 antibody featured herein
can be effective to relieve one or more symptoms of MS, such as one
or more of the symptoms described above. For example,
administration of an anti-.alpha.4 antibody described herein can be
used to treat primary or secondary progressive multiple sclerosis
(PPMS or SPMS, respectively), and treatment with an anti-.alpha.4
antibody can be effective to prevent relapse.
[0148] In addition to or prior to human studies, an animal model
can be used to evaluate the efficacy of using the two agents. An
exemplary animal model for multiple sclerosis is the experimental
autoimmune encephalitis (EAE) mouse model, e.g., as described in
(Tuohy et al. (J. Immunol. (1988) 141: 1126-1130), Sobel et al. (J.
Immunol. (1984) 132: 2393-2401), and Traugott (Cell Immunol. (1989)
119: 114-129). Mice can be administered a first and second agent
described herein prior to EAE induction. Then the mice are
evaluated for characteristic criteria to determine the efficacy of
using the two agents in the model.
[0149] Antibody Generation
[0150] Recombinant antibodies that bind to alpha-4 can be generated
by in vivo or in vitro methods such as phage display. The methods
can be used to supply anti-.alpha.4 CDRs for use in CDR grafted
antibodies described herein. In addition, methods such as phage
display can be used to select such CDRs in the context of the
germline frameworks disclosed herein, such as by using a library
where the framework is a germline framework.
[0151] EP 239 400 (Winter et al.) describes altering antibodies by
substitution (within a given variable region) of their
complementarity determining regions (CDRs) for one species with
those from another. CDR-substituted antibodies can be less likely
to elicit an immune response in humans compared to true chimeric
antibodies because the CDR-substituted antibodies contain
considerably less non-human components. (Riechmann et al., 1988,
Nature 332, 323-327; Verhoeyen et al., 1988, Science 239,
1534-1536). Typically, CDRs of a murine antibody substituted into
the corresponding regions in a human antibody by using recombinant
nucleic acid technology to produce sequences encoding the desired
substituted antibody. Human constant region gene segments of the
desired isotype (usually gamma I for CH and kappa for CL) can be
added and the heavy and light chain genes can be co-expressed in
mammalian cells to produce soluble antibody. Large nonimmunized
phage display libraries may also be used to isolate high affinity
antibodies that can be developed as human therapeutics using
standard phage technology (see, e.g., Hoogenboom et al. (1998)
Immunotechnology 4:1-20; and Hoogenboom et al. (2000) Immunol Today
2:371-8; U.S. 2003-0232333).
[0152] An anti-.alpha.4 antibody or antibody fragment described
herein can recognize epitopes of the .alpha.4 subunit that are
involved in binding to a cognate ligand, e.g., VCAM-1 or
fibronectin. The antibodies described herein can inhibit binding of
to one or more of the cognate ligands (e.g., VCAM-1 and
fibronectin).
[0153] In some embodiments, the antibodies featured herein, can
interact with VLA-4 on cells, e.g., lymphocytes, but do not cause
cell aggregation.
[0154] An exemplary .alpha.4 binding antibody has one or more CDRs,
e.g., all three heavy chain (HC) CDRs and/or all three light chain
(LC) CDRs of a particular antibody disclosed herein, or CDRs that
are, in sum, at least 80, 85, 90, 92, 94, 95, 96, 97, 98, 99%
identical to such an antibody. In one embodiment, the H1 and H2
hypervariable loops have the same canonical structure as those of
an antibody described herein. In one embodiment, the L1 and L2
hypervariable loops have the same canonical structure as those of
an antibody described herein.
[0155] In one embodiment, the amino acid sequence of the HC and/or
LC variable domain sequence is at least 70, 80, 85, 90, 92, 95, 97,
98, 99, or 100% identical to the amino acid sequence of the HC
and/or LC variable domain of an antibody described herein. The
amino acid sequence of the HC and/or LC variable domain sequence
can differ by at least one amino acid, but no more than ten, eight,
six, five, four, three, or two amino acids from the corresponding
sequence of an antibody described herein. For example, the
differences may be primarily or entirely in the framework
regions.
