U.S. patent application number 13/231537 was filed with the patent office on 2011-12-29 for methods for generating anti-il-23 antibodies, compositions, methods and uses.
Invention is credited to Jacqueline Benson, Mark Cunningham, Cynthia Duchala, Jill M. Giles-Komar, Jinquan Luo, Sweet Raymond, Michael A. Rycyzyn.
Application Number | 20110319292 13/231537 |
Document ID | / |
Family ID | 37605195 |
Filed Date | 2011-12-29 |
![](/patent/app/20110319292/US20110319292A1-20111229-D00001.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00002.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00003.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00004.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00005.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00006.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00007.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00008.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00009.png)
![](/patent/app/20110319292/US20110319292A1-20111229-D00010.png)
United States Patent
Application |
20110319292 |
Kind Code |
A1 |
Benson; Jacqueline ; et
al. |
December 29, 2011 |
METHODS FOR GENERATING ANTI-IL-23 ANTIBODIES, COMPOSITIONS, METHODS
AND USES
Abstract
A protein variant of IL-23p19 that has conserved residues in
positions 93-102, 93-110, and/or 127-137 of SEQ ID NO:1, can be
used to generate anti-IL-23p19 antibodies.
Inventors: |
Benson; Jacqueline;
(Malvern, PA) ; Cunningham; Mark; (Kennett Square,
PA) ; Duchala; Cynthia; (North Wales, PA) ;
Giles-Komar; Jill M.; (Downingtown, PA) ; Luo;
Jinquan; (Malvern, PA) ; Rycyzyn; Michael A.;
(Berwyn, PA) ; Raymond; Sweet; (Bala Cynwyd,
PA) |
Family ID: |
37605195 |
Appl. No.: |
13/231537 |
Filed: |
September 13, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12872359 |
Aug 31, 2010 |
|
|
|
13231537 |
|
|
|
|
12370872 |
Feb 13, 2009 |
7807414 |
|
|
12872359 |
|
|
|
|
11479464 |
Jun 30, 2006 |
7491391 |
|
|
12370872 |
|
|
|
|
60695831 |
Jun 30, 2005 |
|
|
|
Current U.S.
Class: |
506/9 ;
530/387.9 |
Current CPC
Class: |
A61P 9/00 20180101; Y02A
50/386 20180101; C07K 2317/56 20130101; A61P 37/00 20180101; C07K
2317/33 20130101; C07K 2319/55 20130101; Y02A 50/41 20180101; A61P
35/00 20180101; G01N 33/6869 20130101; A61K 45/06 20130101; C07K
2317/30 20130101; A61P 19/02 20180101; A61K 39/00 20130101; A61P
17/06 20180101; Y02A 50/30 20180101; C07K 2317/92 20130101; A61P
3/10 20180101; Y02A 50/412 20180101; C07K 2317/565 20130101; A61P
25/00 20180101; A61P 11/00 20180101; A61P 27/02 20180101; A61P 1/00
20180101; A61P 25/02 20180101; A61K 39/3955 20130101; A61K
2039/55544 20130101; A61K 2039/55527 20130101; A61K 2039/505
20130101; A61P 1/04 20180101; Y02A 50/401 20180101; Y02A 50/58
20180101; Y02A 50/57 20180101; C07K 2317/76 20130101; G01N 2333/54
20130101; C07K 16/244 20130101; C07K 2317/34 20130101 |
Class at
Publication: |
506/9 ;
530/387.9 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C07K 16/24 20060101 C07K016/24 |
Claims
1. A method for generating an antibody, comprising panning an
antibody library with an isolated IL-23p19 variant protein,
comprising an amino acid sequence having less than 50% identity to
the amino acid sequence of SEQ ID NO:1 wherein said protein has the
identical amino acid sequence at residues 93-102, 93-110, and/or
127-137 of SEQ ID NO:1, and isolating an antibody or antibody
fragment binding to the IL-23p19 variant protein.
2. The method of claim 1, wherein said protein has less than 60%
identity to the amino acid sequence of SEQ ID NO:1.
3. The method of claim 1, wherein said protein has less than 70%
identity to the amino acid sequence of SEQ ID NO:1.
4. The method of claim 1, wherein said protein has less than 80%
identity to the amino acid sequence of SEQ ID NO:1.
5. The method of claim 1, wherein said protein has less than 90%
identity to the amino acid sequence of SEQ ID NO:1.
6. The method of claim 1, wherein said protein has less than 95%
identity to the amino acid sequence of SEQ ID NO:1.
7. A method for generating an antibody, comprising contacting an
animal producing antibodies with an isolated IL-23p19 variant
protein, comprising an amino acid sequence having less than 50%
identity to the amino acid sequence of SEQ ID NO:1 wherein said
protein has the identical amino acid sequence at residues 93-102,
93-110, and/or 127-137 of SEQ ID NO:1, and isolating an antibody or
antibody fragment binding to the IL-23p19 variant protein.
8. The method of claim 7, wherein said protein has less than 60%
identity to the amino acid sequence of SEQ ID NO:1.
9. The method of claim 7, wherein said protein has less than 70%
identity to the amino acid sequence of SEQ ID NO:1.
10. The method of claim 7, wherein said protein has less than 80%
identity to the amino acid sequence of SEQ ID NO:1.
11. The method of claim 7, wherein said protein has less than 90%
identity to the amino acid sequence of SEQ ID NO:1.
12. The method of claim 7, wherein said protein has less than 95%
identity to the amino acid sequence of SEQ ID NO:1.
13. The method of claim 7, wherein the animal producing antibodies
is selected from the group consisting of mice, rats, rabbits,
camel, goats, cows, horses, sheep, and non-human primates.
14. The method of claim 13, wherein the animal producing antibodies
is a transgenic animal capable of producing antibodies with human
sequences.
15. Any invention described herein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 12/872,359, filed 31 Aug. 2010, currently pending, which is a
divisional of U.S. application Ser. No. 12/370,872, filed 13 Feb.
2009, now U.S. Pat. No. 7,807,414, which is a divisional of U.S.
application Ser. No. 11/479,464, filed 30 Jun. 2006, now U.S. Pat.
No. 7,491,391, which claims the benefit of U.S. Provisional
Application Ser. No. 60/695,831, filed 30 Jun. 2005, the entire
contents of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies, including
specified portions or variants, specific for at least one IL-23
protein or fragment thereof, as well as anti-idiotype antibodies,
and nucleic acids encoding anti-IL-23p19 antibodies, complementary
nucleic acids, vectors, host cells, and methods of making and using
thereof, including therapeutic formulations, administration and
devices.
BACKGROUND OF THE INVENTION
[0003] Interleukin (IL)-12 is a secreted heterodimeric cytokine
comprised of 2 disulfide-linked glycosylated protein subunits,
designated p35 and p40 for their approximate molecular weights.
IL-12 is produced primarily by antigen-presenting cells and drives
cell-mediated immunity by binding to a two-chain receptor complex
that is expressed on the surface of T cells or natural killer (NK)
cells. The IL-12 receptor beta-1 (IL-12R.beta.1) chain binds to the
p40 subunit of IL-12, providing the primary interaction between
IL-12 and its receptor. However, it is IL-12p35 ligation of the
second receptor chain, IL-12R.beta.2, that confers intracellular
signaling (e.g. STAT4 phosphorylation) and activation of the
receptor-bearing cell (Presky et al, 1996). IL-12 signaling
concurrent with antigen presentation is thought to invoke T cell
differentiation towards the T helper 1 (Th1) phenotype,
characterized by interferon gamma (IFN.gamma.) production
(Trinchieri, 2003). Th1 cells are believed to promote immunity to
some intracellular pathogens, generate complement-fixing antibody
isotypes, and contribute to tumor immunosurveillance. Thus, IL-12
is thought to be a significant component to host defense immune
mechanisms.
[0004] It was discovered that the p40 protein subunit of IL-12 can
also associate with a separate protein subunit, designated p19, to
form a novel cytokine, IL-23 (Oppman et al, 2000). IL-23 also
signals through a two-chain receptor complex. Since the p40 subunit
is shared between IL-12 and IL-23, it follows that the
IL-12R.beta.1 chain is also shared between IL-12 and IL-23.
However, it is the IL-23p19 ligation of the second component of the
IL-23 receptor complex, IL-23R, that confers IL-23 specific
intracellular signaling (e.g., STAT3 phosphorylation) and
subsequent IL-17 production by T cells (Parham et al, 2002;
Aggarwal et al. 2003). Recent studies have demonstrated that the
biological functions of IL-23 are distinct from those of IL-12,
despite the structural similarity between the two cytokines
(Langrish et al, 2005).
[0005] Abnormal regulation of IL-12 and Th1 cell populations has
been associated with many immune-mediated diseases since
neutralization of IL-12 by antibodies is effective in treating
animal models of psoriasis, multiple sclerosis (MS), rheumatoid
arthritis, inflammatory bowel disease, insulin-dependent (type 1)
diabetes mellitus, and uveitis (Leonard et al, 1995; Hong et al,
1999; Malfait et al, 1998; Davidson et al, 1998). However, since
these studies targeted the shared p40 subunit, both IL-12 and IL-23
were neutralized in vivo. Therefore, it was unclear whether IL-12
or IL-23 was mediating disease, or if both cytokines needed to be
inhibited to achieve disease suppression. Recent studies have
confirmed through IL-23p19 deficient mice or specific antibody
neutralization of IL-23 that IL-23 inhibition can provide
equivalent benefit as anti-IL-12p40 strategies (Cua et al, 2003,
Murphy et al, 2003, Benson et al 2004). Therefore, there is
increasing evidence for the specific role of IL-23 in
immune-mediated disease. Neutralization of IL-23 without inhibition
of IL-12 pathways could then provide effective therapy of
immune-mediated disease with limited impact on important host
defense immune mechanism. This would represent a significant
improvement over current therapeutic options.
SUMMARY OF THE INVENTION
[0006] The present invention provides isolated mammalian,
including, without limitation, human, antibodies that bind to the
p19 subunit of IL-23, anti-IL-23p19 antibodies (also referred to as
IL-23p19 antibodies), immunoglobulins, fragments, cleavage products
and other specified portions and variants thereof, as well as
anti-IL-23p19 antibody compositions, IL-23p19 anti-idiotype
antibodies, encoding or complementary nucleic acids, vectors, host
cells, compositions, combinations, formulations, devices,
transgenic animals, transgenic plants, and methods of making and
using them.
[0007] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, or hybridizing
to, a polynucleotide encoding specific anti-IL-23p19 antibodies or
anti-idiotype antibodies, comprising at least one specified
sequence, domain, portion or variant thereof. The present invention
further provides recombinant vectors comprising said anti-IL-23p19
antibody nucleic acid molecules, host cells containing such nucleic
acids and/or recombinant vectors, as well as methods of making
and/or using such antibody nucleic acids, vectors and/or host
cells.
[0008] The present invention also provides at least one method for
expressing at least one anti-IL-23p19 antibody, or IL-23p19
anti-idiotype antibody, in a host cell, comprising culturing a host
cell as described herein under conditions wherein at least one
anti-IL-23p19 antibody is expressed in detectable and/or
recoverable amounts.
[0009] The present invention also provides at least one composition
comprising (a) an isolated anti-IL-23p19 antibody encoding nucleic
acid and/or antibody as described herein; and (b) a suitable and/or
pharmaceutically acceptable carrier or diluent.
[0010] The present invention further provides at least one
anti-IL-23p19 antibody method or composition, for administering a
therapeutically effective amount to modulate or treat at least one
IL-23p19 related condition in a cell, tissue, organ, animal or
patient and/or, prior to, subsequent to, or during a related
condition, as known in the art and/or as described herein.
[0011] The present invention also provides at least one
composition, device and/or method of delivery of a therapeutically
or prophylactically effective amount of at least one anti-IL-23p19
antibody, according to the present invention.
[0012] The present invention further provides at least one
anti-IL-23p19 antibody method or composition, for diagnosing at
least one IL-23 related condition in a cell, tissue, organ, animal
or patient and/or, prior to, subsequent to, or during a related
condition, as known in the art and/or as described herein.
