U.S. patent application number 14/950761 was filed with the patent office on 2016-06-23 for tnf-alpha binding proteins.
The applicant listed for this patent is AbbVie Inc.. Invention is credited to Lorenzo Benatuil, Tariq Ghayur, Carrie L. Goodreau, Peter C. Isakson, Jochen Salfeld.
Application Number | 20160176958 14/950761 |
Document ID | / |
Family ID | 44761064 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160176958 |
Kind Code |
A1 |
Benatuil; Lorenzo ; et
al. |
June 23, 2016 |
TNF-ALPHA BINDING PROTEINS
Abstract
Isolated binding proteins, e.g., antibodies or antigen binding
portions thereof, which bind to tumor necrosis factor-alpha
(TNF-.alpha.), e.g., human TNF-.alpha., and related antibody-based
compositions and molecules are disclosed. Also disclosed are
pharmaceutical compositions comprising the antibodies, as well as
therapeutic and diagnostic methods for using the antibodies.
Inventors: |
Benatuil; Lorenzo;
(Northborough, MA) ; Ghayur; Tariq; (Holliston,
MA) ; Goodreau; Carrie L.; (Ludlow, MA) ;
Isakson; Peter C.; (Southborough, MA) ; Salfeld;
Jochen; (No. Grafton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Inc. |
North Chicago |
IL |
US |
|
|
Family ID: |
44761064 |
Appl. No.: |
14/950761 |
Filed: |
November 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13081212 |
Apr 6, 2011 |
9226983 |
|
|
14950761 |
|
|
|
|
61321633 |
Apr 7, 2010 |
|
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Current U.S.
Class: |
435/69.6 ;
435/254.11; 435/254.2; 435/254.21; 435/320.1; 435/328; 435/419;
536/23.53 |
Current CPC
Class: |
A61P 9/04 20180101; A61P
17/00 20180101; A61P 5/14 20180101; A61P 11/02 20180101; A61P 25/16
20180101; C07K 2317/14 20130101; A61P 15/00 20180101; A61P 25/24
20180101; A61P 37/00 20180101; A61P 43/00 20180101; A61P 1/04
20180101; A61P 31/04 20180101; C07K 2317/565 20130101; A61P 1/16
20180101; C07K 2317/24 20130101; C07K 2317/56 20130101; A61K
47/6845 20170801; A61P 3/10 20180101; A61P 25/06 20180101; A61P
27/02 20180101; C07K 2317/52 20130101; A61P 11/08 20180101; A61K
2039/505 20130101; A61P 31/12 20180101; A61P 31/16 20180101; C07K
2317/92 20130101; Y02A 50/30 20180101; A61P 23/00 20180101; A61P
25/04 20180101; A61P 31/20 20180101; A61P 9/06 20180101; A61P 9/10
20180101; A61P 31/18 20180101; Y02A 50/58 20180101; A61P 21/00
20180101; A61P 9/14 20180101; A61P 7/02 20180101; A61P 1/18
20180101; A61P 35/02 20180101; A61P 7/10 20180101; A61P 17/14
20180101; C07K 16/241 20130101; A61P 17/06 20180101; A61P 21/02
20180101; A61P 35/00 20180101; A61P 5/48 20180101; A61P 19/02
20180101; Y02A 50/386 20180101; A61P 11/06 20180101; A61P 37/04
20180101; A61P 1/00 20180101; A61P 11/00 20180101; A61P 25/08
20180101; C07K 2317/76 20130101; A61P 1/02 20180101; A61P 7/04
20180101; A61P 7/06 20180101; A61P 25/14 20180101; A61P 33/00
20180101; A61P 17/04 20180101; A61P 13/12 20180101; A61P 25/28
20180101; G01N 33/6863 20130101; Y02A 50/41 20180101; C07K 2317/567
20130101; A61K 51/1021 20130101; A61P 9/12 20180101; A61P 31/00
20180101; A61P 37/06 20180101; A61P 25/18 20180101; A61P 29/00
20180101; A61P 37/08 20180101; Y02A 50/412 20180101; G01N 2333/7151
20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-48. (canceled)
49. An isolated nucleic acid encoding a humanized binding protein
comprising an antigen binding domain capable of binding human tumor
necrosis factor-alpha (TNF-.alpha.), the antigen binding domain
comprising at least one CDR comprising an amino acid sequence of
residues 31-35 of SEQ ID NO:22; residues 50-65 of SEQ ID NO:22;
residues 98-106 of SEQ ID NO:22; residues 24-34 of SEQ ID NO:23;
residues 50-56 of SEQ ID NO:23; or residues 89-97 of SEQ ID NO:23,
wherein the binding protein comprises a human acceptor framework
and/or an immunoglobulin constant domain.
50. The isolated nucleic acid of claim 49, further comprising an
immunoglobulin constant domain.
51. A vector comprising the isolated nucleic acid of claim 49 or
50.
52. The vector of claim 51, wherein the vector is selected from the
group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and
pBJ.
53. A host cell comprising a vector of claim 51.
54. The host cell of claim 53, wherein the host cell is a
prokaryotic cell.
55. The host cell of claim 53, wherein the host cell is a
eukaryotic cell.
56. The host cell of claim 55, wherein the eukaryotic cell is a
protist cell, an animal cell, a plant cell, a fungal cell, a yeast
cell, a mammalian cell, an avian cell, or an insect cell.
57. The host cell of claim 55, wherein the eukaryotic cell is a CHO
cell, a COS cell, or Saccharomyces cerevisiae.
58. A method of producing a protein that binds TNF-.alpha., the
method comprising the steps of culturing a host cell of claim 53 in
culture medium under conditions sufficient to produce a binding
protein that binds TNF-.alpha..
59-76. (canceled)
77. The nucleic acid of claim 49, wherein the antigen binding
domain comprises a VH region.
78. The nucleic acid of claim 77, wherein the VH region comprises
an amino acid sequence of SEQ ID NOs: 24, 25, 28, 29, 30, 31, 32,
or 33.
79. The nucleic acid of claim 49, wherein the antigen binding
domain comprises a VL region.
80. The nucleic acid of claim 79, wherein the VL region amino acid
sequence is SEQ ID NOs: 26, 27, 34, 35, or 36.
81. The nucleic acid of claim 49, wherein the antigen binding
domain comprises a VH region and a VL region.
82. The nucleic acid of claim 81, wherein the VH region comprises
an amino acid sequence of SEQ ID NO: 24, 25, 28, 29, 30, 31, 32, or
33 and the VL region comprises an amino acid sequence of SEQ ID NO:
26, 27, 34, 35, or 36.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/321,633, filed Apr. 7, 2010, which is hereby
expressly incorporated herein by reference in its entirety for any
purpose.
FIELD OF THE INVENTION
[0002] The present invention relates to TNF-.alpha. binding
proteins and to their uses in the prevention and/or treatment of
acute and chronic immunological diseases such as rheumatoid
arthritis, osteoarthritis, psoriasis, multiple sclerosis, and other
autoimmune diseases.
BACKGROUND OF THE INVENTION
[0003] There is a need in the art for improved antibodies capable
of binding TNF-.alpha. (also referred to as tumor necrosis factor,
tumor necrosis factor-alpha, tumor necrosis factor-.alpha., TNF,
and cachectin). In an embodiment, the antibodies are capable of
neutralizing TNF-.alpha.. The present invention provides a novel
family of binding proteins, CDR grafted antibodies, humanized
antibodies, and fragments thereof, capable of binding TNF-.alpha.,
binding TNF-.alpha. with high affinity, and binding and
neutralizing TNF-.alpha..
SUMMARY OF THE INVENTION
[0004] This invention pertains to TNF-.alpha. binding proteins,
particularly anti-TNF-.alpha. antibodies, or antigen-binding
portions thereof, that bind TNF-.alpha.. In an embodiment, the
antibody, or antigen binding portion thereof, capable of binding
TNF-.alpha. comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: 22-36.
[0005] In one aspect, the invention provides a humanized binding
protein comprising an antigen binding domain capable of binding
human TNF-.alpha., the antigen binding domain comprising at least
one CDR comprising an amino acid sequence selected from the group
consisting of: residues 31-35 of SEQ ID NO:22; residues 50-65 of
SEQ ID NO:22; residues 98-106 of SEQ ID NO:22; residues 24-34 of
SEQ ID NO:23; residues 50-56 of SEQ ID NO:23; and residues 89-97 of
SEQ ID NO:23, wherein the binding protein comprises a human
acceptor framework. In an embodiment, the binding protein comprises
at least 3 CDRs, for example, comprises a variable domain CDR set
selected from the group consisting of: (a) residues 31-35 of SEQ ID
NO:22; residues 50-65 of SEQ ID NO:22; and residues 98-106 of SEQ
ID NO:22; and (b) residues 24-34 of SEQ ID NO:23; residues 50-56 of
SEQ ID NO:23; and residues 89-97 of SEQ ID NO:23. In particular
embodiments, the antigen binding domain comprises an amino acid
sequence comprising residues 31-35 of SEQ ID NO:22; residues 50-65
of SEQ ID NO:22; residues 98-106 of SEQ ID NO:22; residues 24-34 of
SEQ ID NO:23; residues 50-56 of SEQ ID NO:23; and residues 89-97 of
SEQ ID NO:23.
[0006] In an embodiment, the antigen binding domain comprises a VH
region, for example, comprising an amino acid sequence selected
from the group consisting of: SEQ ID NOs: 24, 25, 28, 29, 30, 31,
32, and 33. In another embodiment, the antigen binding domain
comprises a VL region, for example, comprising an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 26, 27,
34, 35, and 36. In a particular embodiment, the antigen binding
domain comprises a VH region and a VL region, for example, wherein
the VH region comprises an amino acid sequence selected from the
group consisting of: SEQ ID NOs: 24, 25, 28, 29, 30, 31, 32, and 33
and the VL region comprises an amino acid sequence selected from
the group consisting of: SEQ ID NOs: 26, 27, 34, 35, and 36.
[0007] In an embodiment, the human acceptor framework comprises at
least one amino acid sequence selected from the group consisting
of: SEQ ID NOs: 6-21. In a particular embodiment, the human
acceptor framework comprises an amino acid sequence selected from
the group consisting of: SEQ IN NOs: 9, 10, 11, 12, 15, 16, 17, and
21. In another embodiment, the human acceptor framework comprises
at least one framework region amino acid substitution, wherein the
amino acid sequence of the framework is at least 65% identical to
the sequence of the human acceptor framework and comprises at least
70 amino acid residues identical to the human acceptor framework.
In another embodiment, the human acceptor framework comprises at
least one framework region amino acid substitution at a key
residue, the key residue selected from the group consisting of: a
residue adjacent to a CDR; a glycosylation site residue; a rare
residue; a residue capable of interacting with human TNF-.alpha.; a
residue capable of interacting with a CDR; a canonical residue; a
contact residue between heavy chain variable region and light chain
variable region; a residue within a Vernier zone; and a residue in
a region that overlaps between a Chothia-defined variable heavy
chain CDR1 and a Kabat-defined first heavy chain framework. In an
embodiment, the key residue is selected from the group consisting
of: H1, H12, H24, H27, H29, H37, H48, H49, H67, H71, H73, H76, H78,
L13, L43, L58, L70, and L80. In an embodiment, the VH mutation is
selected from the group consisting of: Q1E, I12V, A24V, G27F, I29L,
V29F F29L I37V, I48L, V48L, S49G, V67L, F67L, V71K, R71K, T73N,
N76S, L78I, and F78I. In another embodiment, the VL mutation is
selected from the group consisting of: V13L, A435, I58V, E70D, and
S80P. In an embodiment, the binding protein comprises two variable
domains, wherein the two variable domains have amino acid sequences
selected from the group consisting of: SEQ ID NO:24 and SEQ ID
NO:26; SEQ ID NO:24 and SEQ ID NO:27; SEQ ID NO:25 and SEQ ID
NO:26; SEQ ID NO:25 and SEQ ID NO:27.
[0008] In an embodiment, the binding protein binds TNF-.alpha.. In
another embodiment, the binding protein modulates a biological
function of TNF-.alpha.. In another embodiment, the binding protein
neutralizes TNF-.alpha.. In yet another embodiment, the binding
protein diminishes the ability of TNF-.alpha. to bind to its
receptor, for example, the binding protein diminishes the ability
of pro-human TNF-.alpha., mature-human TNF-.alpha., or
truncated-human TNF-.alpha. to bind to its receptor. In yet another
embodiment, the binding protein reduces one or more TNF-.alpha.
biological activities selected from the group consisting of:
TNF-dependent cytokine production; TNF-dependent cell killing;
TNF-dependent inflammation; TNF-dependent bone erosion; and
TNF-dependent cartilage damage.
[0009] In an embodiment, the binding protein has an on rate
constant (K.sub.on) selected from the group consisting of: at least
about 10.sup.2M.sup.-1s.sup.-1; at least about
10.sup.3M.sup.-1s.sup.-1; at least about 10.sup.4M.sup.-1s.sup.-1;
at least about 10.sup.5M.sup.-1s.sup.-1; and at least about
10.sup.6M.sup.-1s.sup.-1; as measured by surface plasmon resonance.
In another embodiment, the binding protein has an off rate constant
(K.sub.off) selected from the group consisting of: at most about
10.sup.-3s.sup.-1; at most about 10.sup.-4s.sup.-1; at most about
10.sup.-5s.sup.-1; and at most about 10.sup.-6s.sup.-1, as measured
by surface plasmon resonance. In yet another embodiment, the
binding protein has a dissociation constant (K.sub.D) selected from
the group consisting of: at most about 10.sup.-7 M; at most about
10.sup.-8 M; at most about 10.sup.-9 M; at most about 10.sup.-10 M;
at most about 10.sup.-11 M; at most about 10.sup.-12M; and at most
10.sup.-13M.
[0010] In an embodiment, the binding protein comprises a heavy
chain immunoglobulin constant domain selected from the group
consisting of: a human IgM constant domain, a human IgG1 constant
domain, a human IgG2 constant domain, a human IgG3 constant domain,
a human IgG4 constant domain, a human IgA constant domain, and a
human IgE constant domain. In a particular embodiment, the heavy
chain immunoglobulin constant region domain is a human IgG1
constant domain. In another embodiment, the binding protein further
comprises a light chain immunoglobulin constant domain selected
from the group consisting of: a human Ig kappa constant domain and
a human Ig lambda constant domain. For example, in an embodiment,
the binding domain comprises an immunoglobulin constant domain
having an amino acid sequence selected from the group consisting
of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a
particular embodiment, the human IgG1 constant domain comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:2 and SEQ ID NO:3. In another embodiment, the light chain
immunoglobulin constant region domain is a human Ig kappa constant
domain comprising an amino acid sequence of SEQ ID NO:4. In yet
another embodiment, the light chain immunoglobulin constant region
domain is a human Ig lambda constant domain comprising an amino
acid sequence SEQ ID NO:5.
[0011] In a particular embodiment, the invention provides a binding
protein capable of binding human TNF-.alpha., the binding protein
comprising: an Ig constant heavy region having an amino acid
sequence selected from the group consisting of SEQ ID NO:2 and SEQ
ID NO: 3; an Ig constant light region having an amino acid sequence
selected from the group consisting of SEQ ID NO:4 and SEQ ID NO: 5;
an Ig variable heavy region having an amino acid sequence selected
from the group consisting of: SEQ ID NO: 24, 25, 28, 29, 30, 31,
32, and 33; and an Ig variable light region having an amino acid
sequence selected from the group consisting of: SEQ ID NO: 26, 27,
34, 35, and 36. In a particular embodiment, the binding protein of
the invention is selected from the group consisting of: an
immunoglobulin molecule, an Fv, a disulfide linked Fv, a monoclonal
antibody, an scFv, a chimeric antibody, a single domain antibody, a
CDR-grafted antibody, a diabody, a humanized antibody, a
multispecific antibody, an Fab, a dual specific antibody, an Fab'
fragment, a bispecific antibody, an F(ab')2 fragment, a DVD-Ig.TM.,
a bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; a Fd fragment consisting of
the VH and CH1 domains; a Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, a dAb fragment, an isolated
complementarity determining region (CDR), and a single chain
antibody.
[0012] In another aspect, the invention provides a crystallized
binding protein comprising a binding protein of the invention,
wherein the binding protein is in the form of a crystal. In an
embodiment, the crystal is a carrier-free pharmaceutical controlled
release crystal. In another embodiment, the binding protein has a
greater half life in vivo than the soluble counterpart of the
binding protein. In another embodiment, the binding protein retains
biological activity.
[0013] In another aspect, the invention provides a composition for
the release of a TNF-.alpha. binding protein, the composition
comprising: (a) a formulation, wherein the formulation comprises a
crystallized binding protein of the invention and an ingredient;
and (b) at least one polymeric carrier. In an embodiment, the
polymeric carrier is a polymer selected from one or more of the
group consisting of: poly acrylic acid, poly cyanoacrylate, a poly
amino acid, a poly anhydride, a poly depsipeptide, a poly ester,
poly lactic acid, poly lactic-co-glycolic acid, poly
b-hydroxybutryate, poly caprolactone, poly dioxanone; poly ethylene
glycol, poly hydroxypropyl methacrylamide, poly organophosphazene,
poly ortho esters, poly vinyl alcohol, poly vinylpyrrolidone,
maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives, collagen,
fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polysaccharides, and blends and
copolymers thereof. In another embodiment, the ingredient is
selected from the group consisting of albumin, sucrose, trehalose,
lactitol, gelatin, hydroxypropyl-.beta.-cyclodextrin,
methoxypolyethylene glycol and polyethylene glycol.
[0014] In another aspect, the invention provides a TNF-.alpha.
binding protein construct comprising the TNF-.alpha. binding
protein of the invention and a polypeptide selected from the group
consisting of a linker and an immunoglobulin constant domain. In an
embodiment, the binding protein possesses a human glycosylation
pattern. In another embodiment, the binding protein construct is a
crystallized TNF-.alpha. binding protein construct. In yet another
embodiment, the crystallized TNF-.alpha. binding protein construct
is a carrier-free pharmaceutical controlled release crystallized
TNF-.alpha. binding protein construct. In a particular embodiment,
the TNF-.alpha. binding protein construct has a greater half life
in vivo than the soluble counterpart of the binding protein
construct. In another embodiment, the binding protein construct
retains biological activity.
[0015] In another aspect, the invention provides a TNF-.alpha.
binding protein conjugate comprising a TNF-.alpha. binding protein
construct of the invention and further comprising an agent selected
from the group consisting of: an immunoadhesion molecule, an
imaging agent, a therapeutic agent, and a cytotoxic agent. In an
embodiment, the agent is an imaging agent selected from the group
consisting of a radiolabel, an enzyme, a fluorescent label, a
luminescent label, a bioluminescent label, a magnetic label, and
biotin. In an embodiment, the imaging agent is a radiolabel
selected from the group consisting of: .sup.3H, .sup.14C, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, and .sup.153Sm. In another embodiment, the agent is a
therapeutic or cytotoxic agent selected from the group consisting
of: an anti-metabolite, an alkylating agent, an antibiotic, a
growth factor, a cytokine, an anti-angiogenic agent, an
anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent.
[0016] In another aspect, the invention provides an isolated
nucleic acid encoding a binding protein of the invention. In
another aspect, the invention provides a vector comprising an
isolated nucleic acid of the invention. In an embodiment, the
vector is selected from the group consisting of pcDNA, pTT, pTT3,
pEFBOS, pBV, pJV, and pBJ. In an embodiment, the invention provides
a host cell comprising a vector of the invention. In another
embodiment, the host cell is a prokaryotic cell, e.g., E. coli. In
another embodiment, the host cell is a eukaryotic cell, e.g., a
protist cell, an animal cell, a plant cell, or a fungal cell. In
another embodiment, the eukaryotic cell is an animal cell selected
from the group consisting of a mammalian cell, an avian cell, and
an insect cell. For example, the host cell is a CHO cell, a COS
cell, a yeast cell, e.g., Saccharomyces cerevisiae, or an insect
Sf9 cell.
[0017] In another aspect, the invention provides a method of
producing a protein that binds TNF-.alpha., the method comprising
the steps of culturing a host cell of the invention in culture
medium under conditions sufficient to produce a binding protein
that binds TNF-.alpha. as well as a TNF-.alpha. binding protein
produced by the method.