[0156] The amino acid sequences of the HC and LC variable domain
sequences can be encoded by a nucleic acid sequence that hybridizes
under high stringency conditions to a nucleic acid sequence
described herein or one that encodes a variable domain or an amino
acid sequence described herein. In one embodiment, the amino acid
sequences of one or more framework regions (e.g., FR1, FR2, FR3,
and/or FR4) of the HC and/or LC variable domain are at least 70,
80, 85, 90, 92, 95, 97, 98, 99, or 100% identical to corresponding
framework regions of the HC and LC variable domains of an antibody
described herein. In one embodiment, one or more heavy or light
chain framework regions (e.g., HC FR1, FR2, and FR3) are at least
70, 80, 85, 90, 95, 96, 97, 98, or 100% identical to the sequence
of corresponding framework regions from a human germline
antibody.
[0157] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) are
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). The optimal alignment is determined as
the best score using the GAP program in the GCG software package
with a Blossum 62 scoring matrix with a gap penalty of 12, a gap
extend penalty of 4, and a frameshift gap penalty of 5. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences.
[0158] As used herein, the term "hybridizes under high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous
and nonaqueous methods are described in that reference and either
can be used. High stringency hybridization conditions include
hybridization in 6.times.SSC at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C., or
substantially similar conditions.
[0159] Antibody Production
[0160] Antibodies can be produced in prokaryotic and eukaryotic
cells. In one embodiment, the antibodies (e.g., scFvs) are
expressed in a yeast cell such as Pichia (see, e.g., Powers et al.
(2001) J. Immunol. Methods 251:123-35), Hanseula, or
Saccharomyces.
[0161] In one embodiment, antibodies, particularly full length
antibodies, e.g., IgGs, are produced in mammalian cells. Exemplary
mammalian host cells for recombinant expression include Chinese
Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in
Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in Kaufman
and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines,
e.g., NS0 myeloma cells and SP2 cells, COS cells, K562, and a cell
from a transgenic animal, e.g., a transgenic mammal. For example,
the cell is a mammary epithelial cell.
[0162] In addition to the nucleic acid sequence encoding the
immunoglobulin domain, the recombinant expression vectors may carry
additional nucleic acid 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). Exemplary selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0163] In an exemplary system for recombinant expression of an
antibody (e.g., a full length antibody or an antigen-binding
portion thereof), a recombinant expression vector encoding both the
antibody heavy chain and the antibody light chain is introduced
into dhfr- CHO cells by calcium phosphate-mediated transfection.
Within the recombinant expression vector, the antibody heavy and
light chain genes are each operatively linked to enhancer/promoter
regulatory elements (e.g., derived from SV40, CMV, adenovirus and
the like, such as a CMV enhancer/AdMLP promoter regulatory element
or an SV40 enhancer/AdMLP promoter regulatory element) to drive
high levels of transcription of the genes. The recombinant
expression vector also carries a DHFR gene, which allows for
selection of CHO cells that have been transfected with the vector
using methotrexate selection/amplification. The selected
transformant host cells are cultured to allow for expression of the
antibody heavy and light chains and intact antibody is recovered
from the culture medium. Standard molecular biology techniques are
used to prepare the recombinant expression vector, to transfect the
host cells, to select for transformants, to culture the host cells,
and to recover the antibody from the culture medium. For example,
some antibodies can be isolated by affinity chromatography with a
Protein A or Protein G. For example, purified .alpha.4-binding
antibodies can be concentrated to about 100 mg/mL to about 200
mg/mL using protein concentration techniques that are known in the
art.
[0164] Antibodies may also include modifications, e.g.,
modifications that alter Fc function, e.g., to decrease or remove
interaction with an Fc receptor or with C1q, or both. For example,
the human IgG4 constant region can have a Ser to Pro mutation at
residue 228 to fix the hinge region. The amino acid sequence of an
IgG4 Fc (hinge+CH2+CH3 domain) is provided in FIG. 5.
[0165] In another example, the human IgG1 constant region can be
mutated at one or more residues, e.g., one or more of residues 234
and 237, e.g., according to the numbering in U.S. Pat. No.