[0013] The present invention also provides at least one
composition, device and/or method of delivery for diagnosing of at
least one anti-IL-23p19 antibody, according to the present
invention.
[0014] Also provided is a medical device, comprising at least one
isolated mammalian anti-IL-23p19 antibody of the invention, wherein
the device is suitable for contacting or administering the at least
one anti-IL-23p19 antibody, IL-23p19 anti-idiotypic antibody,
nucleic acid molecule, compound, protein, and/or composition.
[0015] Also provided is an article of manufacture for human
pharmaceutical or diagnostic use, comprising packaging material and
a container comprising a solution or a lyophilized form of at least
one isolated anti-IL-23p19 antibody of the present invention. The
article of manufacture can optionally have the container as a
component of a delivery device or system.
[0016] The present invention further provides any invention
described herein.
DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows that IL23p19 antibodies bind specifically to
hrIL-23 and not hrIL-12 or hrp40 monomer. An anti-IL12/IL23 p40
antibody is shown to bind IL-23, IL-12 and the p40 monomer. An
anti-IL12 antibody (20C2) is shown to bind IL-12 only.
[0018] FIG. 2 shows the IL-23 binding to plate-immobilized IL-23p19
antibodies of the invention in which all four antibodies tested
show similar binding curves.
[0019] FIG. 3A shows that antibodies C1249 and C1269 block normal
IL-23/IL-23R binding.
[0020] FIG. 3B shows that antibodies C1273 and C1275 block normal
IL-23/IL-23R binding.
[0021] FIG. 4 shows that the IL-23p19 antibodies of the invention
inhibit hrIL-23 mediated IL-17 production.
[0022] FIG. 5 shows the impact of IL-23 Mutations on Binding of
C1249, C1269 and CNTO 209.
[0023] FIG. 6 shows a structural model of human IL-23 in a ribbons
representation.
[0024] FIG. 7 shows the results of competition analysis of
antibodies C1249 and C1269.
[0025] FIG. 8A shows an ELISA analysis of IL-23 mutant proteins
binding to antibody C1249.
[0026] FIG. 8B shows an ELISA analysis of IL-23 mutant proteins
binding to antibody C1269.
[0027] FIG. 9 shows a comparison of the relative binding activity
for IL-23 mutant proteins binding to C1249, C1269 and control
antibody.
DESCRIPTION OF THE INVENTION
[0028] The present invention provides isolated, recombinant and/or
synthetic anti-IL-23p19 antibodies, including, without limitation,
mammalian (e.g., human antibodies) and IL-23p19 anti-idiotype
antibodies thereto, as well as compositions and encoding nucleic
acid molecules comprising at least one polynucleotide encoding at
least one anti-IL-23p19 antibody or anti-idiotype antibody. The
present invention further includes, but is not limited to, methods
of making and using such nucleic acids and antibodies and
anti-idiotype antibodies, including diagnostic and therapeutic
compositions, methods and devices.
[0029] As used herein, an "anti-IL-23p19 antibody," "IL-23p19
antibody," "anti-IL-23p19 antibody portion," or "anti-IL-23p19
antibody fragment" and/or "anti-IL-23p19 antibody variant" and the
like include any protein or peptide containing molecule that
comprises at least a portion of an immunoglobulin molecule, such as
but not limited to, at least one complementarity determining region
(CDR) of a heavy or light chain or a ligand binding portion
thereof, a heavy chain or light chain variable region, a heavy
chain or light chain constant region, a framework region, or any
portion thereof, or at least one portion of an IL-23 receptor or
binding protein, which can be incorporated into an antibody of the
present invention. Such antibody optionally further affects a
specific ligand, such as but not limited to, where such antibody
modulates, decreases, increases, antagonizes, agonizes, mitigates,
alleviates, blocks, inhibits, abrogates and/or interferes with at
least one IL-23 activity or binding, or with IL-23 receptor
activity or binding, in vitro, in situ and/or in vivo. As a
non-limiting example, a suitable anti-IL-23p19 antibody, specified
portion or variant of the present invention can bind at least one
IL-23 molecule, or specified portions, variants or domains thereof.
A suitable anti-IL-23p19 antibody, specified portion, or variant
can also optionally affect at least one of IL-23p19 activity or
function, such as but not limited to, RNA, DNA or protein
synthesis, IL-23 release, IL-23 receptor signaling, membrane IL-23
cleavage, IL-23 activity, IL-23 production and/or synthesis.
[0030] The term "antibody" is further intended to encompass
antibodies, digestion fragments, specified portions and variants
thereof, including, without limitation, antibody mimetics or
comprising portions of antibodies that mimic the structure and/or
function of an antibody or specified fragment or portion thereof,
including, without limitation, single chain antibodies, single
domain antibodies, and fragments thereof. Functional fragments
include antigen-binding fragments that bind to a human IL-23p19.
For example, antibody fragments capable of binding to IL-23p19 or
portions thereof, including, but not limited to, Fab (e.g., by
papain digestion), Fab' (e.g., by pepsin digestion and partial
reduction) and F(ab').sub.2 (e.g., by pepsin digestion), facb
(e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin
digestion), Fd (e.g., by pepsin digestion, partial reduction and
reaggregation), Fv or scFv (e.g., by molecular biology techniques)
fragments, are encompassed by the invention (see, e.g., Colligan,
Immunology, supra).
[0031] Such fragments can be produced by enzymatic cleavage,
synthetic or recombinant techniques, as known in the art and/or as
described herein. Antibodies can also be produced in a variety of
truncated forms using antibody genes in which one or more stop
codons have been introduced upstream of the natural stop site. For
example, a combination gene encoding a F(ab').sub.2 heavy chain
portion can be designed to include DNA sequences encoding the
CH.sub.1 domain and/or hinge region of the heavy chain. The various
portions of antibodies can be joined together chemically by
conventional techniques, or can be prepared as a contiguous protein
using genetic engineering techniques.
[0032] The term "human antibody," as used herein, is intended to
include antibodies having variable and constant regions derived
from or closely matching 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). Thus, as used herein, the
term "human antibody" refers to an antibody in which substantially
every part of the protein (e.g., CDR, framework, C.sub.L, C.sub.H
domains (e.g., C.sub.H1, C.sub.H2, C.sub.H3), hinge, (V.sub.L,
V.sub.H)) is substantially similar to a human germline antibody.
Human antibodies have been classified into groupings based on their
amino acid sequence similarities, see e.g.
http://people.cryst.bbk.ac.uk/.about.ubcg07s/. Thus, using a
sequence similarity search, an antibody with similar linear
sequence can be chosen as a template to create "humanized
antibodies."
[0033] "Humanization" (also called Reshaping or CDR-grafting) is
now a well-established technique for reducing the immunogenicity of
monoclonal antibodies (mAbs) from xenogeneic sources (commonly
rodent) and for improving the effector functions (ADCC, complement
activation, C1q binding). The engineered mAb is engineered using
the techniques of molecular biology, however simple CDR-grafting of
the rodent complementarity-determining regions (CDRs) into human
frameworks often results in loss of binding affinity and/or
specificity of the original mAb. In order to humanize an antibody,
the design of the humanized antibody includes variations such as
conservative amino acid substitutions in residues of the CDRs, and
back substitution of residues from the rodent mAb into the human
framework regions (backmutations). The positions can be discerned
or identified by sequence comparison for structural analysis or by
analysis of a homology model of the variable regions' 3D structure.
The process of affinity maturation has most recently used phage
libraries to vary the amino acids at chosen positions.
[0034] Similarly, many approaches have been used to choose the most
appropriate human frameworks in which to graft the rodent CDRs. As
the datasets of known parameters for antibody structures increases,
so does the sophistication and refinement of these techniques.
Consensus or germline sequences from a single antibody or fragments
of the framework sequences within each light or heavy chain
variable region from several different human mAbs can be used.
Another approach to humanization is to modify only surface residues
of the rodent sequence with the most common residues found in human
mAbs and has been termed "resurfacing" or "veneering." Known human
Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;
www.atcc.org/phage/hdb.html; www.kabatdatabase.com/top.html;
www.antibodyresource.com/onlinecomp.html;
www.appliedbiosystems.com; www.biodesign.com; antibody.bath.ac.uk;
www.unizh.ch; www.cryst.bbk.ac.uk/.about.ubcg07s; Kabat et al.,
Sequences of Proteins of Immunological Interest, U.S. Dept. Health
(1983), each entirely incorporated herein by reference. Often, the
human or humanized antibody is substantially non-immunogenic in
humans.
[0035] Similarly, antibodies designated primate (monkey, baboon,
chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig, hamster,
and the like) and other mammals designate such species, sub-genus,
genus, sub-family, and family specific antibodies. Further,
chimeric antibodies can include any combination of the above. Such
changes or variations optionally and preferably retain or reduce
the immunogenicity in humans or other species relative to
non-modified antibodies. Thus, a human antibody is distinct from a
chimeric or humanized antibody.
[0036] It is pointed out that a human antibody can be produced by a
non-human animal or prokaryotic or eukaryotic cell that is capable
of expressing functionally rearranged human immunoglobulin (e.g.,
heavy chain and/or light chain) genes. Further, when a human
antibody is a single chain or single domain antibody, it can
comprise a linker peptide that is not found in native human
antibodies. For example, an Fv can comprise a linker peptide, such
as two to about eight glycine or other amino acid residues, which
connects the variable region of the heavy chain and the variable
region of the light chain. Such linker peptides are considered to
be of human origin.
[0037] Bispecific, heterospecific, heteroconjugate or similar
antibodies can also be used that are monoclonal, preferably, human
or humanized, antibodies that have binding specificities for at
least two different antigens. In the present case, one of the
binding specificities is for at least one IL-23p19 protein subunit,
the other one is for any other antigen. Methods for making
bispecific antibodies are known in the art. Traditionally, the
recombinant production of bispecific antibodies is based on the
co-expression of two immunoglobulin heavy chain-light chain pairs,
where the two heavy chains have different specificities (Milstein
and Cuello, Nature 305:537 (1983)). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. The purification of the correct molecule is usually done
by affinity chromatography steps. Similar procedures are disclosed,
e.g., in WO 93/08829, U.S. Pat. Nos. 6,210,668, 6,193,967,
6,132,992, 6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530,
5,959,084, 5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706,
5,643,759, 5,601,819, 5,582,996, 5,496,549, 4,676,980, WO 91/00360,
WO 92/00373, EP 03089, Traunecker et al., EMBO J. 10:3655 (1991),
Suresh et al., Methods in Enzymology 121:210 (1986), each entirely
incorporated herein by reference.
[0038] Anti-IL-23p19 antibodies useful in the methods and
compositions of the present invention can optionally be
characterized by high affinity binding to IL-23p19 and, optionally
and preferably, as having low toxicity. In particular, an antibody,
specified fragment or variant of the invention, where the
individual components, such as the variable region, constant region
and framework, individually and/or collectively, optionally and
preferably possess low immunogenicity, is useful in the present
invention. The antibodies that can be used in the invention are
optionally characterized by their ability to treat patients for
extended periods with measurable alleviation of symptoms and low
and/or acceptable toxicity. Low or acceptable immunogenicity and/or
high affinity, as well as other suitable properties, can contribute
to the therapeutic results achieved. "Low immunogenicity" is
defined herein as the incidence of titrable levels of antibodies to
the anti-IL-23p19 antibody in patients treated with anti-IL-23p19
antibody as occurring in less than 25% of patients treated,
preferably, in less than 10% of patients treated with the
recommended dose for the recommended course of therapy during the
treatment period.
[0039] The isolated nucleic acids of the present invention can be
used for production of at least one anti-IL-23p19 antibody or
specified variant thereof, which can be used to measure or effect
in an cell, tissue, organ or animal (including mammals and humans),
to diagnose, monitor, modulate, treat, alleviate, help prevent the
incidence of, or reduce the symptoms of, at least one IL-23 related
condition, selected from, but not limited to, at least one of an
immune disorder or disease, a cardiovascular disorder or disease,
an infectious, malignant, and/or neurologic disorder or disease, or
other known or specified IL-23 related condition.