[0018] In another aspect, the invention provides a pharmaceutical
composition comprising a binding protein of the invention and a
pharmaceutically acceptable carrier. In an embodiment, the
pharmaceutically acceptable carrier functions as an adjuvant useful
to increase the absorption or dispersion of the binding protein. In
another embodiment, the adjuvant is hyaluronidase. In another
embodiment, the pharmaceutical composition further comprises at
least one additional therapeutic agent for treating a disorder in
which TNF-.alpha. activity is detrimental, for example, a
therapeutic agent, an imaging agent, a cytotoxic agent, an
angiogenesis inhibitor, a kinase inhibitor, a co-stimulation
molecule blocker, an adhesion molecule blocker, an anti-cytokine
antibody or functional fragment thereof, methotrexate,
cyclosporine, rapamycin, FK506, a detectable label, a detectable
reporter, a TNF-.alpha. antagonist, an anti-rheumatic; a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteroid, an anabolic steroid, an erythropoietin, an
immunization, an immunoglobulin, an immunosuppressive agent, a
growth hormone, a hormone replacement drug, a radiopharmaceutical,
an antidepressant, an antipsychotic, a stimulant, an asthma
medication, a beta agonist, an inhaled steroid, an oral steroid, an
epinephrine or analog thereof, a cytokine, and a cytokine
antagonist.
[0019] In another aspect, the invention provides a method for
treating a mammal comprising the step of administering to the
mammal an effective amount of the pharmaceutical composition of the
invention. In another embodiment, the invention provides a method
for reducing human TNF-.alpha. activity, the method comprising the
step of: contacting human TNF-.alpha. with the binding protein of
the invention such that human TNF-.alpha. activity is reduced. In
another embodiment, the invention provides a method for reducing
human TNF-.alpha. activity in a human subject suffering from a
disorder in which TNF-.alpha. activity is detrimental, the method
comprising the step of administering to the human subject the
binding protein of the invention such that human TNF-.alpha.
activity in the human subject is reduced. In another embodiment,
the invention provides a method for treating a subject for a
disease or a disorder in which TNF-.alpha. activity is detrimental,
the method comprising the step of administering to the subject the
binding protein of the invention such that treatment is
achieved.
[0020] In another embodiment, the invention provides a method of
treating a patient suffering from a disorder in which TNF-.alpha.
is detrimental comprising the step of administering the binding
protein of the invention before, concurrent, or after the
administration of a second agent, wherein the second agent is
selected from the group consisting of an antibody, or fragment
thereof, capable of binding human IL-12; PGE2; LPA; NGF; CGRP;
SubP; RAGE; histamine; a histamine receptor blocker; bradykinin;
IL-lalpha; IL-1beta; VEGF; PLGF; methotrexate; a corticosteroid, a
glucocorticoid receptor modulator; cyclosporin, rapamycin, FK506, a
non-steroidal anti-inflammatory agent, and sclerostin, In an
embodiment, the disorder is selected from the group consisting of a
respiratory disorder; asthma; allergic and nonallergic asthma;
asthma due to infection; asthma due to infection with respiratory
syncytial virus (RSV); chronic obstructive pulmonary disease
(COPD); a condition involving airway inflammation; eosinophilia;
fibrosis and excess mucus production; cystic fibrosis; pulmonary
fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema;
allergic rhinitis; allergic enterogastritis; an inflammatory and/or
autoimmune condition of the skin; an inflammatory and/or autoimmune
condition of gastrointestinal organs; inflammatory bowel diseases
(IBD); ulcerative colitis; Crohn's disease; an inflammatory and/or
autoimmune condition of the liver; liver cirrhosis; liver fibrosis;
liver fibrosis caused by hepatitis B and/or C virus; scleroderma;
tumors or cancers; hepatocellular carcinoma; glioblastoma;
lymphoma; Hodgkin's lymphoma; a viral infection; a bacterial
infection; a parasitic infection; HTLV-1 infection; suppression of
expression of protective type 1 immune responses, and suppression
of expression of a protective type 1 immune response during
vaccination. In an embodiment, the disorder is selected from the
group consisting of: rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, septic arthritis, Lyme arthritis, psoriatic
arthritis, reactive arthritis, spondyloarthropathy, systemic lupus
erythematosus, Crohn's disease, ulcerative colitis, inflammatory
bowel disease, insulin dependent diabetes mellitus, thyroiditis,
asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft
versus host disease, organ transplant rejection, acute or chronic
immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia areata, seronegative arthropathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthropathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis B, Hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasculitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleostasis, idiosyncratic liver
disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Th1
Type mediated diseases, acute and chronic pain (different forms of
pain), and cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer and hematopoietic
malignancies (leukemia and lymphoma) Abetalipoproteinemia,
Acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti-CD3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aortic and peripheral
aneurysms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chronic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, Dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic arteriosclerotic disease, Diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's Syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, Epstein-Barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hemophagocytic lymphohistiocytosis, fetal thymus implant rejection,
Friedreich's ataxia, functional peripheral arterial disorders,
fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis,
graft rejection of any organ or tissue, gram negative sepsis, gram
positive sepsis, granulomas due to intracellular organisms, hairy
cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis,
hay fever, heart transplant rejection, hemochromatosis,
hemodialysis, hemolytic uremic syndrome/thrombolytic
thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle
arrhythmias, HIV infection/HIV neuropathy, Hodgkin's disease,
hyperkinetic movement disorders, hypersensitivity reactions,
hypersensitivity pneumonitis, hypertension, hypokinetic movement
disorders, hypothalamic-pituitary-adrenal axis evaluation,
idiopathic Addison's disease, idiopathic pulmonary fibrosis,
antibody mediated cytotoxicity, Asthenia, infantile spinal muscular
atrophy, inflammation of the aorta, influenza a, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma,
kidney transplant rejection, legionella, leishmaniasis, leprosy,
lesions of the corticospinal system, lipedema, liver transplant
rejection, lymphedema, malaria, malignant Lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia,
metabolic/idiopathic, migraine headache, mitochondrial multi-system
disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Menzel,
Dejerine-Thomas, Shy-Drager, and Machado-Joseph), myasthenia
gravis, mycobacterium avium intracellulare, mycobacterium
tuberculosis, myelodysplastic syndrome, myocardial infarction,
myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal
chronic lung disease, nephritis, nephrosis, neurodegenerative
diseases, neurogenic I muscular atrophies, neutropenic fever,
non-Hodgkins lymphoma, occlusion of the abdominal aorta and its
branches, occlusive arterial disorders, OKT3.RTM. therapy,
orchitis/epidydimitis, orchitis/vasectomy reversal procedures,
organomegaly, osteoporosis, pancreas transplant rejection,
pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of
malignancy, parathyroid transplant rejection, pelvic inflammatory
disease, perennial rhinitis, pericardial disease, peripheral
atherosclerotic disease, peripheral vascular disorders,
peritonitis, pernicious anemia, pneumocystis carinii pneumonia,
pneumonia, POEMS syndrome (polyneuropathy, organomegaly,
endocrinopathy, monoclonal gammopathy, and skin changes syndrome),
post perfusion syndrome, post pump syndrome, post-MI cardiotomy
syndrome, preeclampsia, Progressive supranucleo Palsy, primary
pulmonary hypertension, radiation therapy, Raynaud's phenomenon and
disease, Raynaud's disease, Refsum's disease, regular narrow QRS
tachycardia, renovascular hypertension, reperfusion injury,
restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea,
Senile Dementia of Lewy body type, seronegative arthropathies,
shock, sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrhythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the
cardiovascular system, systemic anaphylaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, viral
encephalitis/aseptic meningitis, viral-associated hemophagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, acute coronary syndromes, acute
idiopathic polyneuritis, acute inflammatory demyelinating
polyradiculoneuropathy, acute ischemia, adult Still's disease,
alopecia areata, anaphylaxis, anti-phospholipid antibody syndrome,
aplastic anemia, arteriosclerosis, atopic eczema, atopic
dermatitis, autoimmune dermatitis, autoimmune disorder associated
with streptococcus infection, autoimmune enteropathy, autoimmune
hearing loss, autoimmune lymphoproliferative syndrome (ALPS),
autoimmune myocarditis, autoimmune premature ovarian failure,
blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular
disease, catastrophic antiphospholipid syndrome, celiac disease,
cervical spondylosis, chronic ischemia, cicatricial pemphigoid,
clinically isolated syndrome (CIS) with risk for multiple
sclerosis, conjunctivitis, childhood onset psychiatric disorder,
chronic obstructive pulmonary disease (COPD), dacryocystitis,
dermatomyositis, diabetic retinopathy, diabetes mellitus, disk
herniation, disk prolapse, drug induced immune hemolytic anemia,
endocarditis, endometriosis, endophthalmitis, episcleritis,
erythema multiforme, erythema multiforme major, gestational
pemphigoid, Guillain-Barre syndrome (GBS), hay fever, Hughes
syndrome, idiopathic Parkinson's disease, idiopathic interstitial
pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion
body myositis, infectious ocular inflammatory disease, inflammatory
demyelinating disease, inflammatory heart disease, inflammatory
kidney disease, IPF/UIP, iritis, keratitis, keratojunctivitis
sicca, Kussmaul disease or Kussmaul-Meier disease, Landry's
paralysis, Langerhan's cell histiocytosis, livedo reticularis,
macular degeneration, microscopic polyangiitis, morbus bechterev,
motor neuron disorders, mucous membrane pemphigoid, multiple organ
failure, myasthenia gravis, myelodysplastic syndrome, myocarditis,
nerve root disorders, neuropathy, non-A non-B hepatitis, optic
neuritis, osteolysis, ovarian cancer, pauciarticular JRA,
peripheral artery occlusive disease (PAOD), peripheral vascular
disease (PVD), peripheral artery, disease (PAD), phlebitis,
polyarteritis nodosa (or periarteritis nodosa), polychondritis,
polymyalgia rheumatica, poliosis, polyarticular JRA, polyendocrine
deficiency syndrome, polymyositis, polymyalgia rheumatica (PMR),
post-pump syndrome, primary Parkinsonism, prostate and rectal
cancer and hematopoietic malignancies (leukemia and lymphoma),
prostatitis, pure red cell aplasia, primary adrenal insufficiency,
recurrent neuromyelitis optica, restenosis, rheumatic heart
disease, sapho (synovitis, acne, pustulosis, hyperostosis, and
osteitis), scleroderma, secondary amyloidosis, shock lung,
scleritis, sciatica, secondary adrenal insufficiency, silicone
associated connective tissue disease, Sneddon-Wilkinson dermatosis,
spondylitis ankylosans, Stevens-Johnson syndrome (SJS), systemic
inflammatory response syndrome, temporal arteritis, toxoplasmic
retinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS
(tumor necrosis factor receptor associated periodic syndrome), type
1 allergic reaction, type II diabetes, urticaria, usual
interstitial pneumonia (UIP), vasculitis, vernal conjunctivitis,
viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet
macular degeneration, wound healing, yersinia and salmonella
associated arthropathy.
[0021] In another embodiment, the invention provides a method of
treating a patient suffering from a disorder in which TNF-.alpha.
is detrimental, the method comprising the step of administering the
binding protein of the invention before, concurrent, or after the
administration of a second agent, wherein the second agent is
selected from the group consisting of inhaled steroids;
beta-agonists; short-acting or long-acting beta-agonists;
antagonists of leukotrienes or leukotriene receptors; ADVAIR; IgE
inhibitors; anti-IgE antibodies; XOLAIR; phosphodiesterase
inhibitors; PDE4 inhibitors; xanthines; anticholinergic drugs; mast
cell-stabilizing agents; Cromolyn; IL-4 inhibitors; IL-5
inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or
its receptors including H1, H2, H3, and H4; antagonists of
prostaglandin D or its receptors DP1 and CRTH2; TNF antagonists; a
soluble fragment of a TNF receptor; ENBREL; TNF enzyme antagonists;
TNF converting enzyme (TACE) inhibitors; muscarinic receptor
antagonists; TGF-beta antagonists; interferon gamma; perfenidone;
chemotherapeutic agents, methotrexate; leflunomide; sirolimus
(rapamycin) or an analog thereof, CCI-779; COX2 or cPLA2
inhibitors; NSAIDs; immunomodulators; p38 inhibitors; TPL-2, MK-2
and NF.kappa.B inhibitors; budenoside; epidermal growth factor;
corticosteroids; cyclosporine; sulfasalazine; aminosalicylates;
6-mercaptopurine; azathioprine; metronidazole; lipoxygenase
inhibitors; mesalamine; olsalazine; balsalazide; antioxidants;
thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1.beta.
antibodies; anti-IL-6 antibodies; growth factors; elastase
inhibitors; pyridinyl-imidazole compounds; antibodies or agonists
of LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,
IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,
IL-30, IL-31, IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF;
antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,
CD69, CD90 or their ligands; FK506; rapamycin; mycophenolate
mofetil; ibuprofen; prednisolone; phosphodiesterase inhibitors;
adensosine agonists; antithrombotic agents; complement inhibitors;
adrenergic agents; IRAK, NIK, IKK, p38, or MAP kinase inhibitors;
IL-1.beta. converting enzyme inhibitors; TNF-.alpha. converting
enzyme inhibitors; T-cell signaling inhibitors; metalloproteinase
inhibitors; 6-mercaptopurines; angiotensin converting enzyme
inhibitors; soluble cytokine receptors; soluble p55 TNF receptor;
soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R;
anti-inflammatory cytokines; IL-4; IL-10; IL-11; and
TGF-.beta..
[0022] In an embodiment, the binding protein of the invention is
administered to the subject by at least one mode selected from the
group consisting of parenteral, subcutaneous, intramuscular,
intravenous, intra-articular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, and transdermal.
DETAILED DESCRIPTION OF THE INVENTION
[0023] This invention pertains to TNF-.alpha. binding proteins,
particularly anti-TNF-.alpha. antibodies, or antigen-binding
portions thereof, that bind TNF-.alpha. (i.e., tumor necrosis
factor, tumor necrosis factor-alpha, tumor necrosis factor-.alpha.,
TNF, cachectin). Various aspects of the invention relate to
antibodies and antibody fragments, and pharmaceutical compositions
thereof, as well as nucleic acids, recombinant expression vectors
and host cells for making such antibodies and fragments. Methods of
using the antibodies of the invention to detect human TNF-.alpha.,
to inhibit human TNF-.alpha. either in vitro or in vivo, and to
regulate gene expression or TNF-.alpha. related functions are also
encompassed by the invention. Compositions comprising the
antibodies of the present invention, as well as methods of using
such antibodies, are also described.
[0024] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or", unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms of the term, such as "includes" and "included", is not
limiting. Also, terms such as "element" or "component" encompass
both elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0025] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, pathology, oncology,
molecular biology, immunology, microbiology, genetics and protein
and nucleic acid chemistry and hybridization described herein are
those well known and commonly used in the art. The methods and
techniques of the present invention are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification unless
otherwise indicated. Enzymatic reactions and purification
techniques are performed according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0026] That the present invention may be more readily understood,
select terms are defined below.
[0027] The term "polypeptide" refers to any polymeric chain of
amino acids. The terms "peptide" and "protein" are used
interchangeably with the term polypeptide and also refer to a
polymeric chain of amino acids. The term "polypeptide" encompasses
native or artificial proteins, protein fragments and polypeptide
analogs of a protein sequence. A polypeptide may be monomeric or
polymeric.
[0028] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0029] The term "recovering" refers to the process of rendering a
chemical species such as a polypeptide substantially free of
naturally associated components by isolation, e.g., using protein
purification techniques well known in the art.
[0030] The term "human TNF-.alpha." (abbreviated herein as
hTNF-.alpha.), includes a trimeric cytokine protein. The term
includes a homotrimeric protein comprising three 17.5 kD
TNF-.alpha. proteins. The homotrimeric protein is referred to as a
"TNF-.alpha. protein". The term human "TNF-.alpha." is intended to
include recombinant human TNF-.alpha. (rhTNF-.alpha.) which can be
prepared by standard recombinant expression methods. The sequence
of human TNF-.alpha. is shown in Table 1.
TABLE-US-00001 TABLE 1 Sequence of Human TNF.alpha. Sequence
Protein Sequence Identifier 12345678901234567890123456789012 Human
TNF-.alpha. SEQ ID NO.: 1 VRSSSRTPSDKPVAHVVANPQAEGQLQWLNDR
ANALLANGVELRDNQLVVPSEGLYLIYSQVLF KGQGCPSTHVLLTHTISRIAVSYQTKVNLLSA
IKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK GDRLSAEINRPDYLDFAESGQVYFGIIAL
[0031] "Biological activity" refers to all inherent biological
properties of the cytokine. Biological properties of TNF-.alpha.
include but are not limited to binding TNF receptor.
[0032] The terms "specific binding" or "specifically binding", in
reference to the interaction of an antibody, a protein, or a
peptide with a second chemical species, mean that the interaction
is dependent upon the presence of a particular structure (e.g., an
antigenic determinant or epitope) on the chemical species; for
example, an antibody recognizes and binds to a specific protein
structure rather than to proteins generally. If an antibody is
specific for epitope "A", the presence of a molecule containing
epitope A (or free, unlabeled A), in a reaction containing labeled
"A" and the antibody, will reduce the amount of labeled A bound to
the antibody.
[0033] The term "antibody", broadly refers to any immunoglobulin
(Ig) molecule, or antigen binding portion thereof, comprised of
four polypeptide chains, two heavy (H) chains and two light (L)
chains, or any functional fragment, mutant, variant, or derivation
thereof, which retains the essential epitope binding features of an
Ig molecule. Such mutant, variant, or derivative antibody formats
are known in the art. Nonlimiting embodiments of which are
discussed below.
[0034] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CHL CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[0035] The term "antigen-binding portion" or "antigen-binding
region" of an antibody (or simply "antibody portion"), refers to
one or more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., hTNF-.alpha.). The
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also have bispecific, dual specific, or multi-specific formats;
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al. (1989) Nature
341:544-546, Winter et al., PCT publication WO 90/05144 A1), which
comprises a single variable domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see, e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. Other forms of single chain antibodies,
such as diabodies are also encompassed. Diabodies are bivalent,
bispecific antibodies in which VH and VL domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see, e.g.,
Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;
Poljak, et al. (1994) Structure 2:1121-1123). Such antibody binding
portions are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN
3-540-41354-5).
[0036] The term "antibody construct" refers to a polypeptide
comprising one or more antigen-binding portions of the invention
linked to a linker polypeptide or an immunoglobulin constant
domain. Linker polypeptides comprise two or more amino acid
residues joined by peptide bonds and are used to link one or more
antigen binding portions. Such linker polypeptides are well known
in the art (see e.g., Holliger, et al. (1993) Proc. Natl. Acad.
Sci. USA 90:6444-6448; Poljak, et al. (1994) Structure
2:1121-1123). An immunoglobulin constant domain refers to a heavy
or light chain constant domain. Human IgG heavy chain and light
chain constant domain amino acid sequences are known in the art and
represented in Table 2.
TABLE-US-00002 TABLE 2 Sequence of Human IgG Heavy Chain Constant
Domain and Light Chain Constant Sequence Sequence Protein
Identifier 12345678901234567890123456789012 Ig gamma-1 SEQ ID NO.:
2 ASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO.:
3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK Ig Kappa constant SEQ ID NO.: 4
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY region
PREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC Ig Lambda SEQ ID NO.: 5 QPKAAPSVTLFPPSSEELQANKATLVCLISDF
constant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNK
YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS
[0037] An antibody, or antigen-binding portion thereof, may be part
of a larger immunoadhesion molecule, formed by covalent or
noncovalent association of the antibody or antibody portion with
one or more other proteins or peptides. Examples of such
immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, et al.
(1995) Hum. Antibod. Hybridomas 6:93-101) and use of a cysteine
residue, a marker peptide and a C-terminal polyhistidine tag to
make bivalent and biotinylated scFv molecules (Kipriyanov, et al.
(1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab
and F(ab').sub.2 fragments, can be prepared from whole antibodies
using conventional techniques, such as papain or pepsin digestion,
respectively, of whole antibodies. Moreover, antibodies, antibody
portions and immunoadhesion molecules can be obtained using
standard recombinant DNA techniques, as described herein.
[0038] An "isolated antibody" refers to an antibody, or
antigen-binding portion thereof, that is substantially free of
other antibodies having different antigenic specificities (e.g., an
isolated antibody that specifically binds hTNF-.alpha. is
substantially free of antibodies that specifically bind antigens
other than hTNF-.alpha.). An isolated antibody that specifically
binds hTNF-.alpha. may, however, have cross-reactivity to other
antigens, such as TNF-.alpha. molecules from other species.
Moreover, an isolated antibody may be substantially free of other
cellular material and/or chemicals.
[0039] The term "human antibody" includes antibodies, or
antigen-binding portion thereof, that having variable and constant
regions derived from human germline immunoglobulin sequences. The
human antibodies of the invention may include amino acid residues
not encoded by human germline immunoglobulin sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo), for example in the CDRs and
in particular CDR3. However, the term "human antibody", is not
intended to include antibodies in which CDR sequences derived from
the germline of another mammalian species, such as a mouse, have
been grafted onto human framework sequences.