5,648,260. Other exemplary modifications include those described in
U.S. Pat. No. 5,648,260.
[0166] For some antibodies that include an Fc domain, the antibody
production system may be designed to synthesize antibodies in which
the Fc region is glycosylated. In another example, the Fc domain of
IgG molecules is glycosylated at asparagine 297 in the CH2 domain
(see FIG. 5). This asparagine is the site for modification with
biantennary-type oligosaccharides. This glycosylation participates
in effector functions mediated by Fc.gamma. receptors and
complement C1q (Burton and Woof (1992) Adv. Immunol. 51:1-84;
Jefferis et al. (1998) Immunol. Rev. 163:59-76). The Fc domain can
be produced in a mammalian expression system that appropriately
glycosylates the residue corresponding to asparagine 297. The Fc
domain can also include other eukaryotic post-translational
modifications.
[0167] Other suitable Fc domain modifications include those
described in WO2004/029207. For example, the Fc domain can be an
XmAb.RTM. Fc (Xencor, Monrovia, Calif.). The Fc domain, or a
fragment thereof, can have a substitution in an Fc.gamma. Receptor
(Fc.gamma.R) binding region, such as the domains and fragments
described in WO05/063815. In some embodiments, the Fc domain, or a
fragment thereof, has a substitution in a neonatal Fc Receptor
(FcRn) binding region, such as the domains and fragments described
in WO05047327. In other embodiments, the Fc domain is a single
chain, or fragment thereof, or modified version thereof, such as
those described in WO2008143954. Other suitable Fc modifications
are known and described in the art.
[0168] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method for expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acid sequences encoding the antibody of interest, e.g., an
antibody described herein, and a signal sequence for secretion. The
milk produced by females of such transgenic mammals includes,
secreted therein, the antibody of interest, e.g., an antibody
described herein. The antibody can be purified from the milk, or
for some applications, used directly.
[0169] Antibodies can be modified, e.g., with a moiety that
improves its stabilization and/or retention in circulation, e.g.,
in blood, serum, lymph, bronchioalveolar lavage, or other tissues,
e.g., by at least 1.5, 2, 5, 10, or 50 fold.
[0170] For example, a VLA-4 binding antibody can be associated with
a polymer, e.g., a substantially non-antigenic polymer, such as a
polyalkylene oxide or a polyethylene oxide. Suitable polymers will
vary substantially by weight. Polymers having molecular number
average weights ranging from about 200 to about 35,000 daltons (or
about 1,000 to about 15,000, and 2,000 to about 12,500) can be
used.
[0171] For example, a VLA-4 binding antibody can be conjugated to a
water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.
polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides that comprise the saccharide monomers D-mannose, D-
and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0172] Exemplary Second Agents
[0173] In some cases, the formulations described herein, e.g.,
formulations containing an alpha-4 binding antibody, include a
second agent, or are administered in combination with a formulation
containing a second agent.
[0174] In one implementation, the .alpha.4 binding antibody and
second agent is provided as a co-formulation, and the
co-formulation is administered to the subject. It is further
possible, e.g., at least 24 hours before or after administering the
co-formulation, to administer separately one dose of the .alpha.4
binding antibody formulation and then one dose of a formulation
containing the second agent. In another implementation, the
antibody and the second agent are provided as separate
formulations, and the step of administering includes sequentially
administering the antibody and the second agent. The sequential
administrations can be provided on the same day (e.g., within one
hour of one another or at least 3, 6, or 12 hours apart) or on
different days.
[0175] Generally, the antibody and the second agent are each
administered as a plurality of doses separated in time. The
antibody and the second agent are generally each administered
according to a regimen. The regimen for one or both may have a
regular periodicity. The regimen for the antibody can have a
different periodicity from the regimen for the second agent, e.g.,
one can be administered more frequently than the other. In one
implementation, one of the antibody and the second agent is
administered once weekly and the other once monthly. In another
implementation, one of the antibody and the second agent is
administered continuously, e.g., over a period of more than 30
minutes but less than 1, 2, 4, or 12 hours, and the other is
administered as a bolus. The antibody and the second agent can be
administered by any appropriate method, e.g., subcutaneously,
intramuscularly, or intravenously.