[0040] Such a method can comprise administering an effective amount
of a composition or a pharmaceutical composition comprising at
least one anti-IL-23p19 antibody to a cell, tissue, organ, animal
or patient in need of such modulation, treatment, alleviation,
prevention, or reduction in symptoms, effects or mechanisms. The
effective amount can comprise an amount of about 0.001 to 500 mg/kg
per single (e.g., bolus), multiple or continuous administration, or
to achieve a serum concentration of 0.01-5000 .mu.g/ml serum
concentration per single, multiple, or continuous administration,
or any effective range or value therein, as done and determined
using known methods, as described herein or known in the relevant
arts.
[0041] Antibodies of the Present Invention--Production and
Generation
[0042] At least one anti-IL-23p19 antibody of the present invention
can be optionally produced by a cell line, a mixed cell line, an
immortalized cell or clonal population of immortalized cells, as
well known in the art. See, e.g., Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,
N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow
and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.
(1989); Colligan, et al., eds., Current Protocols in Immunology,
John Wiley & Sons, Inc., NY (1994-2001); Colligan et al.,
Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001).
[0043] Antibodies that are specific for human IL-23p19 proteins or
fragments thereof can be raised against an appropriate immunogenic
antigen, such as an isolated IL-23p19 protein and/or a portion
thereof (including synthetic molecules, such as synthetic
peptides). Other specific or general antibodies, including, without
limitation, mammalian antibodies, can be similarly raised.
Preparation of immunogenic antigens, and monoclonal antibody
production can be performed using any suitable technique.
[0044] In one approach, a hybridoma is produced by fusing a
suitable immortal cell line (e.g., a myeloma cell line, such as,
but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5,
L243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA
144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3,
HL-60, MLA 144, NAMALWA, NEURO 2A, or the like, or heteromylomas,
fusion products thereof, or any cell or fusion cell derived
therefrom, or any other suitable cell line as known in the art)
(see, e.g., www.atcc.org, www.lifetech.com., and the like), with
antibody producing cells, such as, but not limited to, isolated or
cloned spleen, peripheral blood, lymph, tonsil, or other immune or
B cell containing cells, or any other cells expressing heavy or
light chain constant or variable or framework or CDR sequences,
either as endogenous or heterologous nucleic acid, as recombinant
or endogenous, viral, bacterial, algal, prokaryotic, amphibian,
insect, reptilian, fish, mammalian, rodent, equine, ovine, goat,
sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial
DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single,
double or triple stranded, hybridized, and the like or any
combination thereof. See, e.g., Ausubel, supra, and Colligan,
Immunology, supra, chapter 2, entirely incorporated herein by
reference.
[0045] Antibody producing cells can also be obtained from the
peripheral blood or, preferably, the spleen or lymph nodes, of
humans or other suitable animals that have been immunized with the
antigen of interest. Any other suitable host cell can also be used
for expressing heterologous or endogenous nucleic acid encoding an
antibody, specified fragment or variant thereof, of the present
invention. The fused cells (hybridomas) or recombinant cells can be
isolated using selective culture conditions or other suitable known
methods, and cloned by limiting dilution or cell sorting, or other
known methods. Cells which produce antibodies with the desired
specificity can be selected by a suitable assay (e.g., ELISA).
[0046] Methods for engineering or humanizing non-human or human
antibodies can also be used and are well known in the art. A
humanized or engineered antibody may have one or more amino acid
residues from a source that is non-human, e.g., but not limited to,
mouse, rat, rabbit, non-human primate or other mammal. These
non-human amino acid residues are replaced by residues that are
often referred to as "import" residues, which are typically taken
from an "import" variable, constant or other domain of a known
human sequence.
[0047] Known human Ig sequences are disclosed, e.g.,
www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;
www.atcc.org/phage/hdb.html; www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php;
www.kabatdatabase.com/top.html; ftp.ncbi.nih.gov/repository/kabat;
www.sciquest.com; www.abcam.com;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research tools.html;
www.whfreeman.com/immunology/CH05/kuby05.htm;
www.hhmi.org/grants/lectures/1996/vlab;
www.path.cam.ac.uk/.about.mrc7/mikeimages.html;
mcb.harvard.edu/BioLinks/Immunology.html; www.immunologylink.com;
pathbox.wustl.edu/.about.hcenter/index.html;
www.appliedbiosystems.com; www.nal.usda.gov/awic/pubs/antibody;
www.m.ehime-u.ac.jp/.about.yasuhito/Elisa.html; www.biodesign.com;
www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org;
baserv.uci.kun.nl/.about.jraats/links1.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu; www.mrc-cpe.cam.ac.uk;
www.ibt.unam.mx/vir/V_mice.html; http://www.bioinf.org.uk/abs;
antibody.bath.ac.uk; www.unizh.ch;
www.cryst.bbk.ac.uk/.about.ubcg07s;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;
www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html; www.jerini.de; Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983), each entirely incorporated herein by reference.
[0048] 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.
[0049] In general, the CDR residues are directly and most
substantially involved in influencing antigen binding. Accordingly,
part or all of the non-human or human CDR sequences are maintained
while the non-human sequences of the variable and constant regions
may be replaced with human or other amino acids.
[0050] Antibodies can also optionally be humanized or engineered or
human antibodies engineered with retention of high affinity for the
antigen and other favorable biological properties. To achieve this
goal, humanized (or human) antibodies can be optionally prepared by
a process of analysis of the parental sequences and various
conceptual humanized and engineered products using
three-dimensional models of the parental, engineered, and humanized
sequences.
[0051] 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.
[0052] In this way, framework (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.
[0053] In addition, the IL-23p19 antibody of the present invention
may comprise a human germline light chain framework. In particular
embodiments, the light chain germline sequence is selected from
human VK sequences including, but not limited to, A1, A10, A11,
A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2,
B3, L1, L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23,
L24, L25, L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4,
and O8. In certain embodiments, this light chain human germline
framework is selected from V1-11, V1-13, V1-16, V1-17, V1-18,
V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1,
V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2,
V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and
V5-6. See PCT WO 2005/005604 for a description of the different
germline sequences.
[0054] In other embodiments, the IL-23 antibody of the present
invention may comprise a human germline heavy chain framework. In
particular embodiments, this heavy chain human germline framework
is selected from VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46,
VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13,
VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35,
VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7,
VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39,
VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81. See PCT WO
2005/005604 for a description of the different germline
sequences.
[0055] In particular embodiments, the light chain variable region
and/or heavy chain variable region comprises a framework region or
at least a portion of a framework region (e.g., containing 2 or 3
subregions, such as FR2 and FR3). In certain embodiments, at least
FRL1, FRL2, FRL3, or FRL4 is fully human. In other embodiments, at
least FRH1, FRH2, FRH3, or FRH4 is fully human. In some
embodiments, at least FRL1, FRL2, FRL3, or FRL4 is a germline
sequence (e.g., human germline) or comprises human consensus
sequences for the particular framework (readily available at the
sources of known human Ig sequences described above). In other
embodiments, at least FRH1, FRH2, FRH3, or FRH4 is a germline
sequence (e.g., human germline) or comprises human consensus
sequences for the particular framework. In preferred embodiments,
the framework region is a human framework region.
[0056] Humanization or engineering of antibodies of the present
invention can be performed using any known method, such as but not
limited to those described in, Winter (Jones et al., Nature 321:522
(1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al.,
Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296
(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et
al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al.,
J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862,
5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886,
5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089,
5,225,539; 4,816,567, PCT/: US98/16280, US96/18978, US91/09630,
US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;
WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirely
incorporated herein by reference, included references cited
therein.
[0057] In certain embodiments, the antibody comprises an altered
(e.g., mutated) Fc region. For example, in some embodiments, the Fc
region has been altered to reduce or enhance the effector functions
of the antibody. In some embodiments, the Fc region is an isotype
selected from IgM, IgA, IgG, IgE, or other isotype.
[0058] Alternatively or additionally, it may be useful to combine
amino acid modifications with one or more further amino acid
modifications that alter C1q binding and/or the complement
dependent cytotoxicity (CDC) function of the Fc region of an
IL-23p19 binding molecule. The binding polypeptide of particular
interest may be one that binds to C1q and displays complement
dependent cytotoxicity.
[0059] Polypeptides with pre-existing C1q binding activity,
optionally further having the ability to mediate CDC may be
modified such that one or both of these activities are enhanced.
Amino acid modifications that alter C1q and/or modify its
complement dependent cytotoxicity function are described, for
example, in WO/0,042,072, which is hereby incorporated by
reference.
[0060] As disclosed above, one can design an Fc region of the
IL-23p19 antibody of the present invention with altered effector
function, e.g., by modifying C1q binding and/or Fc.gamma.R binding
and thereby changing CDC activity and/or ADCC activity. "Effector
functions" are responsible for activating or diminishing a
biological activity (e.g., in a subject). Examples of effector
functions include, but are not limited to: C1q binding; complement
dependent cytotoxicity (CDC); Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g., B cell receptor;
BCR), etc. Such effector functions may require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays (e.g., Fc binding assays,
ADCC assays, CDC assays, etc.).
[0061] For example, one can generate a variant Fc region of the
IL-23p19 antibody with improved C1q binding and improved
Fc.gamma.RIIIbinding (e.g., having both improved ADCC activity and
improved CDC activity). Alternatively, if it is desired that
effector function be reduced or ablated, a variant Fc region can be
engineered with reduced CDC activity and/or reduced ADCC activity.
In other embodiments, only one of these activities may be
increased, and, optionally, also the other activity reduced (e.g.,
to generate an Fc region variant with improved ADCC activity, but
reduced CDC activity and vice versa).
[0062] Fc mutations can also be introduced in engineer to alter
their interaction with the neonatal Fc receptor (FcRn) and improve
their pharmacokinetic properties. A collection of human Fc variants
with improved binding to the FcRn have been described (Shields et
al., (2001). High resolution mapping of the binding site on human
IgG1 for Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII, and FcRn and
design of IgG1 variants with improved binding to the Fc.gamma.R, J.
Biol. Chem. 276:6591-6604).
[0063] Another type of amino acid substitution serves to alter the
glycosylation pattern of the Fc region of the IL-23p19 antibody.
Glycosylation of an Fc region is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. O-linked
glycosylation refers to the attachment of one of the sugars
N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used. The recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain peptide sequences are asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline.
Thus, the presence of either of these peptide sequences in a
polypeptide creates a potential glycosylation site.
[0064] The glycosylation pattern may be altered, for example, by
deleting one or more glycosylation site(s) found in the
polypeptide, and/or adding one or more glycosylation site(s) that
are not present in the polypeptide. Addition of glycosylation sites
to the Fc region of an IL-23p19 antibody is conveniently
accomplished by altering the amino acid sequence such that it
contains one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites). An exemplary glycosylation
variant has an amino acid substitution of residue Asn 297 of the
heavy chain. The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the original polypeptide (for O-linked glycosylation
sites). Additionally, a change of Asn 297 to Ala can remove one of
the glycosylation sites.
[0065] In certain embodiments, the IL-23p19 antibody of the present
invention is expressed in cells that express beta
(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT
III adds GlcNAc to the IL-23p19 antibody. Methods for producing
antibodies in such a fashion are provided in WO/9,954,342,
WO/03011878, patent publication 20030003097A1, and Umana et al.,
Nature Biotechnology, 17:176-180, February 1999. An anti-IL-23p19
antibody can be optionally generated by immunization of a
transgenic animal (e.g., mouse, rat, hamster, non-human primate,
and the like) capable of producing a repertoire of human
antibodies, as described herein and/or as known in the art. Cells
that produce an anti-IL-23p19 antibody can be isolated from such
animals and immortalized using suitable methods, such as the
methods described herein.
[0066] Transgenic mice that can produce a repertoire of human
antibodies that bind to human antigens can be produced by known
methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428,
5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016
and 5,789,650 issued to Lonberg et al.; Jakobovits et al. WO
98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO
98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,
Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151
B1, Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.
5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438
474 B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440
A, Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int.