[0040] The term "recombinant human antibody" is intended to include
all human antibodies, or antigen-binding portions thereof, that are
prepared, expressed, created or isolated by recombinant means, such
as antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial human antibody library (Hoogenboom
(1997) Trends Biotechnol. 15:62-70; Azzazy and Highsmith (2002)
Clin. Biochem. 35:425-445; Gavilondo and Larrick (2000)
BioTechniques 29:128-145; Hoogenboom and Chames (2000) Immunol.
Today 21:371-378), antibodies isolated from an animal (e.g., a
mouse) that is transgenic for human immunoglobulin genes (see,
e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295;
Kellermann and Green (2002) Current Opin. Biotechnol. 13:593-597;
Little et al. (2000) Immunol. Today 21:364-370) or antibodies
prepared, expressed, created or isolated by any other means that
involves splicing of human immunoglobulin gene sequences to other
DNA sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0041] The term "chimeric antibody" refers to antibodies, or
antigen-binding portions thereof, which comprise heavy and light
chain variable region sequences from one species and constant
region sequences from another species, such as antibodies having
murine heavy and light chain variable regions linked to human
constant regions.
[0042] The term "CDR-grafted antibody" refers to antibodies, or
antigen-binding portions thereof, which comprise heavy and light
chain variable region sequences from one species but in which the
sequences of one or more of the CDR regions of VH and/or VL are
replaced with CDR sequences of another species, such as antibodies
having human heavy and light chain variable regions in which one or
more of the human CDRs (e.g., CDR3) has been replaced with murine
CDR sequences.
[0043] The term "humanized antibody" refers to antibodies, or
antigen-binding portions thereof, which comprise heavy and light
chain variable region sequences from a non-human species (e.g., a
mouse) but in which at least a portion of the VH and/or VL sequence
has been altered to be more "human-like", i.e., more similar to
human germline variable sequences. One type of humanized antibody
is a CDR-grafted antibody, in which non-human CDR sequences are
introduced into human VH and VL frameworks.
[0044] The terms "Kabat numbering", "Kabat definitions" and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242). See also, Martin, "Protein Sequence and Structure
Analysis of Antibody Variable Domains," In Kontermann and Dubel,
eds., Antibody Engineering (Springer-Verlag, Berlin, 2001), Chapter
31, especially pages 432-433. For the heavy chain variable region,
the hypervariable region ranges from amino acid positions 31 to 35
for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 106 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0045] The terms "acceptor" and "acceptor antibody" refer to the
antibody or nucleic acid sequence providing or encoding at least
80%, at least 85%, at least 90%, at least 95%, at least 98% or 100%
of the amino acid sequences of one or more of the framework
regions. In some embodiments, the term "acceptor" refers to the
antibody amino acid or nucleic acid sequence providing or encoding
the constant region(s). In yet another embodiment, the term
"acceptor" refers to the antibody amino acid or nucleic acid
sequence providing or encoding one or more of the framework regions
and the constant region(s). In a specific embodiment, the term
"acceptor" refers to a human antibody amino acid or nucleic acid
sequence that provides or encodes at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, or 100% of the amino acid
sequences of one or more of the framework regions. In accordance
with this embodiment, an acceptor may contain at least 1, at least
2, at least 3, least 4, at least 5, or at least 10 amino acid
residues that does (do) not occur at one or more specific positions
of a human antibody. An acceptor framework region and/or acceptor
constant region(s) may be, e.g., derived or obtained from a
germline antibody gene, a mature antibody gene, a functional
antibody (e.g., antibodies well-known in the art, antibodies in
development, or antibodies commercially available).
[0046] The term "CDR" refers to the complementarity determining
region within antibody variable sequences. There are three CDRs in
each of the variable regions of the heavy chain and the light
chain, which are designated CDR1, CDR2 and CDR3, for each of the
variable regions. The term "CDR set" refers to a group of three
CDRs that occur in a single variable region capable of binding the
antigen. The exact boundaries of these CDRs have been defined
differently according to different systems. The system described by
Kabat (Kabat et al., Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987) and
(1991)) not only provides an unambiguous residue numbering system
applicable to any variable region of an antibody, but also provides
precise residue boundaries defining the three CDRs. These CDRs may
be referred to as Kabat CDRs. Chothia and coworkers (Chothia and
Lesk (1987) J. Mol. Biol. 196:901-917) and Chothia et al. (1989)
Nature 342:877-883) found that certain sub-portions within Kabat
CDRs adopt nearly identical peptide backbone conformations, despite
having great diversity at the level of amino acid sequence. These
sub-portions were designated as L1, L2 and L3 or H1, H2 and H3
where the "L" and the "H" designates the light chain and the heavy
chains regions, respectively. These regions may be referred to as
Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
Other boundaries defining CDRs overlapping with the Kabat CDRs have
been described by Padlan (1995) FASEB J. 9:133-139 and MacCallum
(1996) J. Mol. Biol. 262(5):732-745. Still other CDR boundary
definitions may not strictly follow one of the above systems, but
will nonetheless overlap with the Kabat CDRs, although they may be
shortened or lengthened in light of prediction or experimental
findings that particular residues or groups of residues or even
entire CDRs do not significantly impact antigen binding. The
methods used herein may utilize CDRs defined according to any of
these systems, although particular embodiments use Kabat or Chothia
defined CDRs.
[0047] The term "canonical" residue refers to a residue in a CDR or
framework that defines a particular canonical CDR structure as
defined by Chothia et al. (1987) J. Mol. Biol. 196:901-917; Chothia
et al. (1992) J. Mol. Biol. 227:799-817. According to Chothia et
al., critical portions of the CDRs of many antibodies have nearly
identical peptide backbone confirmations despite great diversity at
the level of amino acid sequence. Each canonical structure
specifies primarily a set of peptide backbone torsion angles for a
contiguous segment of amino acid residues forming a loop.
[0048] The terms "donor" and "donor antibody" refer to an antibody
providing one or more CDRs. In a particular embodiment, the donor
antibody is an antibody from a species different from the antibody
from which the framework regions are obtained or derived. In the
context of a humanized antibody, the term "donor antibody" refers
to a non-human antibody providing one or more CDRs.
[0049] The term "framework" or "framework sequence" refers to the
remaining sequences of a variable region minus the CDRs. Because
the exact definition of a CDR sequence can be determined by
different systems, the meaning of a framework sequence is subject
to correspondingly different interpretations. The six CDRs (CDR-L1,
-L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy
chain) also divide the framework regions on the light chain and the
heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each
chain, in which CDR1 is positioned between FR1 and FR2, CDR2
between FR2 and FR3, and CDR3 between FR3 and FR4. Without
specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a
framework region, as referred by others, represents the combined
FR's within the variable region of a single, naturally occurring
immunoglobulin chain. A FR represents one of the four sub-regions,
and FRs represents two or more of the four sub-regions constituting
a framework region.
[0050] Human heavy chain and light chain acceptor sequences are
known in the art. In one embodiment of the invention the human
heavy chain and light chain acceptor sequences are selected from
the sequences listed from V-base (http://vbase.mrc-cpe.cam.ac.uk/)
or from IMGT.RTM., the international ImMunoGeneTics information
System.RTM.
(http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In
another embodiment of the invention the human heavy chain and light
chain acceptor sequences are selected from the sequences described
in Table 3 and Table 4.
TABLE-US-00003 TABLE 3 Heavy Chain Acceptor Sequences Sequence SEQ
ID No. Protein region 12345678901234567890123456789012 SEQ ID NO: 6
VH4-59 FR1 QVQLQESGPGLVKPSETLSLTCTVSGGSISS SEQ ID NO: 7 VH4-59 FR2
WIRQPPGKGLEWIG SEQ ID NO: 8 VH4-59 FR3
RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR SEQ ID NO: 9 VH3-53 FR1
EVQLVESGGGLIQPGGSLRLSCAASGFTVSS SEQ ID NO: 10 VH3-53 FR2
WVRQAPGKGLEWVS SEQ ID NO: 11 VH3-53 FR3
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SEQ ID NO: 12 JH1/JH4/JH5 FR4
WGQGTLVTVSS SEQ ID NO: 13 JH2 FR4 WGRGTLVTVSS SEQ ID NO: 14 JH6 FR4
WGQGTTVTVSS
TABLE-US-00004 TABLE 4 Light Chain Acceptor Sequences Sequence SEQ
ID No. Protein region 12345678901234567890123456789012 SEQ ID NO:
15 1-39/O12 FR1 DIQMTQSPSSLSASVGDRVTITC SEQ ID NO: 16 1-39/O12 FR2
WYQQKPGKAPKLLIY SEQ ID NO: 17 1-39/O12 FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC SEQ ID NO: 18 3-15/L2 FR1
EIVMTQSPATLSVSPGERATLSC SEQ ID NO: 19 3-15/L2 FR2 WYQQKPGQAPRLLIY
SEQ ID NO: 20 3-15/L2 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC SEQ ID
NO: 21 JK2 FR4 FGQGTKLEIKR
[0051] The term "germ line antibody gene" or "gene fragment" refers
to an immunoglobulin sequence encoded by non-lymphoid cells that
have not undergone the maturation process that leads to genetic
rearrangement and mutation for expression of a particular
immunoglobulin (see, e.g., Shapiro et al. (2002) Crit. Rev.
Immunol. 22(3):183-200; Marchalonis et al. (2001) Adv. Exp. Med.
Biol. 484:13-30). One of the advantages provided by various
embodiments of the present invention stems from the recognition
that germ line antibody genes are more likely than mature antibody
genes to conserve essential amino acid sequence structures
characteristic of individuals in the species, hence less likely to
be recognized as from a foreign source when used therapeutically in
that species.
[0052] The term "key" residues refer to certain residues within the
variable region that have more impact on the binding specificity
and/or affinity of an antibody, in particular a humanized antibody.
A key residue includes, but is not limited to, one or more of the
following: a residue that is adjacent to a CDR, a potential
glycosylation site (e.g., N- or O-glycosylation site), a rare
residue, a residue capable of interacting with the antigen, a
residue capable of interacting with a CDR, a canonical residue, a
contact residue between heavy chain variable region and light chain
variable region, a residue within the Vernier zone, and a residue
in the region that overlaps between the Chothia definition of a
variable heavy chain CDR1 and the Kabat definition of the first
heavy chain framework.
[0053] The term "humanized antibody" is an antibody or a variant,
derivative, analog or fragment thereof which immunospecifically
binds to an antigen of interest and which comprises a framework
(FR) region having substantially the amino acid sequence of a human
antibody and a complementary determining region (CDR) having
substantially the amino acid sequence of a non-human antibody. The
term "substantially" in the context of a CDR refers to a CDR having
an amino acid sequence at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 99% identical to the amino acid
sequence of a non-human antibody CDR. A humanized antibody
comprises substantially all of at least one, and typically two,
variable domains (Fab, Fab', F(ab') 2, FabC, Fv) in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. In a particular embodiment, a
humanized antibody also comprises at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. In some embodiments, a humanized antibody contains
both the light chain as well as at least the variable domain of a
heavy chain. The antibody also may include the CH1, hinge, CH2,
CH3, and CH4 regions of the heavy chain. In some embodiments, a
humanized antibody only contains a humanized light chain. In some
embodiments, a humanized antibody only contains a humanized heavy
chain. In specific embodiments, a humanized antibody only contains
a humanized variable domain of a light chain and/or humanized heavy
chain.
[0054] The humanized antibody can be selected from any class of
immunoglobulins, including, e.g., IgM, IgG, IgD, IgA and IgE, and
any isotype, including without limitation, e.g., IgG1, IgG2, IgG3
and IgG4. The humanized antibody may comprise sequences from more
than one class or isotype, and particular constant domains may be
selected to optimize desired effector functions using techniques
well-known in the art.
[0055] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In a particular embodiment, such mutations are not extensive.
Usually, at least 80%, at least 85%, at least 90%, and at least 95%
of the humanized antibody residues will correspond to those of the
parental FR and CDR sequences. The term "consensus framework"
refers to the framework region in the consensus immunoglobulin
sequence. The term "consensus immunoglobulin sequence" refers to
the sequence formed from the most frequently occurring amino acids
(or nucleotides) in a family of related immunoglobulin sequences
(See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft,
Weinheim, Germany 1987). In a family of immunoglobulins, each
position in the consensus sequence is occupied by the amino acid
occurring most frequently at that position in the family. If two
amino acids occur equally frequently, either can be included in the
consensus sequence.
[0056] The term "Vernier" zone refers to a subset of framework
residues that may adjust CDR structure and fine-tune the fit to
antigen as described by Foote and Winter (1992) J. Mol. Biol.
224:487-499. Vernier zone residues form a layer underlying the CDRs
and may impact on the structure of CDRs and the affinity of the
antibody.
[0057] The term "multivalent binding protein" is used in this
specification to denote a binding protein comprising two or more
antigen binding sites. The multivalent binding protein may be
engineered to have the three or more antigen binding sites, and is
generally not a naturally occurring antibody. The term
"multispecific binding protein" refers to a binding protein capable
of binding two or more related or unrelated targets. Dual variable
domain (DVD) binding proteins or immunoglobulins (DVD-Ig) as used
herein, are binding proteins that comprise two or more antigen
binding sites and are tetravalent or multivalent binding proteins.
Such DVD-Igs may be monospecific, i.e., capable of binding one
antigen or multispecific, i.e., capable of binding two or more
antigens. DVD-Ig binding proteins comprising two heavy chain DVD-Ig
polypeptides and two light chain DVD-Ig polypeptides are referred
to a DVD-Ig. Each half of a DVD-Ig comprises a heavy chain DVD-Ig
polypeptide, and a light chain DVD-Ig polypeptide, and two antigen
binding sites. Each binding site comprises a heavy chain variable
domain and a light chain variable domain with a total of 6 CDRs
involved in antigen binding per antigen binding site. DVD binding
proteins and methods of making DVD binding proteins are disclosed
in U.S. Pat. No. 7,612,181.
[0058] One aspect of the invention pertains to a DVD binding
protein comprising binding proteins capable of binding TNF-.alpha..
In a particular embodiment, the DVD binding protein is capable of
binding TNF-.alpha. and a second target.
[0059] The term "neutralizing" refers to neutralization of a
biological activity of a cytokine when a binding protein
specifically binds the cytokine. In a particular embodiment, a
neutralizing binding protein is a neutralizing antibody whose
binding to hTNF-.alpha. results in inhibition of a biological
activity of hTNF-.alpha., e.g., the neutralizing binding protein
binds hTNF-.alpha. and reduces a biologically activity of
hTNF-.alpha. by at least about 20%, 40%, 60%, 80%, 85% or more
Inhibition of a biological activity of hTNF-.alpha. by a
neutralizing binding protein can be assessed by measuring one or
more indicators of hTNF-.alpha. biological activity well known in
the art. For example neutralization of the cytoxicity of
TNF-.alpha. on L929 cells.
[0060] In another embodiment, the term "agonizing" refers to an
increase of a biological activity of TNF-.alpha. when a binding
protein specifically binds TNF-.alpha., e.g., hTNF-.alpha.. In a
particular embodiment, an agonizing binding protein is an agonistic
antibody whose binding to TNF-.alpha. results in the increase of a
biological activity of TNF-.alpha.. In a particular embodiment, the
agonistic binding protein binds TNF-.alpha. and increases a
biologically activity of TNF-.alpha. by at least about 20%, 40%,
60%, 80%, 85%, 90%, 95, 96%, 97%, 98%, 99%, and 100%. An inhibition
of a biological activity of TNF-.alpha. by an agonistic binding
protein can be assessed by measuring one or more indicators of
TNF-.alpha. biological activity well known in the art.
[0061] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-hTNF-.alpha. antibody that binds to a TNF-.alpha. antigen
and/or the neutralizing potency (or agonizing potency) of an
antibody, for example, an anti-hTNF-.alpha. antibody whose binding
to hTNF-.alpha. inhibits the biological activity of hTNF-.alpha.,
e.g., neutralization of the cytoxicity of TNF-.alpha. on L929
cells.
[0062] The terms "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin, e.g., an antibody,
or T-cell receptor binds. In certain embodiments, epitope
determinants include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and/or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody. In certain embodiments, an antibody is said to
specifically bind an antigen when it preferentially recognizes its
target antigen in a complex mixture of proteins and/or
macromolecules. Epitopes can be formed both from contiguous amino
acids or noncontiguous amino acids juxtaposed by tertiary folding
of a protein. Epitopes formed from contiguous amino acids are
typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods for determining what epitopes are
bound by a given antibody (i.e., epitope mapping) are well known in
the art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides from TNF-.alpha. are tested for reactivity with the given
anti-TNF-.alpha. antibody. Methods of determining spatial
conformation of epitopes include techniques in the art and those
described herein, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance (see, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E.
Morris, Ed. (1996)).
[0063] Also, encompassed by the present invention are antibodies
that bind to an epitope on TNF-.alpha. which comprises all or a
portion of an epitope recognized by the particular antibodies
described herein (e.g., the same or an overlapping region or a
region between or spanning the region).
[0064] Also encompassed by the present invention are antibodies
that bind the same epitope and/or antibodies that compete for
binding to TNF-.alpha., e.g., human TNF-.alpha., with the
antibodies described herein. Antibodies that recognize the same
epitope or compete for binding can be identified using routine
techniques. Such techniques include, for example, an immunoassay,
which shows the ability of one antibody to block the binding of
another antibody to a target antigen, i.e., a competitive binding
assay. Competitive binding is determined in an assay in which the
immunoglobulin under test inhibits specific binding of a reference
antibody to a common antigen, such as hTNF-.alpha.. Numerous types
of competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al. (1983) Methods in Enzymol. 9:242); solid phase direct
biotin-avidin EIA (see Kirkland et al. (1986) J. Immunol.
137:3614); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using 1-125 label (see Morel et al. (1988) Mol.
Immunol. 25(1):7); solid phase direct biotin-avidin EIA (Cheung et
al. (1990) Virol. 176:546); and direct labeled RIA. (Moldenhauer et
al. (1990) Scand. J. Immunol. 32:77). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0065] Other techniques include, for example, epitope mapping
methods, such as, x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0066] The term "surface plasmon resonance" refers to an optical
phenomenon that allows for the analysis of real-time biospecific
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example using the BIAcore system
(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For
further descriptions, see Jonsson, et al. (1993) Ann. Biol. Clin.
51:19-26; Jonsson, et al. (1991) Biotechniques 11:620-627;
Johnsson, et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson,
et al. (1991) Anal. Biochem. 198:268-277.
[0067] The term "K.sub.on" refers to the on rate constant for
association of a binding protein (e.g., an antibody) to the antigen
to form, e.g., the antibody/antigen complex as is known in the art.
The "Kon" also is known by the terms "association rate constant",
or "ka", as used interchangeably herein. This value indicating the
binding rate of an antibody to its target antigen or the rate of
complex formation between an antibody and antigen also is shown by
the equation below:
Antibody("Ab")+Antigen("Ag").fwdarw.Ab-Ag
[0068] The term "K.sub.off" refers to the off rate constant for
dissociation, or "dissociation rate constant", of a binding protein
(e.g., an antibody), from the e.g., antibody/antigen complex as is
known in the art. This value indicates the dissociation rate of an
antibody from its target antigen or separation of Ab-Ag complex
over time into free antibody and antigen as shown by the equation
below:
Ab+Ag.rarw.Ab-Ag
[0069] The term "K.sub.D" refers to the "equilibrium dissociation
constant" and refers to the value obtained in a titration
measurement at equilibrium, or by dividing the dissociation rate
constant (koff) by the association rate constant (kon). The
association rate constant, the dissociation rate constant and the
equilibrium dissociation constant are used to represent the binding
affinity of an antibody to an antigen. Methods for determining
association and dissociation rate constants are well known in the
art. Using fluorescence--based techniques offers high sensitivity
and the ability to examine samples in physiological buffers at
equilibrium. Other experimental approaches and instruments such as
a BIAcore.RTM. (biomolecular interaction analysis) assay can be
used (e.g., instrument available from BIAcore International AB, a
GE Healthcare company, Uppsala, Sweden). Additionally, a
KinExA.RTM. (Kinetic Exclusion Assay) assay, available from
Sapidyne Instruments (Boise, Id.) can also be used.
[0070] The term "labeled binding protein" refers to a protein with
a label incorporated that provides for the identification of the
binding protein. In a particular embodiment, the label is a
detectable marker, e.g., incorporation of a radiolabeled amino acid
or attachment to a polypeptide of biotinyl moieties that can be
detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Examples of labels for
polypeptides include, but are not limited to, the following:
radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.90Y .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, or .sup.153Sm); fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), enzymatic labels (e.g.,
horseradish peroxidase, luciferase, alkaline phosphatase);
chemiluminescent markers; biotinyl groups; predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags); and magnetic
agents, such as gadolinium chelates.