[0176] In some embodiments, each of the antibody and the second
agent is administered at the same dose as each is prescribed for
monotherapy. In other embodiments, the antibody is administered at
a dosage that is equal to or less than an amount required for
efficacy if administered alone. Likewise, the second agent can be
administered at a dosage that is equal to or less than an amount
required for efficacy if administered alone.
[0177] Non-limiting examples of second agents for treating multiple
sclerosis in combination with an .alpha.4 binding antibody include:
[0178] interferons, e.g., human interferon beta-1a (e.g.,
AVONEX.RTM. or Rebif.RTM.)) and interferon beta-1b (BETASERON.TM.;
human interferon beta substituted at position 17; Berlex/Chiron);
[0179] glatiramer acetate (also termed Copolymer 1, Cop-1;
COPAXONE.TM.; Teva Pharmaceutical Industries, Inc.); [0180]
Rituxan.RTM. (rituximab) or another anti-CD20 antibody, e.g., one
that competes with or binds an overlapping epitope with rituximab;
[0181] mitoxantrone (NOVANTRONE.RTM., Lederle); [0182] a
chemotherapeutic, e.g., clabribine (LEUSTATIN.RTM.), azathioprine
(IMURAN.RTM.), cyclophosphamide (CYTOXAN.RTM.), cyclosporine-A,
methotrexate, 4-aminopyridine, and tizanidine; [0183] a
corticosteroid, e.g., methylprednisolone (MEDRONE.RTM., Pfizer),
prednisone; [0184] an immunoglobulin, e.g., Rituxan.RTM.
(rituximab); CTLA4 Ig; alemtuzumab (MabCAMPATH.RTM.) or daclizumab
(an antibody that binds CD25); [0185] statins; and [0186] TNF
antagonists.
[0187] Glatiramer acetate is a protein formed from a random chain
of amino acids--glutamic acid, lysine, alanine and tyrosine (hence
GLATiramer). Glatiramer acetate can be synthesized in solution from
these amino acids at a ratio of approximately 5 parts alanine to 3
parts lysine, 1.5 parts glutamic acid and 1 part tyrosine using
N-carboxyamino acid anhydrides.
[0188] Additional second agents include antibodies or antagonists
of other human cytokines or growth factors, for example, TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 IL-15, IL-16, IL-18, EMAP-11,
GM-CSF, FGF, and PDGF. Still other exemplary second agents include
antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,
CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their
ligands. For example, daclizumab is an anti-CD25 antibody that may
ameliorate multiple sclerosis.
[0189] Still other exemplary antibodies include antibodies that
provide an activity of an agent described herein, such as an
antibody that engages an interferon receptor, e.g., an interferon
beta receptor. Typically, in implementations in which the second
agent includes an antibody, it binds to a target protein other than
VLA-4 or other than .alpha.4 integrin, or at least an epitope on
VLA-4 other than one recognized by the first agent.
[0190] Still other additional exemplary second agents include:
FK506, rapamycin, mycophenolate mofetil, leflunomide, non-steroidal
anti-inflammatory drugs (NSAIDs), for example, phosphodiesterase
inhibitors, adenosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, agents that interfere with signaling
by proinflammatory cytokines as described herein, IL-1.beta.
converting enzyme inhibitors (e.g., Vx740), anti-P7s, PSGL, TACE
inhibitors, T-cell signaling inhibitors such as kinase inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors and derivatives thereof, as described
herein, anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-13 and
TGF).
[0191] In some embodiments, a second agent may be used to treat one
or more symptoms or side effects of MS. Such agents include, e.g.,
amantadine, baclofen, papaverine, meclizine, hydroxyzine,
sulfamethoxazole, ciprofloxacin, docusate, pemoline, dantrolene,
desmopressin, dexamethasone, tolterodine, phenyloin, oxybutynin,
bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam,
isoniazid, vardenafil, nitrofurantoin, psyllium hydrophilic
mucilloid, alprostadil, gabapentin, nortriptyline, paroxetine,
propantheline bromide, modafinil, fluoxetine, phenazopyridine,
methylprednisolone, carbamazepine, imipramine, diazepam,
sildenafil, bupropion, and sertraline. Many second agents that are
small molecules have a molecular weight between 150 and 5000
Daltons.