Immunol. 6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21
(1994), Mendez et al., Nature Genetics 15:146-156 (1997), Taylor et
al., Nucleic Acids Research 20(23):6287-6295 (1992), Tuaillon et
al., Proc Natl Acad Sci USA 90(8)3720-3724 (1993), Lonberg et al.,
Int Rev Immunol 13(1):65-93 (1995) and Fishwald et al., Nat
Biotechnol 14(7):845-851 (1996), which are each entirely
incorporated herein by reference). Generally, these mice comprise
at least one transgene comprising DNA from at least one human
immunoglobulin locus that is functionally rearranged, or which can
undergo functional rearrangement. The endogenous immunoglobulin
loci in such mice can be disrupted or deleted to eliminate the
capacity of the animal to produce antibodies encoded by endogenous
genes.
[0067] Screening antibodies for specific binding to similar
proteins or fragments can be conveniently achieved using peptide
display libraries. This method involves the screening of large
collections of peptides for individual members having the desired
function or structure. Antibody screening of peptide display
libraries is well known in the art. The displayed peptide sequences
can be from 3 to 5000 or more amino acids in length, frequently
from 5-100 amino acids long, and often from about 8 to 25 amino
acids long. In addition to direct chemical synthetic methods for
generating peptide libraries, several recombinant DNA methods have
been described. One type involves the display of a peptide sequence
on the surface of a bacteriophage or cell. Each bacteriophage or
cell contains the nucleotide sequence encoding the particular
displayed peptide sequence. Such methods are described in PCT
Patent Publication Nos. 91/17271, 91/18980, 91/19818, and
93/08278.
[0068] Other systems for generating libraries of peptides have
aspects of both in vitro chemical synthesis and recombinant
methods. See, PCT Patent Publication Nos. 92/05258, 92/14843, and
96/19256. See also, U.S. Pat. Nos. 5,658,754; and 5,643,768.
Peptide display libraries, vector, and screening kits are
commercially available from such suppliers as Invitrogen (Carlsbad,
Calif.), and Cambridge Antibody Technologies (Cambridgeshire, UK).
See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778,
5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733,
5,767,260, 5,856,456, assigned to Enzon; 5,223,409, 5,403,484,
5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717,
assigned to Affymax; 5,885,793, assigned to Cambridge Antibody
Technologies; 5,750,373, assigned to Genentech, 5,618,920,
5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to
Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra.
[0069] Antibodies of the present invention can also be prepared
using at least one anti-IL-23p19 antibody encoding nucleic acid to
provide transgenic animals or mammals, such as goats, cows, horses,
sheep, rabbits and the like, that produce such antibodies in their
milk. Such animals can be provided using known methods. See, e.g.,
but not limited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316;
5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of
which is entirely incorporated herein by reference.
[0070] Antibodies of the present invention can additionally be
prepared using at least one anti-IL-23p19 antibody encoding nucleic
acid to provide transgenic plants and cultured plant cells (e.g.,
but not limited to, tobacco and maize) that produce such
antibodies, specified portions or variants in the plant parts or in
cells cultured therefrom. As a non-limiting example, transgenic
tobacco leaves expressing recombinant proteins have been
successfully used to provide large amounts of recombinant proteins,
e.g., using an inducible promoter. See, e.g., Cramer et al., Curr.
Top. Microbol. Immunol. 240:95-118 (1999) and references cited
therein. Also, transgenic maize have been used to express mammalian
proteins at commercial production levels, with biological
activities equivalent to those produced in other recombinant
systems or purified from natural sources. See, e.g., Hood et al.,
Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited
therein. Antibodies have also been produced in large amounts from
transgenic plant seeds including antibody fragments, such as single
chain antibodies (scFv's), including tobacco seeds and potato
tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109
(1998) and references cited therein. Thus, antibodies of the
present invention can also be produced using transgenic plants,
according to known methods. See also, e.g., Fischer et al.,
Biotechnol. Appl. Biochem. 30:99-108 (October, 1999), Ma et al.,
Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol.
109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944
(1994); and references cited therein.
[0071] The antibodies of the invention can bind human IL-23p19 with
a wide range of affinities (K.sub.D). In a preferred embodiment, at
least one mAb of the present invention can optionally bind human
IL-23p19 with high affinity. For example, a human or other mAb can
bind human IL-23p19 with a K.sub.D equal to or less than about
10.sup.-7 M, such as but not limited to, 0.1-9.9 (or any range or
value therein) X 10.sup.-7, 10.sup.-8, 10.sup.-9, 10.sup.-10,
10.sup.-11, 10.sup.-12, 10.sup.-13, 10.sup.-14, 10.sup.-15 or any
range or value therein, as determined by surface plasmon resonance
or the Kinexa method, as practiced by those of skill in the
art.
[0072] In one embodiment, the antibodies of the invention bind
human IL-23, or more specifically, IL-23p19, with a K.sub.D between
about 3.38.times.10.sup.-10 M and about 4.3.times.10.sup.-11 M.
[0073] The affinity or avidity of an antibody for an antigen can be
determined experimentally using any suitable method. (See, for
example, Berzofsky, et al., "Antibody-Antigen Interactions," In
Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York,
N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New
York, N.Y. (1992); and methods described herein). The measured
affinity of a particular antibody-antigen interaction can vary if
measured under different conditions (e.g., salt concentration, pH).
Thus, measurements of affinity and other antigen-binding parameters
(e.g., K.sub.D, K.sub.on, K.sub.off) are preferably made with
standardized solutions of antibody and antigen, and a standardized
buffer, such as the buffer described herein.
[0074] Certain embodiments of the anti-IL-23p19 antibodies of the
invention have the sequences shown in the Sequence Tables below.
For example, an anti-IL-23p19 antibody of the invention has one of
the light chain CDR1 sequences of SEQ ID NOS:9, 19, 29, and 39; one
of the light chain CDR2 sequences of SEQ ID NOS:10, 20, 30, and 40;
one of the light chain CDR3 sequences of SEQ ID NOS:11, 21, 31, and
41; one of the heavy chain CDR1 sequences SEQ ID NOS:4, 14, 24, and
34; one of the heavy chain CDR2 sequences SEQ ID NOS:5, 15, 25, and
35; and/or one of the heavy chain CDR1 sequences SEQ ID NOS:6, 16,
26, and 36.
[0075] Nucleic Acid Molecules
[0076] Using the information provided herein, for example, the
nucleotide sequences encoding at least 70-100% of the contiguous
amino acids of at least one of the light chain variable regions of
SEQ ID NOS:7, 17, 27, and 37 and at least one of the heavy chain
variable regions of SEQ ID NOS:2, 12, 22, and 32, specified
fragments, variants or consensus sequences thereof, or a deposited
vector comprising at least one of these sequences, a nucleic acid
molecule of the present invention encoding at least one
anti-IL-23p19 antibody can be obtained using methods described
herein or as known in the art.
[0077] Nucleic acid molecules of the present invention can be in
the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in
the form of DNA, including, but not limited to, cDNA and genomic
DNA obtained by cloning or produced synthetically, or any
combinations thereof. The DNA can be triple-stranded,
double-stranded or single-stranded, or any combination thereof. Any
portion of at least one strand of the DNA or RNA can be the coding
strand, also known as the sense strand, or it can be the non-coding
strand, also referred to as the anti-sense strand.
[0078] Isolated nucleic acid molecules of the present invention can
include nucleic acid molecules comprising an open reading frame
(ORF), optionally, with one or more introns, e.g., but not limited
to, at least one specified portion of at least one CDR, such as
CDR1, CDR2 and/or CDR3 of at least light chain (9, 10, 11, 19, 20,
21, 29, 30, 31, 39, 40, 41) or at least one heavy chain (SEQ ID
NOS:4, 5, 6, 14, 15, 16, 24, 25, 26, 34, 35, and 36); nucleic acid
molecules comprising the coding sequence for an anti-IL-23p19
antibody or variable region (e.g., light chain variable regions of
SEQ ID NOS:7, 17, 27, and 37 and heavy chain variable regions of
SEQ ID NOS:2, 12, 22, and 32); and nucleic acid molecules which
comprise a nucleotide sequence substantially different from those
described above but which, due to the degeneracy of the genetic
code, still encode at least one anti-IL-23p19 antibody as described
herein and/or as known in the art. Of course, the genetic code is
well known in the art. Thus, it would be routine for one skilled in
the art to generate such degenerate nucleic acid variants that code
for specific anti-IL-23p19 antibodies of the present invention.
See, e.g., Ausubel, et al., supra, and such nucleic acid variants
are included in the present invention.
[0079] As indicated herein, nucleic acid molecules of the present
invention which comprise a nucleic acid encoding an anti-IL-23p19
antibody can include, but are not limited to, those encoding the
amino acid sequence of an antibody fragment, by itself; the coding
sequence for the entire antibody or a portion thereof; the coding
sequence for an antibody, fragment or portion, as well as
additional sequences, such as the coding sequence of at least one
signal leader or fusion peptide, with or without the aforementioned
additional coding sequences, such as at least one intron, together
with additional, non-coding sequences, including but not limited
to, non-coding 5' and 3' sequences, such as the transcribed,
non-translated sequences that play a role in transcription, mRNA
processing, including splicing and polyadenylation signals (for
example, ribosome binding and stability of mRNA); an additional
coding sequence that codes for additional amino acids, such as
those that provide additional functionalities. Thus, the sequence
encoding an antibody can be fused to a marker sequence, such as a
sequence encoding a peptide that facilitates purification of the
fused antibody comprising an antibody fragment or portion.
[0080] Polynucleotides Selectively Hybridizing to a Polynucleotide
as Described Herein
[0081] The present invention provides isolated nucleic acids that
hybridize under selective hybridization conditions to a
polynucleotide disclosed herein. Thus, the polynucleotides of this
embodiment can be used for isolating, detecting, and/or quantifying
nucleic acids comprising such polynucleotides. For example,
polynucleotides of the present invention can be used to identify,
isolate, or amplify partial or full-length clones in a deposited
library. In some embodiments, the polynucleotides are genomic or
cDNA sequences isolated, or otherwise complementary to, a cDNA from
a human or mammalian nucleic acid library.
[0082] Preferably, the cDNA library comprises at least 80%
full-length sequences, preferably, at least 85% or 90% full-length
sequences, and, more preferably, at least 95% full-length
sequences. The cDNA libraries can be normalized to increase the
representation of rare sequences. Low or moderate stringency
hybridization conditions are typically, but not exclusively,
employed with sequences having a reduced sequence identity relative
to complementary sequences. Moderate and high stringency conditions
can optionally be employed for sequences of greater identity. Low
stringency conditions allow selective hybridization of sequences
having about 70% sequence identity and can be employed to identify
orthologous or paralogous sequences.
[0083] Optionally, polynucleotides of this invention will encode at
least a portion of an antibody encoded by the polynucleotides
described herein. The polynucleotides of this invention embrace
nucleic acid sequences that can be employed for selective
hybridization to a polynucleotide encoding an antibody of the
present invention. See, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by reference.
[0084] Construction of Nucleic Acids
[0085] The isolated nucleic acids of the present invention can be
made using (a) recombinant methods, (b) synthetic techniques, (c)
purification techniques, and/or (d) combinations thereof, as
well-known in the art.
[0086] The nucleic acids can conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites can be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences can
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention, excluding the coding sequence, is optionally
a vector, adapter, or linker for cloning and/or expression of a
polynucleotide of the present invention.