[0071] The term "antibody conjugate" refers to a binding protein,
such as an antibody, chemically linked to a second chemical moiety,
such as a therapeutic or cytotoxic agent. The term "agent" denotes
a chemical compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials. In a
particular embodiment, the therapeutic or cytotoxic agents include,
but are not limited to, pertussis toxin, taxol, cytochalasin B,
gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicine, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0072] The terms "crystal" and "crystallized" refers to an
antibody, or antigen binding portion thereof, that exists in the
form of a crystal. Crystals are one form of the solid state of
matter, which is distinct from other forms such as the amorphous
solid state or the liquid crystalline state. Crystals are composed
of regular, repeating, three-dimensional arrays of atoms, ions,
molecules (e.g., proteins such as antibodies), or molecular
assemblies (e.g., antigen/antibody complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships that are well-understood in the field.
The fundamental unit, or building block, that is repeated in a
crystal is called the asymmetric unit. Repetition of the asymmetric
unit in an arrangement that conforms to a given, well-defined
crystallographic symmetry provides the "unit cell" of the crystal.
Repetition of the unit cell by regular translations in all three
dimensions provides the crystal. See Giege et al., Chapter 1, In
Crystallization of Nucleic Acids and Proteins, A Practical
Approach, 2nd ed., (Ducruix and Giege, eds.) (Oxford University
Press, New York, 1999), pp. 1-16.
[0073] The term "polynucleotide" means a polymeric form of two or
more nucleotides, either ribonucleotides (RNAs) or
deoxyribonucleotides (DNAs) or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA but in a particular embodiment is double-stranded DNA.
[0074] The term "isolated polynucleotide" means a polynucleotide
(e.g., of genomic, cDNA, or synthetic origin, or some combination
thereof) that, by virtue of its origin, the "isolated
polynucleotide": is not associated with all or a portion of a
polynucleotide with which the "isolated polynucleotide" is found in
nature; is operably linked to a polynucleotide that it is not
linked to in nature; or does not occur in nature as part of a
larger sequence.
[0075] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked
One type of vector is a "plasmid", which refers to a circular
double stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors"). In general, expression vectors
of utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such
other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0076] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include both expression control sequences that are contiguous with
the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. The term
"expression control sequence" refers to polynucleotide sequences
that are necessary to effect the expression and processing of
coding sequences to which they are ligated. Expression control
sequences include appropriate transcription initiation,
termination, promoter and enhancer sequences; efficient RNA
processing signals such as splicing and polyadenylation signals;
sequences that stabilize cytoplasmic mRNA; sequences that enhance
translation efficiency (i.e., Kozak consensus sequence); sequences
that enhance protein stability; and when desired, sequences that
enhance protein secretion. The nature of such control sequences
differs depending upon the host organism; in prokaryotes, such
control sequences generally include promoter, ribosomal binding
site, and transcription termination sequence; in eukaryotes,
generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences.
[0077] "Transformation" refers to any process by which exogenous
DNA enters a host cell. Transformation may occur under natural or
artificial conditions using various methods well known in the art.
Transformation may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. The method is selected based on the host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, lipofection, and particle bombardment.
Such "transformed" cells include stably transformed cells in which
the inserted DNA is capable of replication either as an
autonomously replicating plasmid or as part of the host chromosome.
They also include cells which transiently express the inserted DNA
or RNA for limited periods of time.
[0078] The term "recombinant host cell" (or simply "host cell")
refers to a cell into which exogenous DNA has been introduced. It
should be understood that such terms are intended to refer not only
to the particular subject cell, but, to the progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term "host cell". In a particular
embodiment, host cells include prokaryotic and eukaryotic cells
selected from any of the Kingdoms of life. Eukaryotic cells include
protist, fungal, plant and animal cells. In a particular
embodiment, host cells include but are not limited to the
prokaryotic cell line E. Coli; mammalian cell lines CHO, HEK 293
and COS; the insect cell line Sf9; and the fungal cell
Saccharomyces cerevisiae.
[0079] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See, e.g., Sambrook et al. Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)).
[0080] "Transgenic organism" refers to an organism having cells
that contain a transgene, wherein the transgene introduced into the
organism (or an ancestor of the organism) expresses a polypeptide
not naturally expressed in the organism. A "transgene" is a DNA
construct, which is stably and operably integrated into the genome
of a cell from which a transgenic organism develops, directing the
expression of an encoded gene product in one or more cell types or
tissues of the transgenic organism.
[0081] The terms "regulate" and "modulate" are used
interchangeably, and, refers to a change or an alteration in the
activity of a molecule of interest (e.g., the biological activity
of hTNF-.alpha.). Modulation may be an increase or a decrease in
the magnitude of a certain activity or function of the molecule of
interest. Exemplary activities and functions of a molecule include,
but are not limited to, binding characteristics, enzymatic
activity, cell receptor activation, and signal transduction.
[0082] Correspondingly, the term "modulator" is a compound capable
of changing or altering an activity or function of a molecule of
interest (e.g., the biological activity of hTNF-.alpha.). For
example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Exemplary inhibitors include, but are
not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies are
described, e.g., in international PCT Publication WO 01/83525.
[0083] The term "agonist" refers to a modulator that, when
contacted with a molecule of interest, causes an increase in the
magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in
the absence of the agonist. Particular agonists of interest may
include, but are not limited to, TNF-.alpha. polypeptides or
polypeptides, nucleic acids, carbohydrates, or any other molecules
that bind to hTNF-.alpha..
[0084] The term "antagonist" or "inhibitor" refers to a modulator
that, when contacted with a molecule of interest causes a decrease
in the magnitude of a certain activity or function of the molecule
compared to the magnitude of the activity or function observed in
the absence of the antagonist. Particular antagonists of interest
include those that block or modulate the biological or
immunological activity of TNF-.alpha., e.g., hTNF-.alpha..
Antagonists and inhibitors of hTNF-.alpha. may include, but are not
limited to, proteins, nucleic acids, carbohydrates, or any other
molecules, which bind to hTNF-.alpha..
[0085] The term "effective amount" refers to the amount of a
therapy which is sufficient to reduce or ameliorate the severity
and/or duration of a disorder or one or more symptoms thereof,
prevent the advancement of a disorder, cause regression of a
disorder, prevent the recurrence, development, onset or progression
of one or more symptoms associated with a disorder, detect a
disorder, or enhance or improve the prophylactic or therapeutic
effect(s) of another therapy (e.g., prophylactic or therapeutic
agent).
[0086] The term "sample" is used in its broadest sense herein. A
"biological sample", includes, but is not limited to, any quantity
of a substance from a living thing or formerly living thing. Such
living things include, but are not limited to, humans, mice, rats,
monkeys, dogs, rabbits and other animals. Such substances include,
but are not limited to, blood, serum, urine, synovial fluid, cells,
organs, tissues, bone marrow, lymph nodes and spleen.
I. Antibodies that Bind Human TNF-.alpha.
[0087] One aspect of the present invention provides isolated murine
monoclonal antibodies, or antigen-binding portions thereof, that
bind to TNF-.alpha. with high affinity, a slow off rate and high
neutralizing capacity. A second aspect of the invention provides
chimeric antibodies that bind TNF-.alpha.. A third aspect of the
invention provides CDR grafted antibodies, or antigen-binding
portions thereof, that bind TNF-.alpha.. A fourth aspect of the
invention provides humanized antibodies, or antigen-binding
portions thereof, that bind TNF-.alpha.. In a particular
embodiment, the antibodies, or portions thereof, are isolated
antibodies. In an embodiment, the antibodies of the invention are
neutralizing human anti-TNF-.alpha. or modulating TNF-.alpha.
functions.
A. Method of Making Anti-TNF-.alpha. Antibodies
[0088] Antibodies of the present invention may be made by any of a
number of techniques known in the art.
1. Anti-TNF-.alpha. Monoclonal Antibodies Using Hybridoma
Technology
[0089] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et
al., eds., "Monoclonal Antibodies and T-Cell Hybridomas," In
Research Monographs in Immunology, vol. 3 (J. L. Turk, General
Editor) (Elsevier, New York, 1981), pp. 563-587. The term
"monoclonal antibody" is not limited to antibodies produced through
hybridoma technology. The term "monoclonal antibody" refers to an
antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced.
[0090] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In one embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein the hybridoma is generated by
fusing splenocytes isolated from a mouse immunized with an antigen
of the invention with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that
secrete an antibody able to bind a polypeptide of the invention.
Briefly, mice can be immunized with a TNF-.alpha. antigen. In a
particular embodiment, the TNF-.alpha. antigen is administered with
an adjuvant to stimulate the immune response. Such adjuvants
include complete or incomplete Freund's adjuvant, RIBI (muramyl
dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants
may protect the polypeptide from rapid dispersal by sequestering it
in a local deposit, or they may contain substances that stimulate
the host to secrete factors that are chemotactic for macrophages
and other components of the immune system. In a particular
embodiment, if a polypeptide is being administered, the
immunization schedule will involve two or more administrations of
the polypeptide, spread out over several weeks.
[0091] After immunization of an animal with a TNF-.alpha. antigen,
antibodies and/or antibody-producing cells may be obtained from the
animal. An anti-TNF-.alpha. antibody-containing serum is obtained
from the animal by bleeding or sacrificing the animal. The serum
may be used as it is obtained from the animal, an immunoglobulin
fraction may be obtained from the serum, or the anti-TNF-.alpha.
antibodies may be purified from the serum. Serum or immunoglobulins
obtained in this manner are polyclonal, thus having a heterogeneous
array of properties.
[0092] Once an immune response is detected, e.g., antibodies
specific for the antigen TNF-.alpha. are detected in the mouse
serum, the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by well-known techniques to any suitable
myeloma cells, for example cells from cell line SP20 available from
the ATCC. Hybridomas are selected and cloned by limited dilution.
The hybridoma clones are then assayed by methods known in the art
for cells that secrete antibodies capable of binding TNF-.alpha..
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0093] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
See, e.g., Harlow and Lane, supra. In a particular embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using TNF-.alpha., or a portion
thereof, or a cell expressing TNF-.alpha.. In a particular
embodiment, the initial screening is performed using an
enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA), or
an ELISA. An example of ELISA screening is provided in
international PCT Publication WO 00/37504.
[0094] Anti-TNF-.alpha. antibody-producing hybridomas are selected,
cloned and further screened for desirable characteristics,
including robust hybridoma growth, high antibody production and
desirable antibody characteristics, as discussed further below.
Hybridomas may be cultured and expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0095] In a particular embodiment, the hybridomas are mouse
hybridomas, as described above. In another particular embodiment,
the hybridomas are produced in a non-human, non-mouse species such
as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human
non-secretory myeloma is fused with a human cell expressing an
anti-TNF-.alpha. antibody.
[0096] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CHI domain of the heavy chain.
2. Anti-TNF-.alpha. Monoclonal Antibodies Using SLAM
[0097] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052; PCT Publication WO
92/02551 and Babcook, et al. (1996) Proc. Natl. Acad. Sci. USA
93:7843-7848. In this method, single cells secreting antibodies of
interest, e.g., lymphocytes derived from any one of the immunized
animals described in Section 1, are screened using an
antigen-specific hemolytic plaque assay, wherein the antigen
TNF-.alpha., a subunit of TNF-.alpha., or a fragment thereof, is
coupled to sheep red blood cells using a linker, such as biotin,
and used to identify single cells that secrete antibodies with
specificity for TNF-.alpha.. Following identification of
antibody-secreting cells of interest, heavy- and light-chain
variable region cDNAs are rescued from the cells by reverse
transcriptase-PCR and these variable regions can then be expressed,
in the context of appropriate immunoglobulin constant regions
(e.g., human constant regions), in mammalian host cells, such as
COS or CHO cells. The host cells transfected with the amplified
immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example by panning the transfected cells to isolate
cells expressing antibodies to TNF-.alpha.. The amplified
immunoglobulin sequences further can be manipulated in vitro, such
as by in vitro affinity maturation methods such as those described
in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
3. Anti-TNF-.alpha. Monoclonal Antibodies Using Transgenic
Animals
[0098] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with a TNF-.alpha. antigen.
In a particular embodiment, the non-human animal is a XENOMOUSE
transgenic mouse, an engineered mouse strain that comprises large
fragments of the human immunoglobulin loci and is deficient in
mouse antibody production. See, e.g., Green et al. (1994) Nature
Genet. 7:13-21 and U.S. Pat. Nos. 5,916,771; 5,939,598; 5,985,615;
5,998,209; 6,075,181; 6,091,001; 6,114,598 and 6,130,364. See also
PCT Publications WO 91/10741, published Jul. 25, 1991; WO 94/02602,
published Feb. 3, 1994; WO 96/34096 and WO 96/33735, both published
Oct. 31, 1996; WO 98/16654, published Apr. 23, 1998; WO 98/24893,
published Jun. 11, 1998; WO 98/50433, published Nov. 12, 1998; WO
99/45031, published Sep. 10, 1999; WO 99/53049, published Oct. 21,
1999; WO 00/09560, published Feb. 24, 2000; and WO 00/37504,
published Jun. 29, 2000. The XENOMOUSE transgenic mouse produces an
adult-like human repertoire of fully human antibodies, and
generates antigen-specific human Mabs. The XENOMOUSE transgenic
mouse contains approximately 80% of the human antibody repertoire
through introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and x light chain loci.
See, Mendez et al. (1997) Nature Genet. 15:146-156; Green and
Jakobovits (1998) J. Exp. Med. 188:483-495.
4. Anti-TNF-.alpha. Monoclonal Antibodies Using Recombinant
Antibody Libraries
[0099] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, U.S. Pat.
No. 5,223,409; PCT Publications WO 92/18619; WO 91/17271; WO
92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and
WO 97/29131; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al. (1990) Nature 348:552-554;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992)
J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nucl. Acid Res. 19:4133-4137; and Barbas
et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and U.S.
Patent Publication No. 2003.0186374.
[0100] The recombinant antibody library may be from a subject
immunized with TNF-.alpha., or a portion of TNF-.alpha..
Alternatively, the recombinant antibody library may be from a naive
subject, i.e., one who has not been immunized with TNF-.alpha.,
such as a human antibody library from a human subject who has not
been immunized with human TNF-.alpha.. Antibodies of the invention
are selected by screening the recombinant antibody library with the
peptide comprising human TNF-.alpha. to thereby select those
antibodies that recognize TNF-.alpha.. Methods for conducting such
screening and selection are well known in the art, such as
described in the references in the preceding paragraph. To select
antibodies of the invention having particular binding affinities
for hTNF-.alpha., such as those that dissociate from human
TNF-.alpha. with a particular k.sub.off rate constant, the
art-known method of surface plasmon resonance can be used to select
antibodies having the desired k.sub.off rate constant. To select
antibodies of the invention having a particular neutralizing
activity for hTNF-.alpha., such as those with a particular an
IC.sub.50, standard methods known in the art for assessing the
inhibition of hTNF-.alpha. activity may be used.
[0101] In one aspect, the invention pertains to an isolated
antibody, or an antigen-binding portion thereof, that binds
TNF-.alpha., e.g., human TNF-.alpha.. In a particular embodiment,
the antibody is a neutralizing antibody. In various embodiments,
the antibody is a recombinant antibody or a monoclonal
antibody.
[0102] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkmann et al. (1995) J. Immunol. Methods 182:41-50; Ames et al.
(1995) J. Immunol. Methods 184:177-186; Kettleborough et al. (1994)
Eur. T. Immunol. 24:952-958; Persic et al. (1997) Gene 187 9-18;
Burton et al. (1994) Adv. Immunol. 57:191-280; PCT Publications WO
90/02809; WO 91/10737; WO 92/01047 (PCT Application No.
PCT/GB91/01134); WO 92/18619; WO 93/11236; WO 95/15982; and WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108.
[0103] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT Publication WO 92/22324; Mullinax et al.
(1992) BioTechniques 12(6):864-869; and Sawai et al. (1995) Am. J.
Reprod. Immunol. 34:26-34; and Better et al. (1998) Science
240:1041-1043. Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods
Enzymol. 203:46-88; Shu et al. (1993) Proc. Natl. Acad Sci. USA
90:7995-7999; and Skerra et al. (1998) Science 240:1038-1041.
[0104] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 and in Roberts and
Szostak (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this
system, a covalent fusion is created between an mRNA and the
peptide or protein that it encodes by in vitro translation of
synthetic mRNAs that carry puromycin, a peptidyl acceptor
antibiotic, at their 3' end. Thus, a specific mRNA can be enriched
from a complex mixture of mRNAs (e.g., a combinatorial library)
based on the properties of the encoded peptide or protein, e.g.,
antibody, or portion thereof, such as binding of the antibody, or
portion thereof, to the dual specificity antigen. Nucleic acid
sequences encoding antibodies, or portions thereof, recovered from
screening of such libraries can be expressed by recombinant means
as described above (e.g., in mammalian host cells) and, moreover,
can be subjected to further affinity maturation by either
additional rounds of screening of mRNA-peptide fusions in which
mutations have been introduced into the originally selected
sequence(s), or by other methods for affinity maturation in vitro
of recombinant antibodies, as described above.
[0105] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed Wittrup et al. U.S.
Pat. No. 6,699,658 and Frenken et al., U.S. Pat. No. 6,114,147.
B. Production of Recombinant TNF-.alpha. Antibodies
[0106] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains is (are) transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is contemplated, for example, in mammalian host
cells, because such eukaryotic cells (and in particular mammalian
cells) are more likely than prokaryotic cells to assemble and
secrete a properly folded and immunologically active antibody.
[0107] Mammalian host cells for expressing the recombinant
antibodies of the invention 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)
J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2
cells. When recombinant expression vectors encoding antibody genes
are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, in particular, secretion of the antibody into the culture
medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using standard protein
purification methods.
[0108] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention and the other heavy and light chain are specific for an
antigen other than the antigens of interest by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0109] In an exemplary system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, 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 CMV enhancer/AdMLP promoter
regulatory elements 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, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
1. Anti hTNF-.alpha. Antibodies
[0110] Table 5 is a list of amino acid sequences of VH and VL
regions (CDR sequences bolded) of anti-hTNF-.alpha. antibodies of
the invention.
TABLE-US-00005 TABLE 5 List of Amino Acid Sequences of Murine
Anti-hTNF-.alpha. Antibody VH And VL Regions SEQ Sequence ID NO.
Protein region 123456789012345678901234567890 22 VH
QVQLKESGPGLVAPSQSLSITCTVSGFSLT MAK195
DYGVNWVRQPPGKGLEWLGMIWGDGSTDYD STLKSRLSISKDNSKSQIFLKMNSLQTDDT
ARYYCAREWHHGPVAYWGQGTLVTVSA VH Residues 31-35 DYGVN MAK195CDR-H1 of
SEQ ID NO: 22 VH Residues 50-65 MIWGDGSTDYDSTLKS MAK195CDR-H2 of
SEQ ID NO: 22 VH Residues 98-106 EWHHGPVAY MAK195CDR-H3 of SEQ ID
NO: 22 23 VL DIVMTQSHKFMSTTVGDRVSITCKASQAVS MAK195
SAVAWYQQKPGQSPKLLIYWASTRHTGVPD RFTGSGSVTDFTLTIHNLQAEDLALYYCQQ
HYSTPFTFGSGTKLEIKR VL Residues 24-34 KASQAVSSAVA MAK195CDR-L1 of
SEQ ID NO: 23 VL Residues 50-56 WASTRHT MAK195CDR-L2 of SEQ ID NO:
23 VL Residues 89-97 QQHYSTPFT MAK195CDR-L3 of SEQ ID NO: 23
2. Anti-hTNF-.alpha. Chimeric Antibodies
[0111] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art and
discussed in detail in Example 2.1. See, e.g., Morrison (1985)
Science 229:1202; Oi et al. (1986) BioTechniques 4:214-221; Gillies
et al. (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397. In addition, techniques
developed for the production of "chimeric antibodies" (Morrison et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger et
al. (1984), Nature 312:604-608; Takeda et al. (1985) Nature
314:452-454; by splicing genes from a mouse antibody molecule of
appropriate antigen specificity together with genes from a human
antibody molecule of appropriate biological activity can be
used.
[0112] In one embodiment, the chimeric antibodies of the invention
are produced by replacing the heavy chain constant region of the
murine monoclonal anti human TNF-.alpha. antibodies described in
section 1 with a human IgG1 constant region.