[0192] Examples of TNF antagonists include chimeric, humanized,
human or in vitro generated antibodies (or antigen-binding
fragments thereof) to TNF (e.g., human TNF .alpha.), such as D2E7,
(human TNF.alpha. antibody, U.S. Pat. No. 6,258,562; BASF),
CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF.alpha. antibody;
Celltech/Pharmacia), cA2 (chimeric anti-TNF.alpha. antibody;
REMICADE.TM., Centocor); anti-TNF antibody fragments (e.g.,
CPD870); soluble fragments of the TNF receptors, e.g., p55 or p75
human TNF receptors or derivatives thereof, e.g., 75 kdTNFR-IgG (75
kD TNF receptor-IgG fusion protein, ENBREL.TM.; Immunex; see e.g.,
Arthritis & Rheumatism (1994) Vol. 37, 5295; J. Invest. Med.
(1996) Vol. 44, 235A), p55 kdTNFR-IgG (55 kD TNF receptor-IgG
fusion protein (LENERCEPT.TM.)); enzyme antagonists, e.g.,
TNF.alpha. converting enzyme (TACE) inhibitors (e.g., an
alpha-sulfonyl hydroxamic acid derivative, WO 01/55112, and
N-hydroxyformamide TACE inhibitor GW 3333, -005, or -022); and
TNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), 5284; Amer. J.
Physiol.--Heart and Circulatory Physiology (1995) Vol. 268, pp.
37-42).
[0193] In addition to a second agent, it is also possible to
deliver other agents to the subject. However, in some embodiments,
no protein or no biologic, other than the .alpha.4 binding antibody
and second agent, are administered to the subject as a
pharmaceutical composition. The .alpha.4 binding antibody and the
second agent may be the only agents that are delivered by
injection. In embodiments in which the second agent is a
recombinant protein, the .alpha.4 binding antibody and second agent
may be the only recombinant agents administered to the subject, or
at least the only recombinant agents that modulate immune or
inflammatory responses. In still other embodiments, the .alpha.4
binding antibody alone is the only recombinant agent or the only
biologic administered to the subject.
[0194] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLE
Example 1
Variant Anti-VLA-4 Antibodies are More Potent than Humanized
HP1/2
[0195] Anti-VLA-4 antibodies were constructed using the germline
framework IGKV4-1 (or design L1 and L2) or germline-engineered
AAH7033.1 (for design L3) for the VL chain and germline framework
IGHV1-f for VH. These antibodies had fewer back mutations than the
humanized HP1/2 antibody described in U.S. Pat. No. 6,602,503.
[0196] Heavy Chain Variations
[0197] The sequences of three variations of the heavy chain are
shown in FIG. 1 as Design H0, Design H1 and Design H2. Each design
has the CDR's of murine HP1/2 grafted into the IGHV1-f framework.
Design H0 includes no back mutations of the framework regions,
while Designs H1 and H2 have various degrees of back mutations in
the framework regions sequences to optimize the affinity of the
humanized antibody.
[0198] Light Chain Variations
[0199] The sequences of four variations of the light chain are
shown in FIG. 2 as Design L0, Design L1, Design L2 and Design L3
(also called L0, L1, L2, L3). Each design has the CDR's of murine
HP1/2 grafted into the germline framework. The IGKV4-1 germline
framework was used for Designs L0, L1, and L2, and the AAH70335
germline engineered framework was used for Design L3. Design L0
includes no back mutations of the framework regions, while Designs
L1, L2, and L3 have various degrees of back mutations in the
framework regions to optimize the affinity of the humanized
antibody.
[0200] The results of competition ELISA assays are shown in Table 1
and FIG. 3. In this experiment .alpha.4.beta.1 was preincubated
with testing mAb and then murine HP1/2 was used as competing
reagent. The results of this experiment indicated that the
antibodies having light chains L2 or L3 were more potent than the
humanized antibody HuHP1/2 described in U.S. Pat. No. 6,602,503.