[0087] Additional sequences can be added to such cloning and/or
expression sequences to optimize their function in cloning and/or
expression, to aid in isolation of the polynucleotide, or to
improve the introduction of the polynucleotide into a cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well
known in the art. (See, e.g., Ausubel, supra; or Sambrook,
supra)
[0088] Recombinant Methods for Constructing Nucleic Acids
[0089] The isolated nucleic acid compositions of this invention,
such as RNA, cDNA, genomic DNA, or any combination thereof, can be
obtained from biological sources using any number of cloning
methodologies known to those of skill in the art. In some
embodiments, oligonucleotide probes that selectively hybridize,
under stringent conditions, to the polynucleotides of the present
invention are used to identify the desired sequence in a cDNA or
genomic DNA library. The isolation of RNA, and construction of cDNA
and genomic libraries, are well known to those of ordinary skill in
the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
[0090] Nucleic Acid Screening and Isolation Methods
[0091] A cDNA or genomic library can be screened using a probe
based upon the sequence of a polynucleotide of the present
invention, such as those disclosed herein. Probes can be used to
hybridize with genomic DNA or cDNA sequences to isolate homologous
genes in the same or different organisms. Those of skill in the art
will appreciate that various degrees of stringency of hybridization
can be employed in the assay; and either the hybridization or the
wash medium can be stringent. As the conditions for hybridization
become more stringent, there must be a greater degree of
complementarity between the probe and the target for duplex
formation to occur. The degree of stringency can be controlled by
one or more of temperature, ionic strength, pH and the presence of
a partially denaturing solvent, such as formamide. For example, the
stringency of hybridization is conveniently varied by changing the
polarity of the reactant solution through, for example,
manipulation of the concentration of formamide within the range of
0% to 50%. The degree of complementarity (sequence identity)
required for detectable binding will vary in accordance with the
stringency of the hybridization medium and/or wash medium. The
degree of complementarity will optimally be 100%, or 70-100%, or
any range or value therein. However, it should be understood that
minor sequence variations in the probes and primers can be
compensated for by reducing the stringency of the hybridization
and/or wash medium.
[0092] Methods of amplification of RNA or DNA are well known in the
art and can be used according to the present invention without
undue experimentation, based on the teaching and guidance presented
herein.
[0093] Known methods of DNA or RNA amplification include, but are
not limited to, polymerase chain reaction (PCR) and related
amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and
4,921,794 to Tabor, et al; 5,142,033 to Innis; 5,122,464 to Wilson,
et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al;
4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067
to Biswas; 4,656,134 to Ringold) and RNA mediated amplification
that uses anti-sense RNA to the target sequence as a template for
double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et
al, with the tradename NASBA), the entire contents of which
references are incorporated herein by reference. (See, e.g.,
Ausubel, supra; or Sambrook, supra.)
[0094] For instance, polymerase chain reaction (PCR) technology can
be used to amplify the sequences of polynucleotides of the present
invention and related genes directly from genomic DNA or cDNA
libraries. PCR and other in vitro amplification methods can also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of the desired mRNA in samples, for
nucleic acid sequencing, or for other purposes. Examples of
techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, supra, Sambrook, supra,
and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No.
4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to
Methods and Applications, Eds., Academic Press Inc., San Diego,
Calif. (1990).
[0095] Commercially available kits for genomic PCR amplification
are known in the art. See, e.g., Advantage-GC Genomic PCR Kit
(Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer
Mannheim) can be used to improve yield of long PCR products.
[0096] Synthetic Methods for Constructing Nucleic Acids
[0097] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by known methods (see,
e.g., Ausubel, et al., supra). Chemical synthesis generally
produces a single-stranded oligonucleotide, which can be converted
into double-stranded DNA by hybridization with a complementary
sequence, or by polymerization with a DNA polymerase using the
single strand as a template. One of skill in the art will recognize
that while chemical synthesis of DNA can be limited to sequences of
about 100 or more bases, longer sequences can be obtained by the
ligation of shorter sequences.
[0098] Recombinant Expression Cassettes
[0099] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence of the present invention, for
example, a cDNA or a genomic sequence encoding an antibody of the
present invention, can be used to construct a recombinant
expression cassette that can be introduced into at least one
desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the present invention operably linked
to transcriptional initiation regulatory sequences that will direct
the transcription of the polynucleotide in the intended host cell.
Both heterologous and non-heterologous (i.e., endogenous) promoters
can be employed to direct expression of the nucleic acids of the
present invention.
[0100] In some embodiments, isolated nucleic acids that serve as
promoter, enhancer, or other elements can be introduced in the
appropriate position (upstream, downstream or in the intron) of a
non-heterologous form of a polynucleotide of the present invention
so as to up or down regulate expression of a polynucleotide of the
present invention. For example, endogenous promoters can be altered
in vivo or in vitro by mutation, deletion and/or substitution.
[0101] Vectors And Host Cells
[0102] The present invention also relates to vectors that include
isolated nucleic acid molecules of the present invention, host
cells that are genetically engineered with the recombinant vectors,
and the production of at least one anti-IL-23p19 antibody by
recombinant techniques, as is well known in the art. See, e.g.,
Sambrook, et al., supra; Ausubel, et al., supra, each entirely
incorporated herein by reference.
[0103] The polynucleotides can optionally be joined to a vector
containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced in a precipitate, such as
a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it can be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0104] The DNA insert should be operatively linked to an
appropriate promoter. The expression constructs will further
contain sites for transcription initiation, termination and, in the
transcribed region, a ribosome binding site for translation. The
coding portion of the mature transcripts expressed by the
constructs will preferably include a translation initiating at the
beginning and a termination codon (e.g., UAA, UGA or UAG)
appropriately positioned at the end of the mRNA to be translated,
with UAA and UAG preferred for mammalian or eukaryotic cell
expression.
[0105] Expression vectors will preferably but optionally include at
least one selectable marker. Such markers include, e.g., but are
not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR,
U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288;
5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic
acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464;
5,770,359; 5,827,739) resistance for eukaryotic cell culture, and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria or prokaryotics (the above patents are
entirely incorporated hereby by reference). Appropriate culture
mediums and conditions for the above-described host cells are known
in the art. Suitable vectors will be readily apparent to the
skilled artisan. Introduction of a vector construct into a host
cell can be effected by calcium phosphate transfection,
DEAE-dextran mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection or other
known methods. Such methods are described in the art, such as
Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters
1, 9, 13, 15, 16.
[0106] At least one antibody of the present invention can be
expressed in a modified form, such as a fusion protein, and can
include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of
additional amino acids, particularly charged amino acids, can be
added to the N-terminus of an antibody to improve stability and
persistence in the host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties can be
added to an antibody of the present invention to facilitate
purification. Such regions can be removed prior to final
preparation of an antibody or at least one fragment thereof. Such
methods are described in many standard laboratory manuals, such as
Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel,
supra, Chapters 16, 17 and 18.
[0107] Those of ordinary skill in the art are knowledgeable in the
numerous expression systems available for expression of a nucleic
acid encoding a protein of the present invention. Alternatively,
nucleic acids of the present invention can be expressed in a host
cell by turning on (by manipulation) in a host cell that contains
endogenous DNA encoding an antibody of the present invention. Such
methods are well known in the art, e.g., as described in U.S. Pat.
Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely
incorporated herein by reference.
[0108] Illustrative of cell cultures useful for the production of
the antibodies, specified portions or variants thereof, are
mammalian cells. Mammalian cell systems often will be in the form
of monolayers of cells although mammalian cell suspensions or
bioreactors can also be used. A number of suitable host cell lines
capable of expressing intact glycosylated proteins have been
developed in the art, and include the COS-1 (e.g., ATCC CRL 1650),
COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO
(e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines,
Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293
cells, HeLa cells and the like, which are readily available from,
for example, American Type Culture Collection, Manassas, Va.
(www.atcc.org). Preferred host cells include cells of lymphoid
origin, such as myeloma and lymphoma cells. Particularly preferred
host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580)
and SP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a
particularly preferred embodiment, the recombinant cell is a
P3X63Ab8.653 or a SP2/0-Ag14 cell.
[0109] Expression vectors for these cells can include one or more
of the following expression control sequences, such as, but not
limited to, an origin of replication; a promoter (e.g., late or
early SV40 promoters, the CMV promoter (U.S. Pat. Nos. 5,168,062;
5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase)
promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at
least one human immunoglobulin promoter; an enhancer, and/or
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site), and transcriptional terminator sequences. See,
e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells
useful for production of nucleic acids or proteins of the present
invention are known and/or available, for instance, from the
American Type Culture Collection Catalogue of Cell Lines and
Hybridomas (www.atcc.org) or other known or commercial sources.
[0110] When eukaryotic host cells are employed, polyadenlyation or
transcription terminator sequences are typically incorporated into
the vector. An example of a terminator sequence is the
polyadenlyation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript can also be
included. An example of a splicing sequence is the VP1 intron from
SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally,
gene sequences to control replication in the host cell can be
incorporated into the vector, as known in the art.
[0111] Purification of an Antibody
[0112] An anti-IL-23p19 antibody can be recovered and purified from
recombinant cell cultures by well-known methods including, but not
limited to, protein A purification, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be employed for
purification. See, e.g., Colligan, Current Protocols in Immunology,
or Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely
incorporated herein by reference.
[0113] Antibodies of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a eukaryotic host,
including, for example, yeast, higher plant, insect and mammalian
cells. Depending upon the host employed in a recombinant production
procedure, the antibody of the present invention can be
glycosylated or can be non-glycosylated, with glycosylated
preferred. Such methods are described in many standard laboratory
manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel,
supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein
Science, supra, Chapters 12-14, all entirely incorporated herein by
reference.
[0114] Anti-IL-23p19 Antibodies
[0115] An anti-IL-23p19 antibody according to the present invention
includes any protein or peptide containing molecule that comprises
at least a portion of an immunoglobulin molecule, such as but not
limited to, at least one ligand binding portion (LBP), such as but
not limited to, a complementarity determining region (CDR) of a
heavy or light chain or a ligand binding portion thereof, a heavy
chain or light chain variable region, a framework region (e.g.,
FR1, FR2, FR3, FR4 or fragment thereof, further optionally
comprising at least one substitution, insertion or deletion), a
heavy chain or light chain constant region, (e.g., comprising at
least one CH1, hinge1, hinge2, hinge3, hinge4, CH2, or CH3 or
fragment thereof, further optionally comprising at least one
substitution, insertion or deletion), or any portion thereof, that
can be incorporated into an antibody of the present invention. An
antibody of the invention can include or be derived from any
mammal, such as but not limited to, a human, a mouse, a rabbit, a
rat, a rodent, a primate, or any combination thereof, and the
like.
[0116] The isolated antibodies of the present invention comprise
the antibody amino acid sequences disclosed herein encoded by any
suitable polynucleotide, or any isolated or prepared antibody.
Preferably, the human antibody or antigen-binding fragment binds
human IL-23p19 and, thereby, partially or substantially neutralizes
at least one biological activity of the protein. An antibody, or
specified portion or variant thereof, that partially or preferably
substantially neutralizes at least one biological activity of at
least one IL-23 protein or fragment can bind the protein or
fragment and thereby inhibit activities mediated through the
binding of IL-23 to one or more of the IL-23 receptors or through
other IL-23-dependent or mediated mechanisms. As used herein, the
term "neutralizing antibody" refers to an antibody that can inhibit
an IL-23-dependent activity by about 20-120%, preferably by at
least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the
assay. The capacity of an anti-IL-23p19 antibody to inhibit an
IL-23-dependent activity is preferably assessed by at least one
suitable IL-23 protein or receptor assay, as described herein
and/or as known in the art. A human antibody of the invention can
be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can
comprise a kappa or lambda light chain. In one embodiment, the
human antibody comprises an IgG heavy chain or defined fragment,
for example, at least one of isotypes, IgG1, IgG2, IgG3 or IgG4
(e.g., .gamma.1, .gamma.2, .gamma.3, or .gamma.4).
[0117] Antibodies of this type can be prepared by employing a
transgenic mouse or other trangenic non-human mammal comprising at
least one human light chain (e.g., IgG, IgA, and IgM) transgenes as
described herein and/or as known in the art. In another embodiment,
the anti-human IL-23p19 antibody comprises an IgG1 heavy chain and
an IgG1 light chain.