3. Anti-TNF-.alpha. CDR-Grafted Antibodies
[0113] CDR-grafted antibodies of the invention comprise heavy and
light chain variable region sequences from a human antibody wherein
one or more of the CDR regions of VH and/or VL are replaced with
CDR sequences of the murine antibodies of the invention. A
framework sequence from any human antibody may serve as the
template for CDR grafting. However, straight chain replacement onto
such a framework often leads to some loss of binding affinity to
the antigen. The more homologous a human antibody is to the
original murine antibody, the less likely the possibility that
combining the murine CDRs with the human framework will introduce
distortions in the CDRs that could reduce affinity. Therefore, in
an embodiment, the human variable framework that is chosen to
replace the murine variable framework apart from the CDRs have at
least a 65% sequence identity with the murine antibody variable
region framework. In a particular embodiment, the human and murine
variable regions apart from the CDRs have at least 70% sequence
identify. In a particular embodiment, the human and murine variable
regions apart from the CDRs have at least 75% sequence identity. In
a particular embodiment, the human and murine variable regions
apart from the CDRs have at least 80% sequence identity. Methods
for producing chimeric antibodies are known in the art and
discussed in detail in Example 2.2. (also see EP Patent No. EP 0
239 400; PCT Publication WO 91/09967; U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106;
EP 0 519 596; Padlan (1991) Mol. Immunol. 28(4/5):489-498;
Studnicka et al. (1994) Protein Eng. 7(6):805-814; Roguska et al.
(1994) Proc. Natl. Acad. Sci. USA 91:969-973), and chain shuffling
(U.S. Pat. No. 5,565,352).
[0114] In a specific embodiment the invention provides CDR grafted
antibodies with VH and/or VL chains as described in Table 6.
TABLE-US-00006 TABLE 6 CDR Grafted Antibodies SEQ Sequence ID No.
Protein region 123456789012345678901234567890 24 hMAK195VH.1z
QVQLQESGPGLVKPSETLSLTCTVSGGSIS DYGVNWIRQPPGKGLEWIGMIWGDGSTDYD
STLKSRVTISVDTSKNQFSLKLSSVTAADT AVYYCAREWHHGPVAYWCQGTLVTVSS 25
hMAK195VH.2z EVQLVESGGGLIQPGGSLRLSCAASGFTVS
DYGVNWVRQAPGKGLEWVSMIWGDGSTDYD STLKSRFTISRDNSKNTLYLQMNSLRAEDT
AVYYCAREWHHGPVAYWGQGTLVTVSS 26 hMAK195Vk.1
DIQMTQSPSSLSASVGDRVTITCKASQAVS SAVAWYQQKPGKAPKLLIYWASTRHTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ HYSTPFTFGQGTKLEIK 27 hMAK195Vk.2z
EIVMTQSPATLSVSPGERATLSCKASQAVS SAVAWYQQKPGQAPRLLIYWASTRHTGIPA
RFSGSGSGTEFTLTISSLQSEDFAVYYCQQ HYSTPFTFGQGTKLEIK
4. Anti-hTNF-.alpha. Humanized Antibodies
[0115] Humanized antibodies are antibody molecules from non-human
species antibody that binds the desired antigen having one or more
complementarity determining regions (CDRs) from the non-human
species and framework regions from a human immunoglobulin molecule.
Known human Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez-/query.fcgi;
www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uni-heidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH-05/kuby05.htm;
www.library.thinkquestorg/12429/Immune/Antibody.html; www hhmi
org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.-html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html-;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/lin-ks.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;
baserv.uci.kun.nl/.about.jraats/linksl html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;
www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.unizh ch/.abouthonegger/AHOsem-inar/SlideOl.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/; www.nimr
mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;
www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983. Such imported sequences can be used to reduce
immunogenicity or reduce, enhance or modify binding, affinity,
on-rate, off-rate, avidity, specificity, half-life, or any other
suitable characteristic, as known in the art.
[0116] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, improve, antigen binding. These framework
substitutions are identified by methods well known in the art,
e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., U.S. Pat. No.
5,585,089; Riechmann et al. (1988) Nature 332:323-327.)
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as but not limited to those described in Jones et al.
(1986) Nature 321:522-525; Verhoeyen et al. (1988) Science
239:1534-1536; Sims et al. (1993) J. Immunol. 151: 2296-2308;
Chothia and Lesk (1987) J. Mol. Biol. 196:901-917; Carter et al.
(1992) Proc. Natl. Acad. Sci. USA 89:4285-4289; Presta et al.
(1993) J. Immunol. 151:2623-2632; Padlan (1991) Mol. Immunol.
28(4/5):489-498; Studnicka et al. (1994) Protein Engineering
7(6):805-814; Roguska. et al. (1994) Proc. Natl. Acad. Sci. USA
91:969-973; PCT Publication Nos. WO 91/09967, WO 99/06834
(International Application No. PCT/US98/16280), WO 97/20032 (Appln.
No. PCT/US96/18978), WO 92/11272 (Appln. No. PCT/US91/09630), WO
92/03461 (Appln. No. PCT/US91/05939), WO 94/18219 (Appln. No.
PCT/US94/01234), WO 92/01047 (Appln. No. PCT/GB91/01134), WO
93/06213 (Appln. No. PCT/GB92/01755), WO 90/14443, WO 90/14424, and
WO 90/14430; European Publication Nos. EP 0 592 106, EP 0 519 596,
and EP 0 239 400, U.S. Pat. Nos. 5,565,332; 5,723,323; 5,976,862;
5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886;
5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089;
5,225,539; and 4,816,567.
C. Production of Antibodies and Antibody-Producing Cell Lines
[0117] In an embodiment, anti-TNF-.alpha. antibodies of the present
invention, exhibit a high capacity to reduce or to neutralize
TNF-.alpha. activity, e.g., as assessed by any one of several in
vitro and in vivo assays known in the art. Alternatively,
anti-TNF-.alpha. antibodies of the present invention, also exhibit
a high capacity to increase or agonize TNF-.alpha. activity.
[0118] In particular embodiments, the isolated antibody, or
antigen-binding portion thereof, binds human TNF-.alpha., wherein
the antibody, or antigen-binding portion thereof, dissociates from
human TNF-.alpha. with a k.sub.off rate constant of about
0.1s.sup.-1 or less, as determined by surface plasmon resonance, or
which inhibits human TNF-.alpha. activity with an IC.sub.50 of
about 1.times.10.sup.-6M or less. Alternatively, the antibody, or
an antigen-binding portion thereof, may dissociate from human
TNF-.alpha. with a k.sub.off rate constant of about
1.times.10.sup.-2s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human TNF-.alpha. activity with an
IC.sub.50 of about 1.times.10.sup.-7M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human TNF-.alpha. with a k.sub.off rate constant of about
1.times.10.sup.-3s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human TNF-.alpha. activity with an
IC.sub.50 of about 1.times.10.sup.-8M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human TNF-.alpha. with a k.sub.off rate constant of about
1.times.10.sup.-4s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human TNF-.alpha. activity with an
IC.sub.50 of about 1.times.10.sup.-9M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human TNF-.alpha. with a k.sub.off rate constant of about
1.times.10.sup.-5s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human TNF-.alpha. activity with an
IC.sub.50 of about 1.times.10.sup.-10 M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from human TNF-.alpha. with a k.sub.off rate constant of about
1.times.10.sup.-5s.sup.-1 or less, as determined by surface plasmon
resonance, or may inhibit human TNF-.alpha. activity with an
IC.sub.50 of about 1.times.10.sup.-11 M or less.
[0119] In certain embodiments, the antibody comprises a heavy chain
constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM
or IgD constant region. In an embodiment, the heavy chain constant
region is an IgG1 heavy chain constant region or an IgG4 heavy
chain constant region. Furthermore, the antibody can comprise a
light chain constant region, either a kappa light chain constant
region or a lambda light chain constant region. In another
embodiment, the antibody comprises a kappa light chain constant
region. Alternatively, the antibody portion can be, for example, a
Fab fragment or a single chain Fv fragment.
[0120] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (See U.S.
Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody
mediates several important effector functions, e.g., cytokine
induction, ADCC, phagocytosis, complement dependent cytotoxicity
(CDC) and half-life/clearance rate of antibody and antigen-antibody
complexes. In some cases these effector functions are desirable for
therapeutic antibody but in other cases might be unnecessary or
even deleterious, depending on the therapeutic objectives. Certain
human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and
CDC via binding to Fc.gamma.Rs and complement C1q, respectively.
Neonatal Fc receptors (FcRn) are the critical components
determining the circulating half-life of antibodies. In still
another embodiment at least one amino acid residue is replaced in
the constant region of the antibody, for example the Fc region of
the antibody, such that effector functions of the antibody are
altered.
[0121] One embodiment provides a labeled binding protein wherein an
antibody or antibody portion of the invention is derivatized or
linked to another functional molecule (e.g., another peptide or
protein). For example, a labeled binding protein of the invention
can be derived by functionally linking an antibody or antibody
portion of the invention (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
associate of the antibody or antibody portion with another molecule
(such as a streptavidin core region or a polyhistidine tag).
[0122] Useful detectable agents with which an antibody or antibody
portion of the invention may be derivatized include fluorescent
compounds. Exemplary fluorescent detectable agents include
fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
[0123] Another embodiment of the invention provides a crystallized
binding protein. In an embodiment, the invention relates to
crystals of whole anti-TNF-.alpha. antibodies and fragments thereof
as disclosed herein, and formulations and compositions comprising
such crystals. In one embodiment the crystallized binding protein
has a greater half-life in vivo than the soluble counterpart of the
binding protein. In another embodiment the binding protein retains
biological activity after crystallization.
[0124] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in PCT
Publication WO 02/72636.
[0125] Another embodiment of the invention provides a glycosylated
binding protein wherein the antibody or antigen-binding portion
thereof comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Protein glycosylation depends on the amino acid
sequence of the protein of interest, as well as the host cell in
which the protein is expressed. Different organisms may produce
different glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (nucleotide sugars)
available. Due to such factors, protein glycosylation pattern, and
composition of glycosyl residues, may differ depending on the host
system in which the particular protein is expressed. Glycosyl
residues useful in the invention may include, but are not limited
to, glucose, galactose, mannose, fucose, n-acetylglucosamine and
sialic acid. In an embodiment, the glycosylated binding protein
comprises glycosyl residues such that the glycosylation pattern is
human.
[0126] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0127] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (U.S. Pat. Nos. 7,449,308 and
7,029,872).
[0128] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. In an embodiment, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
D. Uses of Anti-TNF-.alpha. Antibodies
[0129] Given their ability to bind to human TNF-.alpha., e.g., the
anti-human TNF-.alpha. antibodies, or portions thereof, of the
invention can be used to detect TNF-.alpha. (e.g., in a biological
sample, such as serum or plasma), using a conventional immunoassay,
such as an enzyme linked immunosorbent assays (ELISA), an
radioimmunoassay (RIA) or tissue immunohistochemistry. The
invention provides a method for detecting TNF-.alpha. in a
biological sample comprising contacting a biological sample with an
antibody, or antibody portion, of the invention and detecting
either the antibody (or antibody portion) bound to TNF-.alpha. or
unbound antibody (or antibody portion), to thereby detect
TNF-.alpha. in the biological sample. The antibody is directly or
indirectly labeled with a detectable substance to facilitate
detection of the bound or unbound antibody. Suitable detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177I, .sup.166Ho, or .sup.153Sm.
[0130] Alternative to labeling the antibody, human TNF-.alpha. can
be assayed in biological fluids by a competition immunoassay
utilizing rhTNF-.alpha. standards labeled with a detectable
substance and an unlabeled anti-human TNF-.alpha. antibody. In this
assay, the biological sample, the labeled rhTNF-.alpha. standards
and the anti-human TNF-.alpha. antibody are combined and the amount
of labeled rhTNF-.alpha. standard bound to the unlabeled antibody
is determined. The amount of human TNF-.alpha. in the biological
sample is inversely proportional to the amount of labeled
rhTNF-.alpha. standard bound to the anti-TNF-.alpha. antibody.
Similarly, human TNF-.alpha. can also be assayed in biological
fluids by a competition immunoassay utilizing rhTNF-.alpha.
standards labeled with a detectable substance and an unlabeled
anti-human TNF-.alpha. antibody.
[0131] In an embodiment, the antibodies and antibody portions of
the invention are capable of neutralizing TNF-.alpha. activity,
e.g., human TNF-.alpha. activity, both in vitro and in vivo. In
another embodiment, the antibodies and antibody portions of the
invention are capable of increasing or agonizing human TNF-.alpha.
activity, e.g., human TNF-.alpha. activity. Accordingly, such
antibodies and antibody portions of the invention can be used to
inhibit or increase hTNF-.alpha. activity, e.g., in a cell culture
containing hTNF-.alpha., in human subjects or in other mammalian
subjects having TNF-.alpha. with which an antibody of the invention
cross-reacts. In one embodiment, the invention provides a method
for inhibiting or increasing hTNF-.alpha. activity comprising
contacting hTNF-.alpha. with an antibody or antibody portion of the
invention such that hTNF-.alpha. activity is inhibited or
increased. For example, in a cell culture containing, or suspected
of containing hTNF-.alpha., an antibody or antibody portion of the
invention can be added to the culture medium to inhibit or increase
hTNF-.alpha. activity in the culture.
[0132] In another embodiment, the invention provides a method for
reducing or increasing hTNF-.alpha. activity in a subject,
advantageously from a subject suffering from a disease or disorder
in which TNF-.alpha.-activity is detrimental or, alternatively,
beneficial. The invention provides methods for reducing or
increasing TNF-.alpha. activity in a subject suffering from such a
disease or disorder, which method comprises administering to the
subject an antibody or antibody portion of the invention such that
TNF-.alpha. activity in the subject is reduced or increased. In a
particular embodiment, the TNF-.alpha. is human TNF-.alpha., and
the subject is a human subject. Alternatively, the subject can be a
mammal expressing a TNF-.alpha. to which an antibody of the
invention is capable of binding. Still further the subject can be a
mammal into which TNF-.alpha. has been introduced (e.g., by
administration of TNF-.alpha. or by expression of a TNF-.alpha.
transgene). An antibody of the invention can be administered to a
human subject for therapeutic purposes. Moreover, an antibody of
the invention can be administered to a non-human mammal expressing
a TNF-.alpha. with which the antibody is capable of binding for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention
(e.g., testing of dosages and time courses of administration).
[0133] The term "a disorder in which TNF-.alpha. activity is
detrimental" includes diseases and other disorders in which the
presence of TNF-.alpha. activity in a subject suffering from the
disorder has been shown to be or is suspected of being either
responsible for the pathophysiology of the disorder or a factor
that contributes to a worsening of the disorder. Accordingly, a
disorder in which TNF-.alpha. activity is detrimental is a disorder
in which reduction of TNF-.alpha. activity is expected to alleviate
the symptoms and/or progression of the disorder. Such disorders may
be evidenced, for example, by an increase in the concentration of
TNF-.alpha. in a biological fluid of a subject suffering from the
disorder (e.g., an increase in the concentration of TNF-.alpha. in
serum, plasma, synovial fluid, etc. of the subject), which can be
detected, for example, using an anti-TNF-.alpha. antibody as
described above. Non-limiting examples of disorders that can be
treated with the antibodies of the invention include those
disorders discussed in the section below pertaining to
pharmaceutical compositions of the antibodies of the invention.
[0134] Alternatively, the term "a disorder in which TNF-.alpha.
activity is beneficial" include diseases and other disorders in
which the presence of TNF-.alpha. activity in a subject suffering
from the disorder has been shown to be or is suspected of being
either beneficial for treating the pathophysiology of the disorder
or a factor that contributes to a treatment of the disorder.
Accordingly, a disorder in which TNF-.alpha. activity is beneficial
is a disorder in which an increase of TNF-.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Non-limiting examples of disorders that can be treated
with the antibodies of the invention include those disorders
discussed in the section below pertaining to pharmaceutical
compositions of the antibodies of the invention.
E. Pharmaceutical Compositions
[0135] The invention also provides pharmaceutical compositions
comprising an antibody, or antigen-binding portion thereof, of the
invention and a pharmaceutically acceptable carrier. The
pharmaceutical compositions comprising antibodies of the invention
are for use in, but not limited to, diagnosing, detecting, or
monitoring a disorder, in preventing, treating, managing, or
ameliorating of a disorder or one or more symptoms thereof, and/or
in research. In a specific embodiment, a composition comprises one
or more antibodies of the invention. In another embodiment, the
pharmaceutical composition comprises one or more antibodies of the
invention and one or more prophylactic or therapeutic agents other
than antibodies of the invention for treating a disorder in which
TNF-.alpha. activity is detrimental. In a particular embodiment,
the prophylactic or therapeutic agents known to be useful for or
having been or currently being used in the prevention, treatment,
management, or amelioration of a disorder or one or more symptoms
thereof. In accordance with these embodiments, the composition may
further comprise of a carrier, diluent or excipient.
[0136] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. The term
"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. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, isotonic agents, for
example, sugars, polyalcohols such as mannitol, sorbitol, or sodium
chloride in the composition, may be included. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody or antibody portion.
[0137] Various delivery systems are known and can be used to
administer one or more antibodies of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu
and Wu (1987) J. Biol. Chem. 262:4429-4432), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods
of administering a prophylactic or therapeutic agent of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration,
intratumoral administration, and mucosal administration (e.g.,
intranasal and oral routes). In addition, pulmonary administration
can be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913;
and 5,290,540; and PCT Publication Nos. WO 92/19244, WO 97/32572,
WO 97/44013, WO 98/31346, and WO 99/66903. In one embodiment, an
antibody of the invention, combination therapy, or a composition of
the invention is administered using Alkermes AIR.RTM. pulmonary
drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In a
specific embodiment, prophylactic or therapeutic agents of the
invention are administered intramuscularly, intravenously,
intratumorally, orally, intranasally, pulmonary, or subcutaneously.
The prophylactic or therapeutic agents may be administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local.
[0138] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment; this may be achieved by, for
example, and not by way of limitation, local infusion, by
injection, or by means of an implant, said implant being of a
porous or non-porous material, including membranes and matrices,
such as sialastic membranes, polymers, fibrous matrices (e.g.,
Tissuel.RTM.), or collagen matrices. In one embodiment, an
effective amount of one or more antibodies of the invention
antagonists is administered locally to the affected area to a
subject to prevent, treat, manage, and/or ameliorate a disorder or
a symptom thereof. In another embodiment, an effective amount of
one or more antibodies of the invention is administered locally to
the affected area in combination with an effective amount of one or
more therapies (e.g., one or more prophylactic or therapeutic
agents) other than an antibody of the invention of a subject to
prevent, treat, manage, and/or ameliorate a disorder or one or more
symptoms thereof.
[0139] In another embodiment, the prophylactic or therapeutic agent
can be delivered in a controlled release or sustained release
system. In one embodiment, a pump may be used to achieve controlled
or sustained release (see Langer, supra; Sefton (1987) CRC Crit.
Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980) Surgery
88:507-516; Saudek et al. (1989) N. Engl. J. Med. 321:574-579). In
another embodiment, polymeric materials can be used to achieve
controlled or sustained release of the therapies of the invention
(see e.g., Goodson, J. M., Chapter 6, In Medical Applications of
Controlled Release, Vol. II, Applications and Evaluation, (Langer
and Wise, eds.) (CRC Press, Inc., Boca Raton, 1984), pp. 115-138;
Controlled Drug Bioavailability, Drug Product Design and
Performance, (Smolen and Ball, eds.) (Wiley, New York, 1984);
Langer and Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem.
Phys. C23(1):61-126; see also Levy et al. (1985) Science
228:190-192; During et al. (1989) Ann. Neurol. 25:351-356; Howard
et al. (1989) J. Neurosurg. 71:105-112); U.S. Pat. Nos. 5,679,377;
5,916,597; 5,912,015; 5,989,463; and 5,128,326; and PCT
Publications WO 99/15154 and WO 99/20253. Examples of polymers used
in sustained release formulations include, but are not limited to,
poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a
particular embodiment, the polymer used in a sustained release
formulation is inert, free of leachable impurities, stable on
storage, sterile, and biodegradable. In yet another embodiment, a
controlled or sustained release system can be placed in proximity
of the prophylactic or therapeutic target, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, In Medical
Applications of Controlled Release, Vol. II, supra, pp. 115-138
(1984).
[0140] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; PCT publication WO
91/05548; PCT publication WO 96/20698, Ning et al. (1996)
Radiotherapy Oncol. 39:179-189; Song et al. (1996) PDA J. Pharm.
Sci. Technol. 50:372-377; Cleek et al. (1997) Proceed. Int'l. Symp.
Control. Rel. Bioact. Mater. 24:853-854, and Lam et al. (1997)
Proceed. Int'l. Symp. Control Rel. Bioact. Matter. 24:759-760.
[0141] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, DuPont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al. (1991) Proc. Natl. Acad. Sci.
USA 88:1864-1868). Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression by homologous recombination.
[0142] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0143] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well-known to one of
skill in the art. See, e.g., Remington's Pharmaceutical Sciences
and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack
Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage
forms, viscous to semi-solid or solid forms comprising a carrier or
one or more excipients compatible with topical application and
having a dynamic viscosity greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, optionally in combination with a solid or
liquid inert carrier, is packaged in a mixture with a pressurized
volatile (e.g., a gaseous propellant, such as FREON.RTM.) or in a
squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples
of such additional ingredients are well-known in the art.