The results are shown in Table 1 below, and in FIG. 3. The heavy
chain (H1) in the antibodies for this assay had the "Design H1"
sequence shown in FIG. 1, whereas L1 refers to Design L1 in FIG.
2.
TABLE-US-00001 TABLE 1 Competition Assay by ELISA mAb IC50 nM
Chimeric HP1/2 1.06 H1L0 1.87 H1L1 1.67 H1L2 0.9 H1L3 0.49 HuHP1/2
1.05
[0201] In Table 1, the chimeric mAb is chimerized HP1/2 antibody,
where murine variable heavy and light chains are genetically fused
to human IgG1 constant regions. This antibody is essentially
identical in binding affinity to the original murine HP1/2 antibody
(Sanchez-Madrid et al., Eur. J. Immunol. 16:1343-1349, 1996). The
results of the experiment indicate that it is possible to improve
the affinity of the monoclonal antibody relative to its murine
parental sequence through humanization on germline-engineered
acceptor framework.
[0202] Another competition assay compares the binding affinity of
the new antibodies with the humanized 21.6 anti-.alpha.4 antibody
(Tysabri.RTM. (natalizumab)) described in U.S. Pat. No. 5,840,299.
In this experiment the binding of mixture of mouse HP1/2 with
testing mAb to .alpha.4.beta.1 was assayed. The results of this
experiment are shown in FIG. 4 and in Table 2 below, and indicate
that the newly designed antibodies are about 10-fold more potent
than natalizumab.
TABLE-US-00002 TABLE 2 Competition Assay by ELISA mAb IC50 nM
Chimeric HP1/2 1.64 H1L0 4.46 H1L1 4.55 H1L2 1.34 HuHP1/2 1.41
Tysabri .RTM. 10.9
Example 2
Humanized HP1/2 (HuHP1/2) Binds VLA-4 on Tumor Cell Lines
[0203] Binding of anti-VLA-4 antibody HuHP1/2 to a variety of cell
lines was tested by flow cytometry. Binding was tested on CLL
(chronic lymphocytic leukemic) cell lines Mec1 and JM1; on MM
(multiple myeloma) cell lines U266 and H929; and on AML (acute
myelogenous leukemic) cell lines HL60 and KG1. HuHP1/2 bound all
tumor cell lines tested (FIG. 6). The flow cytometry data was used
to calculate the EC50 values for antibody binding to each of the
different cell lines. This information is shown below in Table
3.
[0204] HuHP1/2 was also found to block adhesion of AML cell lines
to fibronectin (FN) and VCAM1-Ig fusion protein. To test whether
the antibody could block adhesion, AML cell lines HL60 or KG1 were
allowed to adhere to FN-coated wells (FIG. 7A) or VCAM1-Ig-coated
wells (FIG. 7B) in the presence of increasing concentrations of
HP1/2 or isotype control antibody. HuHP1/2 blocked adhesion of both
cell types to FN-coated wells and VCAM1-Ig-coated wells. The
maximal inhibition of HL60 cell binding to both ligands was
achieved with 20 .mu.g/ml HuHP1/2 (FIG. 7C).
[0205] HuHP1/2 was also found to block adhesion of MM cell lines to
FN and VCAM1-Ig fusion protein. The MM cell lines U266 and H929
were allowed to adhere to FN-coated wells (FIG. 8A) or
VCAM1-Ig-coated wells (FIG. 8B) in the presence of increasing
concentrations of HP1/2 or isotype control antibody. HuHP1/2
blocked adhesion of both types of cell lines to FN- and
VCAM1-Ig-coated wells. The maximal inhibition of U266 cell binding
to both ligands was achieved with 20 .mu.g/mL HuHP1/2 (FIG.
8C).
[0206] HuHP1/2 was also found to block adhesion of CLL cell lines
to FN and VCAM1-Ig fusion protein. The CLL cell lines Mec1 and JM1
were allowed to adhere to FN-coated wells (FIG. 9A) or
VCAM1-Ig-coated wells (FIG. 9B) in the presence of increasing
concentrations of HP1/2 or isotype control antibody. HuHP1/2
blocked adhesion of both types of cell lines to FN- and
VCAM1-Ig-coated wells. The maximal inhibition of Mec1 cell binding
to both ligands was achieved with 20 .mu.g/ml HuHP1/2 (FIG.