[0118] At least one antibody of the invention binds at least one
specified epitope specific to at least one IL-23p19 protein,
subunit, fragment, portion or any combination thereof. The at least
one epitope can comprise at least one antibody binding region that
comprises at least one portion of the protein, which epitope is
preferably comprised of at least one extracellular, soluble,
hydrophillic, external or cytoplasmic portion of the protein. The
at least one specified epitope can comprise any combination of at
least one amino acid sequence of at least 1-3 amino acids to the
entire specified portion of contiguous amino acids of amino acid
residues 93-104 and 127-137 of SEQ ID NO:1 (that contains the
initial 19 amino acid signal sequence for the p19 protein subunit)
(or amino acid residues 74-85 and 108-118 of the p19 sequence
without inclusion of the signal sequence), for example, amino acid
residues 93, 93-94, 93-95, 93-96, 97-99, 100-102, 127, 127-128,
128-129, etc. of SEQ ID NO:1, etc. that include any portions or
combinations of these sequences.
[0119] Generally, the antibody or antigen-binding fragment of the
present invention will comprise an antigen-binding region that
comprises at least one complementarity determining region (CDR1,
CDR2 and CDR3) or variant of at least one heavy chain variable
region and at least one complementarity determining region (CDR1,
CDR2 and CDR3) or variant of at least one light chain variable
region. Optionally, the CDR sequences may be derived from human
germline sequences or closely match the germline sequences. For
example, the CDRs from a synthetic library derived from the
original mouse CDRs can be used. These CDRs may be formed by
incorporation of conservative substitutions from the orginal mouse
sequence. As a non-limiting example, the antibody or
antigen-binding portion or variant can comprise at least one of the
heavy chain CDR3, e.g., selected from SEQ ID NOS:6, 16, 26, and 36,
and/or a light chain CDR3, e.g., selected from SEQ ID NOS:11, 21,
31, and 41. In a particular embodiment, the antibody or
antigen-binding fragment can have an antigen-binding region that
comprises at least a portion of at least one heavy chain CDR (i.e.,
CDR1, CDR2 and/or CDR3) (e.g., those disclosed herein). In another
particular embodiment, the antibody or antigen-binding portion or
variant can have an antigen-binding region that comprises at least
a portion of at least one light chain CDR (i.e., CDR1, CDR2 and/or
CDR3) (e.g., those disclosed herein).
[0120] In a preferred embodiment, the three heavy chain CDRs and
the three light chain CDRs of the antibody or antigen-binding
fragment can be prepared by chemically joining together the various
portions (e.g., CDRs, framework) of the antibody using conventional
techniques, by preparing and expressing a (i.e., one or more)
nucleic acid molecule that encodes the antibody using conventional
techniques of recombinant DNA technology or by using any other
suitable method.
[0121] The anti-IL-23p19 antibody can comprise at least one of a
heavy or light chain variable region having a defined amino acid
sequence. For example, in a preferred embodiment, the anti-IL-23p19
antibody comprises at least one of at least one heavy chain
variable region optionally selected from SEQ ID NOS:3, 13, 23, and
33 and/or at least one light chain variable region optionally
selected from SEQ ID NOS:8, 18, 28, and 38. Antibodies that bind to
human IL-23p19 and that comprise a defined heavy or light chain
variable region can be prepared using suitable methods, such as
phage display (Katsube, Y., et al., Int J. Mol. Med, 1(5):863-868
(1998)) or methods that employ transgenic animals, as known in the
art and/or as described herein. For example, a transgenic mouse,
comprising a functionally rearranged human immunoglobulin heavy
chain transgene and a transgene comprising DNA from a human
immunoglobulin light chain locus that can undergo functional
rearrangement, can be immunized with human IL-23 or a fragment
thereof to elicit the production of antibodies.
[0122] If desired, the antibody producing cells can be isolated and
hybridomas or other immortalized antibody-producing cells can be
prepared as described herein and/or as known in the art.
Alternatively, the antibody, specified portion or variant can be
expressed using the encoding nucleic acid or portion thereof in a
suitable host cell.
[0123] Amino Acid Codes
[0124] The amino acids that make up anti-IL-23p19 antibodies of the
present invention are often abbreviated. The amino acid
designations can be indicated by designating the amino acid by its
single letter code, its three letter code, name, or three
nucleotide codon(s) as is well understood in the art (see Alberts,
B., et al., Molecular Biology of The Cell, Third Ed., Garland
Publishing, Inc., New York, 1994) An anti-IL-23p19 antibody of the
present invention can include one or more amino acid substitutions,
deletions or additions, either from natural mutations or human
manipulation, as specified herein. Amino acids in an anti-IL-23p19
antibody of the present invention that are essential for function
can be identified by methods known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,
Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule.
[0125] The resulting mutant molecules are then tested for
biological activity, such as, but not limited to, at least one
IL-23 neutralizing activity. Sites that are critical for antibody
binding can also be identified by structural analysis, such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de
Vos, et al., Science 255:306-312 (1992)).
[0126] Anti-IL-23p19 antibodies of the present invention can
include, but are not limited to, at least one portion, sequence or
combination selected from 5 to all of the contiguous amino acids of
the variable region sequences of SEQ ID NOS:8, 18, 28, and 38 and
SEQ ID NOS:3, 13, 23, and 33.
[0127] Non-limiting variants that can enhance or maintain at least
one of the listed activities include, but are not limited to, any
of the above polypeptides, further comprising at least one mutation
corresponding to at least one substitution in the residues varied
among the disclosed variant amino acid sequences.
[0128] An anti-IL-23p19 antibody can further optionally comprise a
polypeptide with an amino acid sequence that varies from the
sequence of SEQ ID NOS: 3-6, 8-11, 13-16, 18-21, 23-26, 28-31,
33-36, and 38-41 (e.g., one or more conservative substitutions from
the sequences provided herein). Also, the present invention
comprises variants of the amino acid sequence of a light chain
variable region of SEQ ID NOS:8, 18, 28, and 38 or the amino acid
sequence of a heavy chain variable region of SEQ ID NOS:3, 13, 23,
and 33.
[0129] As those of skill will appreciate, the present invention
includes at least one biologically active antibody of the present
invention. Biologically active antibodies have a specific activity
at least 20%, 30%, or 40%, and, preferably, at least 50%, 60%, or
70%, and, most preferably, at least 80%, 90%, or 95%-1000% or more
of that of the native (non-synthetic), endogenous or related and
known antibody. Methods of assaying and quantifying measures of
enzymatic activity and substrate specificity are well known to
those of skill in the art.
[0130] In another aspect, the invention relates to human antibodies
and antigen-binding fragments, as described herein, which are
modified by the covalent attachment of an organic moiety. Such
modification can produce an antibody or antigen-binding fragment
with improved pharmacokinetic properties (e.g., increased in vivo
serum half-life). The organic moiety can be a linear or branched
hydrophilic polymeric group, fatty acid group, or fatty acid ester
group.
[0131] In particular embodiments, the hydrophilic polymeric group
can have a molecular weight of about 800 to about 120,000 Daltons
and can be a polyalkane glycol (e.g., polyethylene glycol (PEG),
polypropylene glycol (PPG)), carbohydrate polymer, amino acid
polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid
ester group can comprise from about eight to about forty carbon
atoms.
[0132] The modified antibodies and antigen-binding fragments of the
invention can comprise one or more organic moieties that are
covalently bonded, directly or indirectly, to the antibody. Each
organic moiety that is bonded to an antibody or antigen-binding
fragment of the invention can independently be a hydrophilic
polymeric group, a fatty acid group or a fatty acid ester group. As
used herein, the term "fatty acid" encompasses mono-carboxylic
acids and di-carboxylic acids. A "hydrophilic polymeric group," as
the term is used herein, refers to an organic polymer that is more
soluble in water than in octane. For example, polylysine is more
soluble in water than in octane. Thus, an antibody modified by the
covalent attachment of polylysine is encompassed by the invention.
Hydrophilic polymers suitable for modifying antibodies of the
invention can be linear or branched and include, for example,
polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol
(mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose,
oligosaccharides, polysaccharides and the like), polymers of
hydrophilic amino acids (e.g., polylysine, polyarginine,
polyaspartate and the like), polyalkane oxides (e.g., polyethylene
oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
Preferably, the hydrophilic polymer that modifies the antibody of
the invention has a molecular weight of about 800 to about 150,000
Daltons as a separate molecular entity. For example, PEG.sub.5000
and PEG.sub.20,000, wherein the subscript is the average molecular
weight of the polymer in Daltons, can be used. The hydrophilic
polymeric group can be substituted with one to about six alkyl,
fatty acid or fatty acid ester groups. Hydrophilic polymers that
are substituted with a fatty acid or fatty acid ester group can be
prepared by employing suitable methods. For example, a polymer
comprising an amine group can be coupled to a carboxylate of the
fatty acid or fatty acid ester, and an activated carboxylate (e.g.,
activated with N,N-carbonyl diimidazole) on a fatty acid or fatty
acid ester can be coupled to a hydroxyl group on a polymer.
[0133] Fatty acids and fatty acid esters suitable for modifying
antibodies of the invention can be saturated or can contain one or
more units of unsaturation. Fatty acids that are suitable for
modifying antibodies of the invention include, for example,
n-dodecanoate (C.sub.12, laurate), n-tetradecanoate (C14,
myristate), n-octadecanoate (C.sub.18, stearate), n-eicosanoate
(C.sub.20, arachidate), n-docosanoate (C.sub.22, behenate),
[0134] n-triacontanoate (C.sub.30), n-tetracontanoate (C.sub.40),
cis-.DELTA.9-octadecanoate (C.sub.18, oleate), all
cis-.DELTA.5,8,11,14-eicosatetraenoate (C.sub.20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid,
docosanedioic acid, and the like. Suitable fatty acid esters
include mono-esters of dicarboxylic acids that comprise a linear or
branched lower alkyl group. The lower alkyl group can comprise from
one to about twelve, preferably, one to about six, carbon
atoms.
[0135] The modified human antibodies and antigen-binding fragments
can be prepared using suitable methods, such as by reaction with
one or more modifying agents. A "modifying agent" as the term is
used herein, refers to a suitable organic group (e.g., hydrophilic
polymer, a fatty acid, a fatty acid ester) that comprises an
activating group. An "activating group" is a chemical moiety or
functional group that can, under appropriate conditions, react with
a second chemical group thereby forming a covalent bond between the
modifying agent and the second chemical group. For example,
amine-reactive activating groups include electrophilic groups, such
as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),
N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups
that can react with thiols include, for example, maleimide,
iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic
acid thiol (TNB-thiol), and the like. An aldehyde functional group
can be coupled to amine- or hydrazide-containing molecules, and an
azide group can react with a trivalent phosphorous group to form
phosphoramidate or phosphorimide linkages. Suitable methods to
introduce activating groups into molecules are known in the art
(see for example, Hermanson, G. T., Bioconjugate Techniques,
Academic Press: San Diego, Calif. (1996)). An activating group can
be bonded directly to the organic group (e.g., hydrophilic polymer,
fatty acid, fatty acid ester), or through a linker moiety, for
example, a divalent C.sub.1-C.sub.12 group wherein one or more
carbon atoms can be replaced by a heteroatom, such as oxygen,
nitrogen or sulfur. Suitable linker moieties include, for example,
tetraethylene glycol, --(CH2).sub.3--,
--NH--(CH.sub.2).sub.6--NH--, --(CH.sub.2).sub.2--NH-- and
--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH--NH--.
Modifying agents that comprise a linker moiety can be produced, for
example, by reacting a mono-Boc-alkyldiamine (e.g.,
mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid
in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) to form an amide bond between the free amine and the fatty
acid carboxylate. The Boc protecting group can be removed from the
product by treatment with trifluoroacetic acid (TFA) to expose a
primary amine that can be coupled to another carboxylate, as
described, or can be reacted with maleic anhydride and the
resulting product cyclized to produce an activated maleimido
derivative of the fatty acid. (See, for example, Thompson, et al.,
WO 92/16221, the entire teachings of which are incorporated herein
by reference.)
[0136] The modified antibodies of the invention can be produced by
reacting a human antibody or antigen-binding fragment with a
modifying agent. For example, the organic moieties can be bonded to
the antibody in a non-site specific manner by employing an
amine-reactive modifying agent, for example, an NHS ester of PEG.