[0144] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0145] If the method of the invention comprises oral
administration, compositions can be formulated orally in the form
of tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0146] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; and 5,290,540; and PCT Publications WO 92/19244, WO
97/32572, WO 97/44013, WO 98/31346, and WO 99/66903. In a specific
embodiment, an antibody of the invention, combination therapy,
and/or composition of the invention is administered using Alkermes
AIR.RTM. pulmonary drug delivery technology (Alkermes, Inc.,
Cambridge, Mass. US).
[0147] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0148] The methods of the invention may additionally comprise of
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0149] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0150] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0151] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. In an embodiment,
one or more of the prophylactic or therapeutic agents or
pharmaceutical compositions of the invention is supplied as a dry
sterile lyophilized powder in a hermetically sealed container at a
unit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at
least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at
least 75 mg, or at least 100 mg. The lyophilized prophylactic or
therapeutic agents or pharmaceutical compositions of the invention
should be stored at between 2.degree. C. and 8.degree. C. in its
original container and the prophylactic or therapeutic agents, or
pharmaceutical compositions of the invention should be administered
within 1 week, within 5 days, within 72 hours, within 48 hours,
within 24 hours, within 12 hours, within 6 hours, within 5 hours,
within 3 hours, or within 1 hour after being reconstituted. In an
alternative embodiment, one or more of the prophylactic or
therapeutic agents or pharmaceutical compositions of the invention
is supplied in liquid form in a hermetically sealed container
indicating the quantity and concentration of the agent. In an
embodiment, the liquid form of the administered composition is
supplied in a hermetically sealed container at least 0.25 mg/ml, at
least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg/ml, at least 5
mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at
least 25 mg/ml, at least 50 mg/ml, at least 75 mg/ml or at least
100 mg/ml. The liquid form should be stored at between 2.degree. C.
and 8.degree. C. in its original container.
[0152] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. In an embodiment, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampoule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ
surfactants.
[0153] The compositions of this invention may be in a variety of
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 particular form depends
on the intended mode of administration and therapeutic application.
Typical compositions are in the form of injectable or infusible
solutions, such as compositions similar to those used for passive
immunization of humans with other antibodies. The mode of
administration includes parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a particular
embodiment, the antibody is administered by intravenous infusion or
injection. In another particular embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0154] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. 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 the active compound (i.e., antibody or antibody
portion) 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 the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the methods of preparation include vacuum
drying and spray-drying that yields a powder of the active
ingredient 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.
[0155] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, for example, the
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, 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 to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0156] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0157] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which TNF-.alpha. activity is detrimental.
For example, an anti-hTNF-.alpha. antibody or antibody portion of
the invention may be coformulated and/or coadministered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies.
[0158] In certain embodiments, an antibody to TNF-.alpha. or
fragment thereof is linked to a half-life extending vehicle known
in the art. Such vehicles include, but are not limited to, the Fc
domain, polyethylene glycol, and dextran. Such vehicles are
described, e.g., in U.S. Pat. No. 6,660,843.
[0159] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding an antibody of the invention or
another prophylactic or therapeutic agent of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded antibody or
prophylactic or therapeutic agent of the invention that mediates a
prophylactic or therapeutic effect.
[0160] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al. (1993) Clin.
Pharm. 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev
(1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993)
Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev.
Biochem. 62:191-217; Robinson (1993) Trends Biotechnol. 11(5):155.
Methods commonly known in the art of recombinant DNA technology
which can be used are described in Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
and Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY (1990). Detailed descriptions of various methods
of gene therapy are disclosed in U.S. Patent Publication No.
2009/0297514.
[0161] TNF-.alpha. plays a critical role in the pathology
associated with a variety of diseases involving immune and
inflammatory elements. These diseases include, but are not limited
to, Acquired Immunodeficiency Disease Syndrome; Acquired
Immunodeficiency Related Diseases; acquired pernicious anaemia;
acute coronary syndromes; acute and chronic pain (different forms
of pain); acute idiopathic polyneuritis; acute immune disease
associated with organ transplantation; acute or chronic immune
disease associated with organ transplantation; acute inflammatory
demyelinating polyradiculoneuropathy; acute ischemia; acute liver
disease; acute rheumatic fever; acute transverse myelitis;
Addison's disease; adult (acute) respiratory distress syndrome;
Adult Still's Disease; alcoholic cirrhosis; alcohol-induced liver
injury; allergic diseases; allergy; alopecia; alopecia areata;
Alzheimer's disease; anaphylaxis; ankylosing spondylitis;
ankylosing spondylitis associated lung disease; Anti-Phospholipid
Antibody Syndrome; aplastic anemia; arteriosclerosis; arthropathy;
asthma; atheromatous disease/arteriosclerosis; atherosclerosis;
atopic allergy; atopic eczema; atopic dermatitis; atrophic
autoimmune hypothyroidism; autoimmune bullous disease; autoimmune
dermatitis; autoimmune diabetes; autoimmune disorder associated
with Streptococcus infection; autoimmune enteropathy; autoimmune
haemolytic anaemia; autoimmune hepatitis; autoimmune hearing loss;
Autoimmune Lymphoproliferative Syndrome (ALPS); autoimmune mediated
hypoglycaemia; autoimmune myocarditis; autoimmune neutropenia;
autoimmune premature ovarian failure; autoimmune thrombocytopenia
(AITP); autoimmune thyroid disease; autoimmune uveitis;
bronchiolitis obliterans; Behcet's disease; blepharitis;
bronchiectasis; bullous pemphigoid; cachexia; cardiovascular
disease; catastrophic antiphospholipid syndrome; celiac disease;
cervical spondylosis; chlamydia; choleostasis; chronic active
hepatitis; chronic eosinophilic pneumonia; chronic fatigue
syndrome; chronic immune disease associated with organ
transplantation; chronic ischemia; chronic liver diseases; chronic
mucocutaneous candidiasis; cicatricial pemphigoid; clinically
isolated syndrome (CIS) with risk for multiple sclerosis; common
varied immunodeficiency (common variable hypogammaglobulinaemia);
connective tissue disease associated interstitial lung disease;
conjunctivitis; Coombs positive haemolytic anaemia; childhood onset
psychiatric disorder; chronic obstructive pulmonary disease (COPD);
Crohn's disease; cryptogenic autoimmune hepatitis; cryptogenic
fibrosing alveolitis; dacryocystitis; depression; dermatitis
scleroderma; dermatomyositis; dermatomyositis/polymyositis
associated lung disease; diabetic retinopathy; diabetes mellitus;
dilated cardiomyopathy; discoid lupus erythematosus; disk
herniation; disk prolapse; disseminated intravascular coagulation;
drug-induced hepatitis; drug-induced interstitial lung disease;
drug induced immune hemolytic anemia; endocarditis; endometriosis;
endophthalmitis; enteropathic synovitis; episcleritis; erythema
multiforme; erythema multiforme major; female infertility;
fibrosis; fibrotic lung disease; gestational pemphigoid; giant cell
arteritis (GCA); glomerulonephritides; goitrous autoimmune
hypothyroidism (Hashimoto's disease); Goodpasture's syndrome; gouty
arthritis; graft versus host disease (GVHD); Grave's disease; group
B streptococci (GBS) infection; Guillain-Barre syndrome (GBS);
haemosiderosis associated lung disease; hay fever; heart failure;
hemolytic anemia; Henoch-Schoenlein purpurea; hepatitis B;
hepatitis C; Hughes Syndrome; Huntington's chorea; hyperthyroidism;
hypoparathyroidism; idiopathic leucopaenia; idiopathic
thrombocytopaenia; idiopathic Parkinson's Disease; idiopathic
interstitial pneumonia; idiosyncratic liver disease; IgE-mediated
Allergy; Immune hemolytic anemiae; inclusion body myositis;
infectious diseases; Infectious ocular inflammatory disease;
inflammatory bowel disease; Inflammatory demyelinating disease;
Inflammatory heart disease; Inflammatory kidney disease; insulin
dependent diabetes mellitus; interstitial pneumonitis; IPF/UIP;
iritis; juvenile chronic arthritis; juvenile pernicious anaemia;
juvenile rheumatoid arthritis; Kawasaki's disease; keratitis;
keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meier
Disease; Landry's paralysis; Langerhan's cell histiocytosis; linear
IgA disease; livedo reticularis; Lyme arthritis; lymphocytic
infiltrative lung disease; macular degeneration; male infertility
idiopathic or NOS; malignancies; microscopic vasculitis of the
kidneys; microscopic polyangiitis; mixed connective tissue disease
associated lung disease; Morbus Bechterev; motor neuron disorders;
mucous membrane pemphigoid; multiple sclerosis (all subtypes:
primary progressive, secondary progressive, relapsing remitting,
etc.); multiple organ failure; myalgic encephalitis/Royal Free
Disease; Myasthenia Gravis; myelodysplastic syndrome; myocardial
infarction; myocarditis; nephrotic syndrome; nerve root disorders;
neuropathy; non-alcoholic steatohepatitis; non-A non-B hepatitis;
optic neuritis; organ transplant rejection; osteoarthritis;
osteolysis; ovarian cancer; ovarian failure; pancreatitis;
parasitic diseases; Parkinson's disease; pauciarticular JRA;
pemphigoid; pemphigus foliaceus; pemphigus vulgaris; peripheral
artery occlusive disease (PAOD); peripheral vascular disease (PVD);
peripheral artery disease (PAD); phacogenic uveitis; phlebitis;
polyarteritis nodosa (or periarteritis nodosa); polychondritis;
polymyalgia rheumatica; poliosis; polyarticular JRA; polyendocrine
deficiency syndrome; polymyositis; polyglandular deficiency type I
and polyglandular deficiency type II; polymyalgia rheumatica (PMR);
postinfectious interstitial lung disease; post-inflammatory
interstitial lung disease; post-pump syndrome; premature ovarian
failure; primary biliary cirrhosis; primary myxoedema; primary
Parkinsonism; primary sclerosing cholangitis; primary sclerosing
hepatitis; primary vasculitis; prostate and rectal cancer and
hematopoietic malignancies (leukemia and lymphoma); prostatitis;
psoriasis; psoriasis type 1; psoriasis type 2; psoriatic arthritis;
psoriatic arthropathy; pulmonary hypertension secondary to
connective tissue disease; pulmonary manifestation of polyarteritis
nodosa; pure red cell aplasia; primary adrenal insufficiency;
radiation fibrosis; reactive arthritis; Reiter's disease; recurrent
neuromyelitis optica; renal disease NOS; restenosis; rheumatoid
arthritis; rheumatoid arthritis associated interstitial lung
disease; rheumatic heart disease; SAPHO (synovitis, acne,
pustulosis, hyperostosis, and osteitis); sarcoidosis;
schizophrenia; Schmidt's syndrome; scleroderma; secondary
amyloidosis; shock lung; scleritis; sciatica; secondary adrenal
insufficiency; sepsis syndrome; septic arthritis; septic shock;
seronegative arthropathy; silicone associated connective tissue
disease Sjogren's disease associated lung disease; Sjorgren's
syndrome; Sneddon-Wilkinson dermatosis; sperm autoimmunity;
spondyloarthropathy; spondylitis ankylosans; Stevens-Johnson
Syndrome (SJS); Still's disease; stroke; sympathetic ophthalmia;
systemic inflammatory response syndrome; systemic lupus
erythematosus; systemic lupus erythematosus associated lung
disease; systemic sclerosis; systemic sclerosis associated
interstitial lung disease; Takayasu's disease/arteritis; temporal
arteritis; Th2 Type and Th1 Type mediated diseases; thyroiditis;
toxic shock syndrome; toxoplasmic retinitis; toxic epidermal
necrolysis; transverse myelitis; TRAPS (tumor-necrosis factor
receptor type 1 (TNFR)-Associated Periodic Syndrome); type B
insulin resistance with acanthosis nigricans; Type 1 allergic
reaction; type-1 autoimmune hepatitis (classical autoimmune or
lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibody
hepatitis)e; Type II diabetes; ulcerative colitic arthropathy;
ulcerative colitis; urticaria; usual interstitial pneumonia (UIP);
uveitis; vasculitic diffuse lung disease; vasculitis; vernal
conjunctivitis; viral retinitis; vitiligo; Vogt-Koyanagi-Harada
syndrome (VKH syndrome); Wegener's granulomatosis; Wet macular
degeneration; wound healing; yersinia and salmonella associated
arthropathy.
[0162] The antibodies, and antibody portions of the invention can
be used to treat humans suffering from autoimmune diseases, in
particular those associated with inflammation, rheumatoid
arthritis, osteoarthritis, psoriasis, multiple sclerosis, and other
autoimmune diseases.
[0163] An antibody, or antibody portion, of the invention also can
be administered with one or more additional therapeutic agents
useful in the treatment of autoimmune and inflammatory
diseases.
[0164] Antibodies of the invention, or antigen binding portions
thereof can be used alone or in combination to treat such diseases.
It should be understood that the antibodies of the invention, or
antigen binding portion thereof, can be used alone or in
combination with an additional agent, e.g., a therapeutic agent,
said additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent that imparts a
beneficial attribute to the therapeutic composition, e.g., an agent
that affects the viscosity of the composition.
[0165] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed
composition can perform its intended function.
[0166] Particular combinations are non-steroidal anti-inflammatory
drug(s) also referred to as NSAIDS which include drugs like
ibuprofen. Other combinations are corticosteroids including
prednisolone; the well known side-effects of steroid use can be
reduced or even eliminated by tapering the steroid dose required
when treating patients in combination with the anti TNF-.alpha.
antibodies of this invention. Non-limiting examples of therapeutic
agents for rheumatoid arthritis with which an antibody, or antibody
portion, of the invention can be combined include the following:
cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies
to or antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-15, IL-16, IL-17, IL-18, IL-21, interferons, EMAP-II, GM-CSF,
FGF, and PDGF. Antibodies of the invention, or antigen binding
portions thereof, can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,
CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands
including CD154 (gp39 or CD40L).
[0167] Particular combinations of therapeutic agents may interfere
at different points in the autoimmune and subsequent inflammatory
cascade; particular examples include TNF antagonists like chimeric,
humanized or human TNF antibodies, D2E7, (PCT Publication No. WO
97/29131), CA2 (Remicade.TM.), CDP 571, and soluble p55 or p75 TNF
receptors, derivatives, thereof, (p75TNFR1gG (Enbrel.TM.) or
p55TNFR1gG (Lenercept), and also TNF-.alpha. converting enzyme
(TACE) inhibitors; similarly IL-1 inhibitors
(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may be
effective for the same reason. Other combinations are with
Interleukin 11. Yet other combinations are with other key players
of the autoimmune response which may act in parallel to,
dependently on or in concert with TNF-.alpha. function. Yet other
combinations are with non-depleting anti-CD4 inhibitors. Yet other
combinations are with antagonists of the co-stimulatory pathway
CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors
or antagonistic ligands.
[0168] The antibodies of the invention, or antigen binding portions
thereof, may also be combined with agents, such as methotrexate,
6-MP, azathioprine sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, colchicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signaling by
proinflammatory cytokines such as TNF-.alpha. or IL-1 (e.g., IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF-.alpha. converting enzyme (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 (e.g., soluble
p55 or p75 TNF receptors and the derivatives p75TNFRIgG (Enbrel.TM.
and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, sIL-6R),
antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGF.beta.), celecoxib, folic acid, hydroxychloroquine sulfate,
rofecoxib, etanercept, infliximab, naproxen, valdecoxib,
sulfasalazine, methylprednisolone, meloxicam, methylprednisolone
acetate, gold sodium thiomalate, aspirin, triamcinolone acetonide,
propoxyphene napsylate/apap, folate, nabumetone, diclofenac,
piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hcl,
hydrocodone bitartrate/apap, diclofenac sodium/misoprostol,
fentanyl, anakinra, human recombinant, tramadol hcl, salsalate,
sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate
sodium, prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl,
sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram. Particular combinations
include methotrexate or leflunomide and in moderate or severe
rheumatoid arthritis cases, cyclosporine.
[0169] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may be determined by a person
skilled in the art and may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the antibody or antibody portion to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody,
or antibody portion, are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0170] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on (a) the
unique characteristics of the active compound and the particular
therapeutic or prophylactic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0171] It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0172] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention may be made using suitable equivalents without departing
from the scope of the invention or the embodiments disclosed
herein. Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included for purposes of illustration only and
are not intended to be limiting of the invention.
Example 1
Recombinant Anti-Human TNF-.alpha. Antibodies
Example 1.1
Construction and Expression of Recombinant Chimeric Anti-Human
TNF-.alpha. Antibodies
[0173] The DNA encoding the heavy chain constant region of murine
anti-human TNF-.alpha. monoclonal antibody MAK195 was replaced by a
cDNA fragment encoding the human IgG1 constant region containing 2
hinge-region amino acid mutations by homologous recombination in
bacteria. These mutations are a leucine to alanine change at
position 234 (EU numbering) and a leucine to alanine change at
position 235 (Lund et al. (1991) J. Immunol. 147:2657-2662). The
light chain constant region of each of these antibodies was
replaced by a human kappa constant region. Full-length chimeric
antibodies were transiently expressed in HEK293 cells by
co-transfection of chimeric heavy and light chain cDNAs ligated
into the pHybE expression plasmid. Cell supernatants containing
recombinant chimeric antibody were purified by protein A sepharose
chromatography and bound antibody was eluted by addition of acid
buffer. Antibodies were neutralized and dialyzed into PBS.
[0174] The purified chimeric anti-human TNF-.alpha. monoclonal
antibodies were then tested for their ability to bind the
hTNF-.alpha. protein by ELISA to confirm antigen binding.
Example 1.2
Construction of CDR Grafted Anti-Human TNF-.alpha. Antibodies
[0175] By applying standard methods well known in the art, the CDR
sequences of VH and VL chains of monoclonal antibody MAK195 (see
Table 5 above) were grafted into different human heavy and light
chain acceptor sequences.
[0176] Based on sequence VH and VL alignments with the VH and VL
sequences of monoclonal antibody MAK195 of the present invention
the following known human sequences were selected: [0177] a) VH4-59
(IGHV4-59) and VH3-53 (IGHV3-53) as well as the joining sequences
hJH4 for constructing heavy chain acceptor sequences [0178] b)
1-39/O12 and 3-15/L2 as well as hJK2 for constructing light chain
acceptor sequences
[0179] By grafting the corresponding VH and VL CDRs of MAK195 into
said acceptor sequences, the CDR-grafted, humanized, and modified
VH and VL sequences were prepared (see also Table 6, above).
Example 1.3
Construction of Framework Back Mutations in CDR-Grafted
Antibodies
[0180] To generate humanized antibody framework back mutations,
mutations are introduced into the CDR-grafted antibody sequences as
prepared according to Example 1.2, by de novo synthesis of the
variable domain and/or using mutagenic primers and PCR, and methods
well known in the art (see, e.g., WO 2007/042261; WO 99/54440;
Traunecker et al. (1987) EMBO J., 10(12):3655-9 and Lanzavecchia
and Scheidegger (1987) Eur. J. Immunol., 17(1):105-11. Different
combinations of back mutations and other mutations are constructed
for each of the CDR-grafts as follows. A summary of the proposed
design versions of each humanized antibody is set forth below.
Residue numbers for these mutations are based on the Kabat
numbering system. [0181] For heavy chains hMAK195VH.1z, one or more
of the following Vernier and VH/VL interfacing residues were back
mutated as follows: G27.fwdarw.F, I29.fwdarw.L, I37.fwdarw.V,
I48.fwdarw.L V67.fwdarw.L, V71.fwdarw.K, T73.fwdarw.N,
N76.fwdarw.S, and F78.fwdarw.I. [0182] Additional mutations include
the following: Q1.fwdarw.E. [0183] For heavy chains hMAK195VH.2z,
one or more of the following Vernier and VH/VL interfacing residues
are back mutated as follows: A24.fwdarw.V, F29.fwdarw.L,
V48.fwdarw.L, F67.fwdarw.L, R71.fwdarw.K, S49.fwdarw.G,
N76.fwdarw.S, and L78.fwdarw.I. [0184] Additional mutations include
the following: Q1.fwdarw.E, I12.fwdarw.N, and V29.fwdarw.F. [0185]
For light chain hMAK195Vk.1 one or more of the following Vernier
and VH/VL interfacing residues are back mutated as follows:
A43.fwdarw.S. [0186] For light chain hMAK195Vk.2z one or more of
the following Vernier and VH/VL interfacing residues are back
mutated as follows: A43.fwdarw.S, 158.fwdarw.N. [0187] Additional
mutation include the following: V13.fwdarw.L, E70.fwdarw.D, and
S80.fwdarw.P.