9C).
[0207] The IC50 values for HuHP1/2 binding to the tumor cell lines
were calculated from the data shown in FIGS. 7-9. These data are
shown in Table 3.
TABLE-US-00003 TABLE 3 Quantitation of HuHP1/2 on tumor cell lines
IC.sub.50 (nM) EC.sub.50 (nM) Fibronectin VCAM CLL Mec1 0.11 0.10
0.07 JM1 0.21 -- 0.12 MM U266 0.46 0.14 0.13 H929 0.91 0.21 1.35
AML HL60 0.11 0.16 0.91 KG1 0.19 0.05 0.1
[0208] Other embodiments are in the claims.
Sequence CWU 1
1
151121PRTMus sp. 1Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val
Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly
Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys Gln Arg
Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala
Ser Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Val Lys Ala
Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Trp 65 70 75 80 Leu Gln
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100
105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 298PRTHomo
sapiens 2Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Leu Val Asp Pro Glu Asp Gly
Glu Thr Ile Tyr Ala Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr
3121PRTArtificial SequenceSynthetic polypeptide 3Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30
Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Arg Ile Asp Pro Ala Ser Gly Asp Thr Lys Tyr Asp Pro Lys
Phe 50 55 60 Gln Val Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Met Trp Val Ser Thr Gly
Tyr Ala Leu Asp Phe Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 4121PRTArtificial SequenceSynthetic polypeptide
4Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Ser Gly Asp Thr Lys
Tyr Asp Pro Lys Phe 50 55 60 Gln Val Arg Val Thr Ile Thr Ala Asp
Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asp Gly Met Trp
Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 5121PRTArtificial SequenceSynthetic
polypeptide 5Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Phe
Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Ser
Gly Asp Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Val Arg Ala Thr
Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Leu Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Asp Gly Met Trp Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp Gly 100 105
110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 6107PRTMus sp. 6Ser
Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp
20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr
Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys
Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 7101PRTHomo sapiens 7Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro 100
8107PRTArtificial SequenceSynthetic polypeptide 8Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp
Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 9107PRTArtificial SequenceSynthetic polypeptide
9Ser Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Thr Asn
Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro
Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Phe
Cys Gln Gln Asp Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 10107PRTArtificial
SequenceSynthetic polypeptide 10Ser Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys
Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala
Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser
Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70
75 80 Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
11107PRTArtificial SequenceSynthetic polypeptide 11Ser Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser
Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser
Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Asp
Tyr Ser Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 12107PRTHomo sapiens 12Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Asp Ile Lys Thr Tyr 20 25 30 Leu Ser Trp
Phe Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile 35 40 45 Ser
Asp Ala Ser Gly Phe Gln Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Tyr Gly Thr Asp Phe Ser Phe Thr Ile Thr Ser Leu Arg Pro
65 70 75 80 Asp Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Lys Val
Pro Phe 85 90 95 Thr Phe Gly Pro Gly Lys Val Gly Phe Asn Arg 100
105 13107PRTArtificial SequenceSynthetic polypeptide 13Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Lys Thr Tyr 20 25
30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Gly Phe Gln Pro Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln
Tyr Glu Lys Val Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 14228PRTArtificial SequenceSynthetic
polypeptide 14Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala
Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105
110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu
Ser Leu Gly 225 15236PRTHomo sapiens 15Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg
Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser 35 40 45 Gln
Asp Ile Lys Thr Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys 50 55
60 Pro Pro Lys Leu Leu Ile Ser Asp Ala Ser Gly Phe Gln Pro Gly Val
65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe Ser
Phe Thr 85 90 95 Ile Thr Ser Leu Arg Pro Asp Asp Thr Ala Thr Tyr
Tyr Cys Gln Gln 100 105 110 Tyr Glu Lys Val Pro Phe Thr Phe Gly Pro
Gly Thr Lys Val Gly Phe 115 120 125 Asn Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185
190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
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