Modified human antibodies or antigen-binding fragments can also be
prepared by reducing disulfide bonds (e.g., intra-chain disulfide
bonds) of an antibody or antigen-binding fragment. The reduced
antibody or antigen-binding fragment can then be reacted with a
thiol-reactive modifying agent to produce the modified antibody of
the invention. Modified human antibodies and antigen-binding
fragments comprising an organic moiety that is bonded to specific
sites of an antibody of the present invention can be prepared using
suitable methods, such as reverse proteolysis (Fisch et al.,
Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate
Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.
6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68
(1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463
(1997)), and the methods described in Hermanson, G. T.,
Bioconjugate Techniques, Academic Press: San Diego, Calif.
(1996).
[0137] Anti-Idiotype Antibodies to Anti-IL-23p19 Antibody
Compositions
[0138] In addition to monoclonal anti-IL-23p19 antibodies, the
present invention is also directed to an anti-idiotypic (anti-Id)
antibody specific for such antibodies of the invention. An anti-Id
antibody is an antibody which recognizes unique determinants
generally associated with the antigen-binding region of another
antibody. The anti-Id can be prepared by immunizing an animal of
the same species and genetic type (e.g., mouse strain) as the
source of the Id antibody with the antibody or a CDR containing
region thereof. The immunized animal will recognize and respond to
the idiotypic determinants of the immunizing antibody and produce
an anti-Id antibody. The anti-Id antibody may also be used as an
"immunogen" to induce an immune response in yet another animal,
producing a so-called anti-anti-Id antibody.
[0139] The present invention also provides at least one
anti-IL-23p19 antibody composition comprising at least one, at
least two, at least three, at least four, at least five, at least
six or more anti-IL-23p19 antibodies thereof, as described herein
and/or as known in the art that are provided in a non-naturally
occurring composition, mixture or form. Such compositions comprise
non-naturally occurring compositions comprising at least one or two
full length, C- and/or N-terminally deleted variants, domains,
fragments, or specified variants, of the anti-IL-23p19 antibody
amino acid sequence selected from the group consisting of 70-100%
of the contiguous amino acids of SEQ ID NOS:3-6, 8-11, 13-16,
18-21, 23-26, 28-31, 33-36, and 38-41, or specified fragments,
domains or variants thereof. Preferred anti-IL-23p19 antibody
compositions include at least one or two full length, fragments,
domains or variants of at least one CDR or LBP containing portions
of the anti-IL-23p19 antibody sequence described herein, for
example, 70-100% of SEQ ID NOS: 3-6, 8-11, 13-16, 18-21, 23-26,
28-31, 33-36, and 38-41, or specified fragments, domains or
variants thereof. Further preferred compositions comprise, for
example, 40-99% of at least one of 70-100% of SEQ ID NOS: 3-6,
8-11, 13-16, 18-21, 23-26, 28-31, 33-36, and 38-41, or specified
fragments, domains or variants thereof. Such composition
percentages are by weight, volume, concentration, molarity, or
molality as liquid or dry solutions, mixtures, suspension,
emulsions, particles, powder, or colloids, as known in the art or
as described herein.
[0140] Antibody Compositions Comprising Further Therapeutically
Active Ingredients
[0141] The antibody compositions of the invention can optionally
further comprise an effective amount of at least one compound or
protein selected from at least one of an anti-infective drug, a
cardiovascular (CV) system drug, a central nervous system (CNS)
drug, an autonomic nervous system (ANS) drug, a respiratory tract
drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug
for fluid or electrolyte balance, a hematologic drug, an
antineoplastic, an immunomodulation drug, an ophthalmic, otic or
nasal drug, a topical drug, a nutritional drug or the like. Such
drugs are well known in the art, including formulations,
indications, dosing and administration for each presented herein
(see, e.g., Nursing 2001 Handbook of Drugs, 21.sup.st edition,
Springhouse Corp., Springhouse, Pa., 2001; Health Professional's
Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc,
Upper Saddle River, N.J.; Pharmcotherapy Handbook, Wells et al.,
ed., Appleton & Lange, Stamford, Conn., each entirely
incorporated herein by reference).
[0142] The anti-infective drug can be at least one selected from
amebicides or at least one antiprotozoals, anthelmintics,
antifungals, antimalarials, antituberculotics or at least one
antileprotics, aminoglycosides, penicillins, cephalosporins,
tetracyclines, sulfonamides, fluoroquinolones, antivirals,
macrolide anti-infectives, and miscellaneous anti-infectives. The
CV drug can be at least one selected from inotropics,
antiarrhythmics, antianginals, antihypertensives, antilipemics, and
miscellaneous cardiovascular drugs. The CNS drug can be at least
one selected from normarcotic analgesics or at least one selected
from antipyretics, nonsteroidal anti-inflammatory drugs, narcotic
or at least one opiod analgesics, sedative-hypnotics,
anticonvulsants, antidepressants, antianxiety drugs,
antipsychotics, central nervous system stimulants,
antiparkinsonians, and miscellaneous central nervous system drugs.
The ANS drug can be at least one selected from cholinergics
(parasympathomimetics), anticholinergics, adrenergics
(sympathomimetics), adrenergic blockers (sympatholytics), skeletal
muscle relaxants, and neuromuscular blockers. The respiratory tract
drug can be at least one selected from antihistamines,
bronchodilators, expectorants or at least one antitussive, and
miscellaneous respiratory drugs. The GI tract drug can be at least
one selected from antacids or at least one adsorbent or at least
one antiflatulent, digestive enzyme or at least one gallstone
solubilizer, antidiarrheals, laxatives, antiemetics, and antiulcer
drugs. The hormonal drug can be at least one selected from
corticosteroids, androgens or at least one anabolic steroid,
estrogen or at least one progestin, gonadotropin, antidiabetic drug
or at least one glucagon, thyroid hormone, thyroid hormone
antagonist, pituitary hormone, and parathyroid-like drug. The drug
for fluid and electrolyte balance can be at least one selected from
diuretics, electrolytes or at least one replacement solution,
acidifier or at least one alkalinizer. The hematologic drug can be
at least one selected from hematinics, anticoagulants, blood
derivatives, and thrombolytic enzymes. The antineoplastics can be
at least one selected from alkylating drugs, antimetabolites,
antibiotic antineoplastics, antineoplastics that alter hormone
balance, and miscellaneous antineoplastics. The immunomodulation
drug can be at least one selected from immunosuppressants, vaccines
or at least one toxoid, antitoxin or at least one antivenin, immune
serum, and biological response modifier. The ophthalmic, otic, and
nasal drugs can be at least one selected from ophthalmic
anti-infectives, ophthalmic anti-inflammatories, miotics,
mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,
otics, and nasal drugs. The topical drug can be at least one
selected from local anti-infectives, scabicides or at least one
pediculicide or topical corticosteroid. The nutritional drug can be
at least one selected from vitamins, minerals, or calorics. See,
e.g., contents of Nursing 2001 Drug Handbook, supra.
[0143] The at least one amebicide or antiprotozoal can be at least
one selected from atovaquone, chloroquine hydrochloride,
chloroquine phosphate, metronidazole, metronidazole hydrochloride,
and pentamidine isethionate.
[0144] The at least one anthelmintic can be at least one selected
from mebendazole, pyrantel pamoate, and thiabendazole. The at least
one antifungal can be at least one selected from amphotericin B,
amphotericin B cholesteryl sulfate complex, amphotericin B lipid
complex, amphotericin B liposomal, fluconazole, flucytosine,
griseofulvin microsize, griseofulvin ultramicrosize, itraconazole,
ketoconazole, nystatin, and terbinafine hydrochloride. The at least
one antimalarial can be at least one selected from chloroquine
hydrochloride, chloroquine phosphate, doxycycline,
hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine
phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. The
at least one antituberculotic or antileprotic can be at least one
selected from clofazimine, cycloserine, dapsone, ethambutol
hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin,
rifapentine, and streptomycin sulfate. The at least one
aminoglycoside can be at least one selected from amikacin sulfate,
gentamicin sulfate, neomycin sulfate, streptomycin sulfate, and
tobramycin sulfate. The at least one penicillin can be at least one
selected from amoxcillin/clavulanate potassium, amoxicillin
trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate,
ampicillin sodium/sulbactam sodium, cloxacillin sodium,
dicloxacillin sodium, mezlocillin sodium, nafcillin sodium,
oxacillin sodium, penicillin G benzathine, penicillin G potassium,
penicillin G procaine, penicillin G sodium, penicillin V potassium,
piperacillin sodium, piperacillin sodium/tazobactam sodium,
ticarcillin disodium, and ticarcillin disodium/clavulanate
potassium. The at least one cephalosporin can be at least one
selected from cefaclor, cefadroxil, cefazolin sodium, cefdinir,
cefepime hydrochloride, cefixime, cefmetazole sodium, cefonicid
sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium,
cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime,
ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime
axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin
monohydrate, cephradine, and loracarbef. The at least one
tetracycline can be at least one selected from demeclocycline
hydrochloride, doxycycline calcium, doxycycline hyclate,
doxycycline hydrochloride, doxycycline monohydrate, minocycline
hydrochloride, and tetracycline hydrochloride. The at least one
sulfonamide can be at least one selected from co-trimoxazole,
sulfadiazine, sulfamethoxazole, sulfisoxazole, and sulfisoxazole
acetyl. The at least one fluoroquinolone can be at least one
selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin,
levofloxacin, lomefloxacin hydrochloride, nalidixic acid,
norfloxacin, ofloxacin, sparfloxacin, and trovafloxacin mesylate.
The at least one fluoroquinolone can be at least one selected from
alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin,
lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin,
sparfloxacin, and trovafloxacin mesylate. The at least one
antiviral can be at least one selected from abacavir sulfate,
acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir,
delavirdine mesylate, didanosine, efavirenz, famciclovir,
fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir
sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate,
nevirapine, oseltamivir phosphate, ribavirin, rimantadine
hydrochloride, ritonavir, saquinavir, saquinavir mesylate,
stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and
zidovudine. The at least one macroline anti-infective can be at
least one selected from azithromycin, clarithromycin,
dirithromycin, erythromycin base, erythromycin estolate,
erythromycin ethylsuccinate, erythromycin lactobionate, and
erythromycin stearate. The at least one miscellaneous
anti-infective can be at least one selected from aztreonam,
bacitracin, chloramphenicol sodium sucinate, clindamycin
hydrochloride, clindamycin palmitate hydrochloride, clindamycin
phosphate, imipenem and cilastatin sodium, meropenem,
nitrofurantoin macrocrystals, nitrofurantoin microcrystals,
quinupristin/dalfopristin, spectinomycin hydrochloride,
trimethoprim, and vancomycin hydrochloride. (See, e.g., pp. 24-214
of Nursing 2001 Drug Handbook.)
[0145] The at least one inotropic can be at least one selected from
aminone lactate, digoxin, and milrinone lactate. The at least one
antiarrhythmic can be at least one selected from adenosine,
amiodarone hydrochloride, atropine sulfate, bretylium tosylate,
diltiazem hydrochloride, disopyramide, disopyramide phosphate,
esmolol hydrochloride, flecamide acetate, ibutilide fumarate,
lidocaine hydrochloride, mexiletine hydrochloride, moricizine
hydrochloride, phenyloin, phenyloin sodium, procainamide
hydrochloride, propafenone hydrochloride, propranolol
hydrochloride, quinidine bisulfate, quinidine gluconate, quinidine
polygalacturonate, quinidine sulfate, sotalol, tocamide
hydrochloride, and verapamil hydrochloride. The at least one
antianginal can be at least one selected from amlodipidine
besylate, amyl nitrite, bepridil hydrochloride, diltiazem
hydrochloride, isosorbide dinitrate, isosorbide mononitrate,
nadolol, nicardipine hydrochloride, nifedipine, nitroglycerin,
propranolol hydrochloride, verapamil, and verapamil hydrochloride.