Example 1.4
Humanized Anti-hTNF-.alpha. Heavy and Light Chains Containing
Framework Back Mutations
TABLE-US-00007 [0188] Sequence SEQ ID No. Protein region
123456789012345678901234567890 SEQ ID NO: 28 hMAK195VH.1
EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVNWIRQPPGKGLEWIGMIWGDGSTDYD
STLKSRVTISVDTSKNQFSLKLSSVTAADT AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID
NO: 29 hMAK195VH.1a EVQLQESGPGLVKPSETLSLTCTVSGFSLS
DYGVNWVRQPPGKGLEWLGMIWGDGSTDYD STLKSRLTISKDNSKSQISLKLSSVTAADT
AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID NO: 30 hMAK195VH.1b
EVQLQESGPGLVKPSETLSLTCTVSGFSLS DYGVNWIRQPPGKGLEWIGMIWGDGSTDYD
STLKSRVTISKDTSKNQFSLKLSSVTAADT AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID
NO: 31 hMAK195VH.2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS
DYGVNWVRQAPGKGLEWVSMIWGDGSTDYD STLKSRFTISRDNSKNTLYLQMNSLRAEDT
AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID NO: 32 hMAK195VH.2a
EVQLVESGGGLVQPGGSLRLSCAVSGFTLS DYGVNWVRQAPGKGLEWLGMIWGDGSTDYD
STLKSRLTISKDNSKSTIYLQMNSLRAEDT AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID
NO: 33 hMAK195VH.2b EVQLVESGGGLVQPGGSLRLSCAASGFTLS
DYGVNWVRQAPGKGLEWVSMIWGDGSTDYD STLKSRFTISKDNSKNTLYLQMNSLRAEDT
AVYYCAREWHHGPVAYWGQGTLVTVSS SEQ ID NO: 34 hMAK195Vk.1a
DIQMTQSPSSLSASVGDRVTITCKASQAVS SAVAWYQQKPGKSPKLLIYWASTRHTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ HYSTPFTFGQGTKLEIK SEQ ID NO: 35
hMAK195Vk.2 EIVMTQSPATLSLSPGERATLSCKASQAVS
SAVAWYQQKPGQAPRLLIYWASTRHTGIPA RFSGSGSGTDFTLTISSLQPEDFAVYYCQQ
HYSTPFTFGQGTKLEIK SEQ ID NO: 36 hMAK195Vk.2a
EIVMTQSPATLSLSPGERATLSCKASQAVS SAVAWYQQKPGQSPRLLIYWASTRHTGVPA
RFSGSGSGTDFTLTISSLQPEDFAVYYCQQ HYSTPFTFGQGTKLEIK
Example 1.5
Humanized Anti-hTNF-.alpha. hMAK195 Antibody VH/VL Pairings
TABLE-US-00008 [0189] Antibody Designation VH VL AB240 hMAK195VH.1
hMAK195VL.1 AB241 hMAK195VH.1 hMAK195VL.1a AB242 hMAK195VH.1
hMAK195VL.2 AB243 hMAK195VH.1 hMAK195VL.2a AB244 hMAK195VH.1a
hMAK195VL.1 AB245 hMAK195VH.1a hMAK195VL.1a AB246 hMAK195VH.1a
hMAK195VL.2 AB247 hMAK195VH.1a hMAK195VL.2a AB248 hMAK195VH.1b
hMAK195VL.1 AB249 hMAK195VH.1b hMAK195VL.1a AB250 hMAK195VH.1b
hMAK195VL.2 AB251 hMAK195VH.1b hMAK195VL.2a AB252 hMAK195VH.2
hMAK195VL.1 AB253 hMAK195VH.2 hMAK195VL.1a AB254 hMAK195VH.2
hMAK195VL.2 AB255 hMAK195VH.2 hMAK195VL.2a AB256 hMAK195VH.2a
hMAK195VL.1 AB257 hMAK195VH.2a hMAK195VL.1a AB258 hMAK195VH.2a
hMAK195VL.2 AB259 hMAK195VH.2a hMAK195VL.2a AB260 hMAK195VH.2b
hMAK195VL.1 AB261 hMAK195VH.2b hMAK195VL.1a AB262 hMAK195VH.2b
hMAK195VL.2 AB263 hMAK195VH.2b hMAK195VL.2a
Example 1.6
Affinity Determination Using BIACORE Technology
TABLE-US-00009 [0190] TABLE 7 Reagent Used in Biacore Analyses
Antigen Vendor Designation Vendor Catalog # TNF.alpha. Recombinant
Human TNF- R&D 210-TA .alpha./TNFSF1A systems
BIACORE Methods:
[0191] The BIACORE assay (Biacore, Inc. Piscataway, N.J.)
determines the affinity of antibodies with kinetic measurements of
on-rate and off-rate constants. Binding of antibodies to a target
antigen (for example, a purified recombinant target antigen) is
determined by surface plasmon resonance-based measurements with a
Biacore.RTM. 1000 or 3000 instrument (Biacore.RTM. AB, Uppsala,
Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3
mM EDTA, and 0.005% surfactant P20) at 25.degree. C. All chemicals
are obtained from Biacore.RTM. AB (Uppsala, Sweden) or otherwise
from a different source as described in the text. For example,
approximately 5000 RU of goat anti-mouse IgG, (Fey), fragment
specific polyclonal antibody (Pierce Biotechnology Inc, Rockford,
Ill., US) diluted in 10 mM sodium acetate (pH 4.5) is directly
immobilized across a CM5 research grade biosensor chip using a
standard amine coupling kit according to manufacturer's
instructions and procedures at 25 .mu.g/ml. Unreacted moieties on
the biosensor surface are blocked with ethanolamine. Modified
carboxymethyl dextran surface in flowcell 2 and 4 is used as a
reaction surface. Unmodified carboxymethyl dextran without goat
anti-mouse IgG in flow cell 1 and 3 is used as the reference
surface. For kinetic analysis, rate equations derived from the 1:1
Langmuir binding model are fitted simultaneously to association and
dissociation phases of all eight injections (using global fit
analysis) with the use of Biaevaluation 4.0.1 software. Purified
antibodies are diluted in HEPES-buffered saline for capture across
goat anti-mouse IgG specific reaction surfaces. Antibodies to be
captured as a ligand (25 .mu.g/ml) are injected over reaction
matrices at a flow rate of 5 .mu.l/minute. The association and
dissociation rate constants, k.sub.on (M.sup.-1s.sup.-1) and
k.sub.off (s.sup.-1), are determined under a continuous flow rate
of 25 .mu.l/minute. Rate constants are derived by making kinetic
binding measurements at different antigen concentrations ranging
from 10-200 nM. The equilibrium dissociation constant (M) of the
reaction between antibodies and the target antigen is then
calculated from the kinetic rate constants by the following
formula: K.sub.D=k.sub.off/k.sub.on. Binding is recorded as a
function of time and kinetic rate constants are calculated. In this
assay, on-rates as fast as 10.sup.6M.sup.-1s.sup.-1 and off-rates
as slow as 10.sup.-6 s.sup.-1 can be measured.
TABLE-US-00010 TABLE 8 BIACORE Analysis of Anti-hTNF-.alpha.
Antibodies Antibody k.sub.on k.sub.off k.sub.D Designation VH VL
(M-1s-1) (s-1) (M) AB240 hMAK195VH.1 hMAK195VL.1 1.64E+06 2.28E-03
1.40E-09 AB241 hMAK195VH.1 hMAK195VL.1a 2.27E+06 1.30E-03 5.73E-10
AB242 hMAK195VH.1 hMAK195VL.2 1.79E+06 1.42E-03 7.96E-10 AB243
hMAK195VH.1 hMAK195VL.2a 1.72E+06 2.70E-03 1.62E-09 AB244
hMAK195VH.1a hMAK195VL.1 3.80E+06 3.35E-04 8.91E-11 AB245
hMAK195VH.1a hMAK195VL.1a 3.32E+06 3.30E-04 9.94E-11 AB246
hMAK195VH.1a hMAK195VL.2 2.61E+06 1.19E-04 4.58E-11 AB247
hMAK195VH.1a hMAK195VL.2a 2.62E+06 2.60E-04 9.96E-11 AB248
hMAK195VH.1b hMAK195VL.1 1.82E+06 3.16E-04 1.74E-10 AB249
hMAK195VH.1b hMAK195VL.1a 2.04E+06 3.15E-04 1.56E-10 AB250
hMAK195VH.1b hMAK195VL.2 2.00E+06 3.56E-04 1.79E-10 AB251
hMAK195VH.1b hMAK195VL.2a 1.71E+06 4.25E-04 2.50E-10 AB252
hMAK195VH.2 hMAK195VL.1 2.92E+06 2.52E-04 8.66E-11 AB253
hMAK195VH.2 hMAK195VL.1a 3.02E+06 2.21E-04 7.33E-11 AB254
hMAK195VH.2 hMAK195VL.2 2.40E+06 2.87E-04 1.20E-10 AB255
hMAK195VH.2 hMAK195VL.2a 2.62E+06 3.84E-04 1.48E-10 AB256
hMAK195VH.2a hMAK195VL.1 3.54E+06 1.77E-04 5.05E-11 AB257
hMAK195VH.2a hMAK195VL.1a 3.56E+06 2.17E-04 6.20E-11 AB258
hMAK195VH.2a hMAK195VL.2 3.04E+06 2.44E-04 8.16E-11 AB259
hMAK195VH.2a hMAK195VL.2a 3.00E+06 2.03E-04 6.77E-11 AB260
hMAK195VH.2b hMAK195VL.1 2.27E+06 2.30E-04 1.01E-10 AB261
hMAK195VH.2b hMAK195VL.1a 2.36E+06 2.85E-04 1.22E-10 AB262
hMAK195VH.2b hMAK195VL.2 2.14E+06 2.90E-04 1.36E-10 AB263
hMAK195VH.2b hMAK195VL.2a 1.61E+06 2.73E-04 1.69E-10
[0192] Binding of all humanized constructs characterized by Biacore
technology was maintained and comparable to that of the murine
parental antibody.
Example 1.7
Neutralization of Human TNF-.alpha.
[0193] L929 cells were grown to a semi-confluent density and
harvested using 0.25% trypsin (Gibco#25300). The cells were washed
with PBS, counted and resuspended at 1E6 cells/mL in assay media
containing 4 .mu.g/mL actinomycin D. The cells were seeded in a
96-well plate (Costar#3599) at a volume of 100 .mu.L and 5E4
cells/well. The antibodies and control IgG were diluted to a
4.times. concentration in assay media and serial 1:4 dilutions were
performed. The huTNF-.alpha. was diluted to 400 .mu.g/mL in assay
media. Antibody sample (200 .mu.L) was added to the huTNF-.alpha.
(200 .mu.L) in a 1:2 dilution scheme and allowed to incubate for
0.5 hour at room temperature.
[0194] The antibody/human TNF-.alpha. solution was added to the
plated cells at 100 .mu.L for a final concentration of 100 pg/mL
huTNF-.alpha. and 150 nM-0.0001 nM antibody. The plates were
incubated for 20 hours at 37.degree. C., 5% CO.sub.2. To quantitate
viability, 100 .mu.L was removed from the wells and 10 .mu.L of
WST-1 reagent (Roche cat#11644807001) was added. Plates were
incubated under assay conditions for 3.5 hours. The plates were
read at OD 420-600 nm on a Spectromax 190 ELISA plate reader. An
average EC50 from several assays is included in Table 9.
TABLE-US-00011 TABLE 9 Human TNF-.alpha. Neutralization Assay with
Humanized anti-hTNF-.alpha. Antibodies TNF-.alpha. Antibody
neutralization assay Designation VH VL IC.sub.50 (nM) AB240
hMAK195VH.1 hMAK195VL.1 14.190 AB241 hMAK195VH.1 hMAK195VL.1a 5.325
AB242 hMAK195VH.1 hMAK195VL.2 25.150 AB243 hMAK195VH.1 hMAK195VL.2a
50.710 AB244 hMAK195VH.1a hMAK195VL.1 0.155 AB245 hMAK195VH.1a
hMAK195VL.1a 0.072 AB246 hMAK195VH.1a hMAK195VL.2 0.099 AB247
hMAK195VH.1a hMAK195VL.2a 0.186 AB248 hMAK195VH.1b hMAK195VL.1
0.213 AB249 hMAK195VH.1b hMAK195VL.1a 0.245 AB250 hMAK195VH.1b
hMAK195VL.2 0.301 AB251 hMAK195VH.1b hMAK195VL.2a 0.350 AB252
hMAK195VH.2 hMAK195VL.1 0.222 AB253 hMAK195VH.2 hMAK195VL.1a 0.279
AB254 hMAK195VH.2 hMAK195VL.2 0.799 AB255 hMAK195VH.2 hMAK195VL.2a
0.477 AB256 hMAK195VH.2a hMAK195VL.1 0.058 AB257 hMAK195VH.2a
hMAK195VL.1a 0.077 AB258 hMAK195VH.2a hMAK195VL.2 0.078 AB259
hMAK195VH.2a hMAK195VL.2a 0.071 AB260 hMAK195VH.2b hMAK195VL.1
0.093 AB261 hMAK195VH.2b hMAK195VL.1a 0.187 AB262 hMAK195VH.2b
hMAK195VL.2 0.218 AB263 hMAK195VH.2b hMAK195VL.2a 0.193
[0195] All anti-hTNF-.alpha. antibodies showed neutralization in
the TNF-.alpha. neutralization assay.
Example 1.8
Physicochemical and In Vitro Stability Analysis of Humanized
Monoclonal Antibodies
Size Exclusion Chromatography
[0196] Antibodies were diluted to 2.5 mg/mL with water and 20 mL
analyzed on a Shimadzu HPLC system using a TSK gel G3000 SWXL
column (Tosoh Bioscience, cat# k5539-05k). Samples were eluted from
the column with 211 mM sodium sulfate, 92 mM sodium phosphate, pH
7.0, at a flow rate of 0.3 mL/minutes. The HPLC system operating
conditions were the following:
[0197] Mobile phase: 211 mM Na.sub.2SO.sub.4, 92 mM
Na.sub.2HPO.sub.4*7H.sub.2O, pH 7.0
[0198] Gradient: Isocratic
[0199] Flow rate: 0.3 mL/minute
[0200] Detector wavelength: 280 nm
[0201] Autosampler cooler temp: 4.degree. C.
[0202] Column oven temperature: ambient
[0203] Run time: 50 minutes
[0204] Table 10 contains purity data of antibody constructs
expressed as percent monomer (unaggregated protein of the expected
molecular weight) as determined by the above protocol.
TABLE-US-00012 TABLE 10 Purity of anti-hTNF-.alpha. Antibodies as
Determined by Size Exclusion Chromatography Antibody % monomer
Designation VH VL (purity) AB240 hMAK195VH.1 hMAK195VL.1 88.6 AB241
hMAK195VH.1 hMAK195VL.1a 93.4 AB242 hMAK195VH.1 hMAK195VL.2 89.8
AB243 hMAK195VH.1 hMAK195VL.2a 92.2 AB244 hMAK195VH.1a hMAK195VL.1
100 AB245 hMAK195VH.1a hMAK195VL.1a 99 AB246 hMAK195VH.1a
hMAK195VL.2 98.8 AB247 hMAK195VH.1a hMAK195VL.2a 99 AB248
hMAK195VH.1b hMAK195VL.1 93.5 AB249 hMAK195VH.1b hMAK195VL.1a 96
AB250 hMAK195VH.1b hMAK195VL.2 93.8 AB251 hMAK195VH.1b hMAK195VL.2a
95.4 AB252 hMAK195VH.2 hMAK195VL.1 98.4 AB253 hMAK195VH.2
hMAK195VL.1a 98.6 AB254 hMAK195VH.2 hMAK195VL.2 97.5 AB255
hMAK195VH.2 hMAK195VL.2a 97.7 AB256 hMAK195VH.2a hMAK195VL.1 95.7
AB257 hMAK195VH.2a hMAK195VL.1a 96.7 AB258 hMAK195VH.2a hMAK195VL.2
95.6 AB259 hMAK195VH.2a hMAK195VL.2a 96.6 AB260 hMAK195VH.2b
hMAK195VL.1 99.1 AB261 hMAK195VH.2b hMAK195VL.1a 100 AB262
hMAK195VH.2b hMAK195VL.2 98.6 AB263 hMAK195VH.2b hMAK195VL.2a
98.9
[0205] Anti-hTNF-.alpha. antibodies showed an excellent SEC profile
with most showing >95% monomer.
Sodium Dodecyl Sulfate--Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
[0206] Antibodies are analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both
reducing and non-reducing conditions. Adalimumab lot AFPO4C is used
as a control. For reducing conditions, the samples are mixed 1:1
with 2.times. tris glycine SDS-PAGE sample buffer (Invitrogen, cat#
LC2676, lot#1323208) with 100 mM DTT, and heated at 60.degree. C.
for 30 minutes. For non-reducing conditions, the samples are mixed
1:1 with sample buffer and heated at 100.degree. C. for 5 minutes.
The reduced samples (10 mg per lane) are loaded on a 12% pre-cast
tris-glycine gel (Invitrogen, cat# EC6005box, lot#6111021), and the
non-reduced samples (10 mg per lane) are loaded on an 8%-16%
pre-cast tris-glycine gel (Invitrogen, cat# EC6045box,
lot#6111021). SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot#1351542)
is used as a molecular weight marker. The gels are run in a XCell
SureLock mini cell gel box (Invitrogen, cat# EI0001) and the
proteins are separated by first applying a voltage of 75 to stack
the samples in the gel, followed by a constant voltage of 125 until
the dye front reached the bottom of the gel. The running buffer
used is 1.times. tris glycine SDS buffer, prepared from a 10.times.
tris glycine SDS buffer (ABC, MPS-79-080106)). The gels are stained
overnight with colloidal blue stain (Invitrogen cat#46-7015,
46-7016) and destained with Milli-Q water until the background is
clear. The stained gels are then scanned using an Epson Expression
scanner (model 1680, S/N DASX003641).
Sedimentation Velocity Analysis
[0207] Antibodies are loaded into the sample chamber of each of
three standard two-sector carbon epon centerpieces. These
centerpieces have a 1.2 cm optical path length and are built with
sapphire windows. PBS is used for a reference buffer and each
chamber contained 140 .mu.L. All samples are examined
simultaneously using a 4-hole (AN-60Ti) rotor in a Beckman
ProteomeLab XL-I analytical ultracentrifuge (serial #
PL106C01).
[0208] Run conditions are programmed and centrifuge control is
performed using ProteomeLab (v5.6). The samples and rotor are
allowed to thermally equilibrate for one hour prior to analysis
(20.0.+-.0.1.degree. C.). Confirmation of proper cell loading is
performed at 3000 rpm and a single scan is recorded for each cell.
The sedimentation velocity conditions are the following:
[0209] Sample Cell Volume: 420 mL
[0210] Reference Cell Volume: 420 mL
[0211] Temperature: 20.degree. C.
[0212] Rotor Speed: 35,000 rpm
[0213] Time: 8:00 hours
[0214] UV Wavelength: 280 nm
[0215] Radial Step Size: 0.003 cm
[0216] Data Collection: One data point per step without signal
averaging.
[0217] Total Number of Scans: 100
LC-MS Molecular Weight Measurement of Intact Antibodies
[0218] Molecular weights of intact antibodies are analyzed by
LC-MS. Each antibody is diluted to approximately 1 mg/mL with
water. An 1100 HPLC (Agilent) system with a protein microtrap
(Michrom Bioresources, Inc, cat#004/25109/03) is used to desalt and
introduce 5 mg of the sample into an API QSTAR Pulsar i mass
spectrometer (Applied Biosystems). A short gradient is used to
elute the samples. The gradient is run with mobile phase A (0.08%
FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FA and 0.02%
TFA in acetonitrile) at a flow rate of 50 mL/minute. The mass
spectrometer is operated at 4.5 kvolts spray voltage with a scan
range from 2000 to 3500 mass to charge ratio.
LC-MS Molecular Weight Measurement of Antibody Light and Heavy
Chains
[0219] Molecular weight measurement of antibody light chains (LC),
heavy chains (HC) and deglycosylated HC are analyzed by LC-MS.
Antibody is diluted to 1 mg/mL with water and the sample is reduced
to LC and HC with a final concentration of 10 mM DTT for 30 minutes
at 37.degree. C. To deglycosylate the antibody, 100 mg of the
antibody is incubated with 2 mL of PNGase F, 5 mL of 10%
N-octylglucoside in a total volume of 100 mL overnight at
37.degree. C. After deglycosylation the sample is reduced with a
final concentration of 10 mM DTT for 30 minutes at 37.degree. C. An
Agilent 1100 HPLC system with a C4 column (Vydac, cat#214TP5115,
S/N 060206537204069) is used to desalt and introduce the sample (5
mg) into an API QSTAR Pulsar i mass spectrometer (Applied
Biosystems). A short gradient is used to elute the sample. The
gradient is run with mobile phase A (0.08% FA, 0.02% TFA in HPLC
water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile)
at a flow rate of 50 mL/minute. The mass spectrometer is operated
at 4.5 kvolts spray voltage with a scan range from 800 to 3500 mass
to charge ratio.
Peptide Mapping
[0220] Antibody is denatured for 15 minutes at room temperature
with a final concentration of 6 M guanidine hydrochloride in 75 mM
ammonium bicarbonate. The denatured samples are reduced with a
final concentration of 10 mM DTT at 37.degree. C. for 60 minutes,
followed by alkylation with 50 mM iodoacetic acid (IAA) in the dark
at 37.degree. C. for 30 minutes. Following alkylation, the sample
is dialyzed overnight against four liters of 10 mM ammonium
bicarbonate at 4.degree. C. The dialyzed sample is diluted to 1
mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mg of
antibody is either digested with trypsin (Promega, cat# V5111) or
Lys-C(Roche, cat#11 047 825 001) at a 1:20 (w/w)
trypsin/Lys-C:antibody ratio at 37.degree. C. for 4 hrs. Digests
are quenched with 1 mL of 1 N HCl. For peptide mapping with mass
spectrometer detection, 40 mL of the digests are separated by
reverse phase high performance liquid chromatography (RPHPLC) on a
C18 column (Vydac, cat#218TP51, S/N NE9606 10.3.5) with an Agilent
1100 HPLC system. The peptide separation is run with a gradient
using mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water)
and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a
flow rate of 50 mL/minutes. The API QSTAR Pulsar i mass
spectrometer is operated in positive mode at 4.5 kvolts spray
voltage and a scan range from 800 to 2500 mass to charge ratio.
Disulfide Bond Mapping
[0221] To denature the antibody, 100 mL of the antibody is mixed
with 300 mL of 8 M guanidine HCl in 100 mM ammonium bicarbonate.
The pH is checked to ensure that it is between 7 and 8 and the
samples are denatured for 15 minutes at room temperature in a final
concentration of 6 M guanidine HCl. A portion of the denatured
sample (100 mL) is diluted to 600 mL with Milli-Q water to give a
final guanidine-HCl concentration of 1 M. The sample (220 mg) is
digested with either trypsin (Promega, cat # V5111, lot#22265901)
or Lys-C(Roche, cat#11047825001, lot#12808000) at a 1:50 trypsin or
1:50 Lys-C: antibody (w/w) ratios (4.4 mg enzyme: 220 mg sample) at
37.degree. C. for approximately 16 hours. An additional 5 mg of
trypsin or Lys-C is added to the samples and digestion is allowed
to proceed for an additional 2 hours at 37.degree. C. Digestions
are stopped by adding 1 mL of TFA to each sample. Digested samples
are separated by RPHPLC using a C18 column (Vydac, cat#218TP51 S/N
NE020630-4-1A) on an Agilent HPLC system. The separation is run
with the same gradient used for peptide mapping using mobile phase
A (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B
(0.02% TFA and 0.08% FA in acetonitrile) at a flow rate of 50
mL/minute. The HPLC operating conditions are the same as those used
for peptide mapping. The API QSTAR Pulsar i mass spectrometer is
operated in positive mode at 4.5 kvolts spray voltage and a scan
range from 800 to 2500 mass-to-charge ratio. Disulfide bonds are
assigned by matching the observed MWs of peptides with the
predicted MWs of tryptic or Lys-C peptides linked by disulfide
bonds.
Free Sulfhydryl Determination
[0222] The method used to quantify free cysteines in an antibody is
based on the reaction of Ellman's reagent, 5,5'-dithio-bis
(2-nitrobenzoic acid) (DTNB), with sulfhydryl groups (SH) which
gives rise to a characteristic chromophoric product,
5-thio-(2-nitrobenzoic acid) (TNB). The reaction is illustrated in
the formula:
DTNB+RSH.fwdarw.RS-TNB+TNB.sup.-+H.sup.+
[0223] The absorbance of the TNB.sup.- is measured at 412 nm using
a Cary 50 spectrophotometer. An absorbance curve is plotted using
dilutions of 2-mercaptoethanol (.beta.-ME) as the free SH standard
and the concentrations of the free sulfhydryl groups in the protein
are determined from absorbance at 412 nm of the sample.
[0224] The .beta.-ME standard stock is prepared by a serial
dilution of 14.2 M .beta.-ME with HPLC grade water to a final
concentration of 0.142 mM. Then standards in triplicate for each
concentration are prepared. Antibody is concentrated to 10 mg/mL
using an amicon ultra 10,000 MWCO centrifugal filter (Millipore,
cat# UFC801096, lot# L3KN5251) and the buffer is changed to the
formulation buffer used for adalimumab (5.57 mM sodium phosphate
monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07
mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, pH 5.2,
0.1% (w/v) Tween.RTM.). The samples are mixed on a shaker at room
temperature for 20 minutes. Then 180 mL of 100 mM Tris buffer, pH
8.1 is added to each sample and standard followed by the addition
of 300 mL of 2 mM DTNB in 10 mM phosphate buffer, pH 8.1. After
thorough mixing, the samples and standards are measured for
absorption at 412 nm on a Cary 50 spectrophotometer. The standard
curve is obtained by plotting the amount of free SH and OD.sub.412
nm of the .beta.-ME standards. Free SH content of samples are
calculated based on this curve after subtraction of the blank
Weak Cation Exchange Chromatography
[0225] Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate,
pH 6.0. Charge heterogeneity is analyzed using a Shimadzu HPLC
system with a WCX-10 ProPac analytical column (Dionex, cat#054993,
S/N 02722). The samples are loaded on the column in 80% mobile
phase A (10 mM sodium phosphate, pH 6.0) and 20% mobile phase B (10
mM sodium phosphate, 500 mM NaCl, pH 6.0) and eluted at a flow rate
of 1.0 mL/minute.
Oligosaccharide Profiling
[0226] Oligosaccharides released after PNGase F treatment of
antibody are derivatized with 2-aminobenzamide (2-AB) labeling
reagent. The fluorescent-labeled oligosaccharides are separated by
normal phase high performance liquid chromatography (NPHPLC) and
the different forms of oligosaccharides are characterized based on
retention time comparison with known standards.
[0227] The antibody is first digested with PNGaseF to cleave
N-linked oligosaccharides from the Fc portion of the heavy chain.
The antibody (200 mg) is placed in a 500 mL Eppendorf tube along
with 2 mL PNGase F and 3 mL of 10% N-octylglucoside. Phosphate
buffered saline is added to bring the final volume to 60 mL. The
sample is incubated overnight at 37.degree. C. in an Eppendorf
thermomixer set at 700 RPM. Adalimumab lot AFPO4C is also digested
with PNGase F as a control.
[0228] After PNGase F treatment, the samples are incubated at
95.degree. C. for 5 minutes in an Eppendorf thermomixer set at 750
RPM to precipitate out the proteins, then the samples are placed in
an Eppendorf centrifuge for 2 minutes at 10,000 RPM to spin down
the precipitated proteins. The supernatent containing the
oligosaccharides are transferred to a 500 mL Eppendorf tube and
dried in a speed-vac at 65.degree. C.
[0229] The oligosaccharides are labeled with 2AB using a 2AB
labeling kit purchased from Prozyme (cat# GKK-404, lot#132026). The
labeling reagent is prepared according to the manufacturer's
instructions. Acetic acid (150 mL, provided in kit) is added to the
DMSO vial (provided in kit) and mixed by pipeting the solution up
and down several times. The acetic acid/DMSO mixture (100 mL) is
transferred to a vial of 2-AB dye (just prior to use) and mixed
until the dye is fully dissolved. The dye solution is then added to
a vial of reductant (provided in kit) and mixed well (labeling
reagent). The labeling reagent (5 mL) is added to each dried
oligosaccharide sample vial, and mixed thoroughly. The reaction
vials are placed in an Eppendorf thermomixer set at 65.degree. C.
and 700-800 RPM for 2 hours of reaction.
[0230] After the labeling reaction, the excess fluorescent dye is
removed using GlycoClean S Cartridges from Prozyme (cat# GKI-4726).
Prior to adding the samples, the cartridges are washed with 1 mL of
Milli-Q water followed with 5 washes of 1 mL 30% acetic acid
solution. Just prior to adding the samples, 1 mL of acetonitrile
(Burdick and Jackson, cat# AH015-4) is added to the cartridges.
[0231] After all of the acetonitrile passed through the cartridge,
the sample is spotted onto the center of the freshly washed disc
and allowed to adsorb onto the disc for 10 minutes. The disc is
washed with 1 mL of acetonitrile followed by five washes of 1 mL of
96% acetonitrile. The cartridges are placed over a 1.5 mL Eppendorf
tube and the 2-AB labeled oligosaccharides are eluted with 3 washes
(400 mL each wash) of Milli-Q water.
[0232] The oligosaccharides are separated using a Glycosep N HPLC
(cat# GKI-4728) column connected to a Shimadzu HPLC system. The
Shimadzu HPLC system consisted of a system controller, degasser,
binary pumps, autosampler with a sample cooler, and a fluorescent
detector.
Stability at Elevated Temperatures
[0233] The final concentration of the antibodies is adjusted to 2
mg/mL with the appropriate buffers, surfactants, stabilizers,
and/or sugars. The antibody solutions are then filter sterilized
and 0.25 mL aliquots are prepared under sterile conditions. The
aliquots are left at either -80.degree. C., 5.degree. C.,
25.degree. C., or 40.degree. C. for 1, 2 or 3 weeks. At the end of
the incubation period, the samples are analyzed by size exclusion
chromatography and SDS-PAGE.
[0234] The stability samples are analyzed by SDS-PAGE under both
reducing and non-reducing conditions. The procedure used is the
same as described herein. The gels are stained overnight with
colloidal blue stain (Invitrogen cat#46-7015, 46-7016) and
destained with Milli-Q water until the background is clear. The
stained gels are then scanned using an Epson Expression scanner
(model 1680, S/N DASX003641). To obtain more sensitivity, the same
gels are silver stained using silver staining kit (Owl Scientific,
Gel Company, San Francisco, Calif. US) and the recommended
procedures given by the manufacturer is used.
Example 1.9
Transfection and Expression in HEK 293-6E Cells
[0235] The anti-hTNF-.alpha. antibody vector constructs were
transfected into 293 cells for production of protein. The 293
transient transfection procedure used is a modification of the
methods published in Durocher et al. (2002) Nucl. Acids Res.
30(2e9):1-9 and Pham et al. (2005) Biotech. Bioeng. 90(3):332-44.
Reagents that were used in the transfection included: [0236] HEK
293-6E cells (human embryonic kidney cell line stably expressing
EBNA1; obtained from National Research Council Canada) cultured in
disposable Erlenmeyer flasks in a humidified incubator set at 130
rpm, 37.degree. C. and 5% CO.sub.2. [0237] Culture medium:
FreeStyle 293 Expression Medium (Invitrogen 12338-018) plus 25
.mu.g/mL Geneticin (G418) (Invitrogen 10131-027) and 0.1% Pluronic
F-68 (Invitrogen 24040-032). [0238] Transfection medium: FreeStyle
293 Expression Medium plus 10 mM HEPES (Invitrogen 15630-080).
[0239] Polyethylenimine (PEI) stock: 1 mg/mL sterile stock
solution, pH 7.0, prepared with linear 25 kDa PEI (Polysciences)
and stored at less than -15.degree. C. [0240] Tryptone Feed Medium:
5% w/v sterile stock of Tryptone N1 (Organotechnie, 19554) in
[0241] FreeStyle 293 Expression Medium.
Cell Preparation for Transfection:
[0242] Approximately 2-4 hours prior to transfection, HEK 293-6E
cells were harvested by centrifugation and resuspended in culture
medium at a cell density of approximately 1 million viable cells
per mL. For each transfection, 40 mL of the cell suspension were
transferred into a disposable 250-mL Erlenmeyer flask and incubated
for 2-4 hours.
Transfection:
[0243] The transfection medium and PEI stock were prewarmed to room
temperature (RT). For each transfection, 25 .mu.g of plasmid DNA
and 50 .mu.g of polyethylenimine (PEI) were combined in 5 mL of
transfection medium and incubated for 15-20 minutes at RT to allow
the DNA:PEI complexes to form. For the BR3-Ig transfections, 25
.mu.g of BR3-Ig plasmid was used per transfection. Each 5-mL
DNA:PEI complex mixture was added to a 40-mL culture prepared
previously and returned to the humidified incubator set at 130 rpm,
37.degree. C. and 5% CO.sub.2. After 20-28 hours, 5 mL of Tryptone
Feed Medium was added to each transfection and the cultures were
continued for six days.
[0244] Table 11 contains the yield data for parent antibodies
expressed as milligrams per liter in HEK 293-6E cells.
TABLE-US-00013 TABLE 11 Transient Expression in Yields of
anti-hTNF-.alpha. Antibodies in HEK 293-6E Cells Antibody
Expression Designation VH VL yield (mg/L) AB240 hMAK195VH.1
hMAK195VL.1 101.4 AB241 hMAK195VH.1 hMAK195VL.1a 100.4 AB242
hMAK195VH.1 hMAK195VL.2 66.4 AB243 hMAK195VH.1 hMAK195VL.2a 89
AB244 hMAK195VH.1a hMAK195VL.1 87.6 AB245 hMAK195VH.1a hMAK195VL.1a
83.8 AB246 hMAK195VH.1a hMAK195VL.2 58.6 AB247 hMAK195VH.1a
hMAK195VL.2a 73 AB248 hMAK195VH.1b hMAK195VL.1 83.2 AB249
hMAK195VH.1b hMAK195VL.1a 89 AB250 hMAK195VH.1b hMAK195VL.2 65.4
AB251 hMAK195VH.1b hMAK195VL.2a 77.2 AB252 hMAK195VH.2 hMAK195VL.1
21.4 AB253 hMAK195VH.2 hMAK195VL.1a 52 AB254 hMAK195VH.2
hMAK195VL.2 5.8 AB255 hMAK195VH.2 hMAK195VL.2a 12.56 AB256
hMAK195VH.2a hMAK195VL.1 99.6 AB257 hMAK195VH.2a hMAK195VL.1a 102.8
AB258 hMAK195VH.2a hMAK195VL.2 62 AB259 hMAK195VH.2a hMAK195VL.2a
78.2 AB260 hMAK195VH.2b hMAK195VL.1 69.4 AB261 hMAK195VH.2b
hMAK195VL.1a 98.4 AB262 hMAK195VH.2b hMAK195VL.2 30.8 AB263
hMAK195VH.2b hMAK195VL.2a 49.2
[0245] All antibodies expressed well in HEK 293-6E cells. In most
cases >50 mg/L purified antibody could be obtained easily from
supernatants of HEK 293-6E cells.
INCORPORATION BY REFERENCE
[0246] The present invention incorporates by reference in their
entirety techniques well known in the field of molecular biology
and drug delivery. These techniques include, but are not limited
to, techniques described in the following publications: Ausubel et
al. (eds.), Current Protocols in Molecular Biology, John Wiley
& Sons, NY (1993); Ausubel, F. M. et al. eds., Short Protocols
In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY.
(ISBN 0-471-32938-X). Controlled Drug Bioavailability Drug Product
Design and Performance, Smolen and Ball (eds.), Wiley, New York
(1984); Giege et al., Chapter 1, In Crystallization of Nucleic
Acids and Proteins, A Practical Approach, 2nd ed., (Ducruix and
Giege, eds.) (Oxford University Press, New York, 1999) pp. 1-16;
Goodson, J. M., Chapter 6, In Medical Applications of Controlled
Release, Vol. II, Applications and Evaluation, (Langer and Wise,
eds.) (CRC Press, Inc., Boca Raton, 1984), pp. 115-138; Hammerling
et al., eds., "Monoclonal Antibodies and T-Cell Hybridomas," In
Research Monographs in Immunology, vol. 3 (J. L. Turk, General
Editor) (Elsevier, New York, 1981), pp. 563-587; Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987); 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; Kontermann and
Dubel, eds., Antibody Engineering (2001) Springer-Verlag. New York.
790 pp. (ISBN 3-540-41354-5); Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990); Lu and
Weiner eds., Cloning and Expression Vectors for Gene Function
Analysis (2001) BioTechniques Press. Westborough, Mass. 298 pp.
(ISBN 1-881299-21-X); Goodson, J. M., Medical Applications of
Controlled Release, (Langer and Wise, eds.) (CRC Press, Boca Raton,
1974); Old and Primrose, Principles of Gene Manipulation: An
Introduction To Genetic Engineering (3d Ed. 1985) Blackwell
Scientific Publications, Boston; Studies in Microbiology, V.2:409
pp. (ISBN 0-632-01318-4); Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory
Press, NY. Vols. 1-3 (ISBN 0-87969-309-6); Sustained and Controlled
Release Drug Delivery Systems, (J. R. Robinson, ed.) (Marcel
Dekker, Inc., New York, 1978); Winnacker, E. L. From Genes To
Clones: Introduction To Gene Technology (1987) VCH Publishers, N.Y.
(translated by Horst Ibelgaufts), 634 pp. (ISBN 0-89573-614-4).
[0247] The contents of all cited references (including literature
references, patents, patent applications, and websites) that are
cited throughout this application are hereby expressly incorporated
by reference in their entirety, as are the references cited
therein. The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of immunology,
molecular biology and cell biology, which are well known in the
art.
EQUIVALENTS
[0248] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 1
1
361157PRTHomo sapiens 1Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln
Leu Gln Trp Leu Asn Asp Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn
Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu
Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln
Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90
95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys
100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu
Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp
Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile
Ile Ala Leu 145 150 155 2330PRTHomo sapiens 2Ala Ser Thr Lys Gly
Pro Ser Val Phe Phe Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
3330PRTHomo sapiens 3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 4106PRTHomo sapiens 4Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10
15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 5105PRTHomo sapiens 5Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40
45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100
105 631PRTHomo sapiens 6Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Ser 20 25 30 714PRTHomo sapiens 7Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 832PRTHomo
sapiens 8Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala Arg 20 25 30 931PRTHomo sapiens 9Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser 20 25 30
1014PRTHomo sapiens 10Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser 1 5 10 1132PRTHomo sapiens 11Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30
1211PRTHomo sapiens 12Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 1311PRTHomo sapiens 13Trp Gly Arg Gly Thr Leu Val Thr Val Ser
Ser 1 5 10 1411PRTHomo sapiens 14Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 1 5 10 1523PRTHomo sapiens 15Asp 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 20 1615PRTHomo sapiens 16Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 1732PRTHomo sapiens 17Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10
15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
20 25 30 1823PRTHomo sapiens 18Glu Ile Val Met Thr Gln Ser Pro Ala
Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
20 1915PRTHomo sapiens 19Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile Tyr 1 5 10 15 2032PRTHomo sapiens 20Gly Ile Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr
Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30
2111PRTHomo sapiens 21Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 1
5 10 22117PRTMus sp. 22Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly
Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60 Ser Arg Leu
Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Ile Phe Leu 65 70 75 80 Lys
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala 85 90
95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ala 115 23108PRTMus sp. 23Asp Ile Val
Met Thr Gln Ser His Lys Phe Met Ser Thr Thr Val Gly 1 5 10 15 Asp
Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Ala Val Ser Ser Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Ser Val Thr Asp Phe Thr Leu Thr Ile His
Asn Leu Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln
His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 24117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 24Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asp Tyr 20 25 30 Gly Val
Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50
55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp
Cys Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
25117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Val Ser Asp Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Trp Gly
Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60 Ser Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 26107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
26Asp 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 Lys Ala Ser Gln Ala Val Ser Ser
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 27107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ala Val Ser Ser
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln His Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 28117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
28Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asp
Tyr 20 25 30 Gly Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr
Asp Ser Thr Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Trp His His
Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 29117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp
Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr
Asp Ser Thr Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn
Ser Lys Ser Gln Ile Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Trp His His
Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 30117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 30Glu Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr 20 25 30 Gly Val Asn Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Met
Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60
Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65
70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
31117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met Ile Trp Gly
Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60 Ser Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 32117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Asp
Tyr 20 25 30 Gly Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr
Asp Ser Thr Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Asn
Ser Lys Ser Thr Ile Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Trp His His
Gly Pro Val Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 33117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 33Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Leu Ser Asp Tyr 20 25 30 Gly Val Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Met
Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asp Ser Thr Leu Lys 50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Glu Trp His His Gly Pro Val Ala Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
34107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 34Asp 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 Lys Ala
Ser Gln Ala Val Ser Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr
Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
35107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 35Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala
Ser Gln Ala Val Ser Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr
Arg His Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
36107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 36Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala
Ser Gln Ala Val Ser Ser Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Arg Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr
Arg His Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
* * * * *
References