The at least one antihypertensive can be at least one selected from
acebutolol hydrochloride, amlodipine besylate, atenolol, benazepril
hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,
candesartan cilexetil, captopril, carteolol hydrochloride,
carvedilol, clonidine, clonidine hydrochloride, diazoxide,
diltiazem hydrochloride, doxazosin mesylate, enalaprilat, enalapril
maleate, eprosartan mesylate, felodipine, fenoldopam mesylate,
fosinopril sodium, guanabenz acetate, guanadrel sulfate, guanfacine
hydrochloride, hydralazine hydrochloride, irbesartan, isradipine,
labetalol hydrchloride, lisinopril, losartan potassium, methyldopa,
methyldopate hydrochloride, metoprolol succinate, metoprolol
tartrate, minoxidil, moexipril hydrochloride, nadolol, nicardipine
hydrochloride, nifedipine, nisoldipine, nitroprusside sodium,
penbutolol sulfate, perindopril erbumine, phentolamine mesylate,
pindolol, prazosin hydrochloride, propranolol hydrochloride,
quinapril hydrochloride, ramipril, telmisartan, terazosin
hydrochloride, timolol maleate, trandolapril, valsartan, and
verapamil hydrochloride. The at least one antilipemic can be at
least one selected from atorvastatin calcium, cerivastatin sodium,
cholestyramine, colestipol hydrochloride, fenofibrate (micronized),
fluvastatin sodium, gemfibrozil, lovastatin, niacin, pravastatin
sodium, and simvastatin. The at least one miscellaneous CV drug can
be at least one selected from abciximab, alprostadil, arbutamine
hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole,
eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine
hydrochloride, and tirofiban hydrochloride. (See, e.g., pp. 215-336
of Nursing 2001 Drug Handbook.)
[0146] The at least one normarcotic analgesic or antipyretic can be
at least one selected from acetaminophen, aspirin, choline
magnesium trisalicylate, diflunisal, and magnesium salicylate. The
at least one nonsteroidal anti-inflammatory drug can be at least
one selected from celecoxib, diclofenac potassium, diclofenac
sodium, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen,
indomethacin, indomethacin sodium trihydrate, ketoprofen, ketorolac
tromethamine, nabumetone, naproxen, naproxen sodium, oxaprozin,
piroxicam, rofecoxib, and sulindac. The at least one narcotic or
opiod analgesic can be at least one selected from alfentanil
hydrochloride, buprenorphine hydrochloride, butorphanol tartrate,
codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl
transdermal system, fentanyl transmucosal, hydromorphone
hydrochloride, meperidine hydrochloride, methadone hydrochloride,
morphine hydrochloride, morphine sulfate, morphine tartrate,
nalbuphine hydrochloride, oxycodone hydrochloride, oxycodone
pectinate, oxymorphone hydrochloride, pentazocine hydrochloride,
pentazocine hydrochloride and naloxone hydrochloride, pentazocine
lactate, propoxyphene hydrochloride, propoxyphene napsylate,
remifentanil hydrochloride, sufentanil citrate, and tramadol
hydrochloride. The at least one sedative-hypnotic can be at least
one selected from chloral hydrate, estazolam, flurazepam
hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital
sodium, secobarbital sodium, temazepam, triazolam, zaleplon, and
zolpidem tartrate. The at least one anticonvulsant can be at least
one selected from acetazolamide sodium, carbamazepine, clonazepam,
clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde,
fosphenyloin sodium, gabapentin, lamotrigine, magnesium sulfate,
phenobarbital, phenobarbital sodium, phenyloin, phenyloin sodium,
phenyloin sodium (extended), primidone, tiagabine hydrochloride,
topiramate, valproate sodium, and valproic acid. The at least one
antidepressant can be at least one selected from amitriptyline
hydrochloride, amitriptyline pamoate, amoxapine, bupropion
hydrochloride, citalopram hydrobromide, clomipramine hydrochloride,
desipramine hydrochloride, doxepin hydrochloride, fluoxetine
hydrochloride, imipramine hydrochloride, imipramine pamoate,
mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride,
paroxetine hydrochloride, phenelzine sulfate, sertraline
hydrochloride, tranylcypromine sulfate, trimipramine maleate, and
venlafaxine hydrochloride. The at least one antianxiety drug can be
at least one selected from alprazolam, buspirone hydrochloride,
chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepate
dipotassium, diazepam, doxepin hydrochloride, hydroxyzine embonate,
hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam,
mephrobamate, midazolam hydrochloride, and oxazepam. The at least
one antipsychotic drug can be at least one selected from
chlorpromazine hydrochloride, clozapine, fluphenazine decanoate,
fluephenazine enanthate, fluphenazine hydrochloride, haloperidol,
haloperidol decanoate, haloperidol lactate, loxapine hydrochloride,
loxapine succinate, mesoridazine besylate, molindone hydrochloride,
olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine
fumarate, risperidone, thioridazine hydrochloride, thiothixene,
thiothixene hydrochloride, and trifluoperazine hydrochloride. The
at least one central nervous system stimulant can be at least one
selected from amphetamine sulfate, caffeine, dextroamphetamine
sulfate, doxapram hydrochloride, methamphetamine hydrochloride,
methylphenidate hydrochloride, modafinil, pemoline, and phentermine
hydrochloride. The at least one antiparkinsonian can be at least
one selected from amantadine hydrochloride, benztropine mesylate,
biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,
carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,
pramipexole dihydrochloride, ropinirole hydrochloride, selegiline
hydrochloride, tolcapone, and trihexyphenidyl hydrochloride. The at
least one miscellaneous central nervous system drug can be at least
one selected from bupropion hydrochloride, donepezil hydrochloride,
droperidol, fluvoxamine maleate, lithium carbonate, lithium
citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine
transdermal system, propofol, rizatriptan benzoate, sibutramine
hydrochloride monohydrate, sumatriptan succinate, tacrine
hydrochloride, and zolmitriptan. (See, e.g., pp. 337-530 of Nursing
2001 Drug Handbook.)
[0147] The at least one cholinergic (e.g., parasymathomimetic) can
be at least one selected from bethanechol chloride, edrophonium
chloride, neostigmine bromide, neostigmine methylsulfate,
physostigmine salicylate, and pyridostigmine bromide. The at least
one anticholinergic can be at least one selected from atropine
sulfate, dicyclomine hydrochloride, glycopyrrolate, hyoscyamine,
hyoscyamine sulfate, propantheline bromide, scopolamine,
scopolamine butylbromide, and scopolamine hydrobromide. The at
least one adrenergic (sympathomimetics) can be at least one
selected from dobutamine hydrochloride, dopamine hydrochloride,
metaraminol bitartrate, norepinephrine bitartrate, phenylephrine
hydrochloride, pseudoephedrine hydrochloride, and pseudoephedrine
sulfate. The at least one adrenergic blocker (sympatholytic) can be
at least one selected from dihydroergotamine mesylate, ergotamine
tartrate, methysergide maleate, and propranolol hydrochloride. The
at least one skeletal muscle relaxant can be at least one selected
from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine
hydrochloride, dantrolene sodium, methocarbamol, and tizanidine
hydrochloride. The at least one neuromuscular blocker can be at
least one selected from atracurium besylate, cisatracurium
besylate, doxacurium chloride, mivacurium chloride, pancuronium
bromide, pipecuronium bromide, rapacuronium bromide, rocuronium
bromide, succinylcholine chloride, tubocurarine chloride, and
vecuronium bromide. (See, e.g., pp. 531-84 of Nursing 2001 Drug
Handbook.)
[0148] The at least one antihistamine can be at least one selected
from brompheniramine maleate, cetirizine hydrochloride,
chlorpheniramine maleate, clemastine fumarate, cyproheptadine
hydrochloride, diphenhydramine hydrochloride, fexofenadine
hydrochloride, loratadine, promethazine hydrochloride, promethazine
theoclate, and triprolidine hydrochloride. The at least one
bronchodilator can be at least one selected from albuterol,
albuterol sulfate, aminophylline, atropine sulfate, ephedrine
sulfate, epinephrine, epinephrine bitartrate, epinephrine
hydrochloride, ipratropium bromide, isoproterenol, isoproterenol
hydrochloride, isoproterenol sulfate, levalbuterol hydrochloride,
metaproterenol sulfate, oxtriphylline, pirbuterol acetate,
salmeterol xinafoate, terbutaline sulfate, and theophylline. The at
least one expectorant or antitussive can be at least one selected
from benzonatate, codeine phosphate, codeine sulfate,
dextramethorphan hydrobromide, diphenhydramine hydrochloride,
guaifenesin, and hydromorphone hydrochloride. The at least one
miscellaneous respiratory drug can be at least one selected from
acetylcysteine, beclomethasone dipropionate, beractant, budesonide,
calfactant, cromolyn sodium, dornase alfa, epoprostenol sodium,
flunisolide, fluticasone propionate, montelukast sodium, nedocromil
sodium, palivizumab, triamcinolone acetonide, zafirlukast, and
zileuton. (See, e.g., pp. 585-642 of Nursing 2001 Drug
Handbook.)
[0149] The at least one antacid, adsorbent, or antiflatulent can be
at least one selected from aluminum carbonate, aluminum hydroxide,
calcium carbonate, magaldrate, magnesium hydroxide, magnesium
oxide, simethicone, and sodium bicarbonate. The at least one
digestive enzyme or gallstone solubilizer can be at least one
selected from pancreatin, pancrelipase, and ursodiol. The at least
one antidiarrheal can be at least one selected from attapulgite,
bismuth subsalicylate, calcium polycarbophil, diphenoxylate
hydrochloride and atropine sulfate, loperamide, octreotide acetate,
opium tincture, and opium tincture (camphorated). The at least one
laxative can be at least one selected from bisocodyl, calcium
polycarbophil, cascara sagrada, cascara sagrada aromatic
fluidextract, cascara sagrada fluidextract, castor oil, docusate
calcium, docusate sodium, glycerin, lactulose, magnesium citrate,
magnesium hydroxide, magnesium sulfate, methylcellulose, mineral
oil, polyethylene glycol or electrolyte solution, psyllium, senna,
and sodium phosphates.
[0150] The at least one antiemetic can be at least one selected
from chlorpromazine hydrochloride, dimenhydrinate, dolasetron
mesylate, dronabinol, granisetron hydrochloride, meclizine
hydrochloride, metocloproamide hydrochloride, ondansetron
hydrochloride, perphenazine, prochlorperazine, prochlorperazine
edisylate, prochlorperazine maleate, promethazine hydrochloride,
scopolamine, thiethylperazine maleate, and trimethobenzamide
hydrochloride. The at least one antiulcer drug can be at least one
selected from cimetidine, cimetidine hydrochloride, famotidine,
lansoprazole, misoprostol, nizatidine, omeprazole, rabeprozole
sodium, rantidine bismuth citrate, ranitidine hydrochloride, and
sucralfate. (See, e.g., pp. 643-95 of Nursing 2001 Drug
Handbook.)
[0151] The at least one corticosteroid can be at least one selected
from betamethasone, betamethasone acetate or betamethasone sodium
phosphate, betamethasone sodium phosphate, cortisone acetate,
dexamethasone, dexamethasone acetate, dexamethasone sodium
phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone
acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate,
hydrocortisone sodium succinate, methylprednisolone,
methylprednisolone acetate, methylprednisolone sodium succinate,
prednisolone, prednisolone acetate, prednisolone sodium phosphate,
prednisolone tebutate, prednisone, triamcinolone, triamcinolone
acetonide, and triamcinolone diacetate. The at least one androgen
or anabolic steroid can be at least one selected from danazol,
fluoxymesterone, methyltestosterone, nandrolone decanoate,
nandrolone phenpropionate, testosterone, testosterone cypionate,
testosterone enanthate, testosterone propionate, and testosterone
transdermal system. The at least one estrogen or progestin can be
at least one selected from esterified estrogens, estradiol,
estradiol cypionate, estradiol/norethindrone acetate transdermal
system, estradiol valerate, estrogens (conjugated), estropipate,
ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl
estradiol and ethynodiol diacetate, ethinyl estradiol and
desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl
estradiol and levonorgestrel, ethinyl estradiol and norethindrone,
ethinyl estradiol and norethindrone acetate, ethinyl estradiol and
norgestimate, ethinyl estradiol and norgestrel, ethinyl estradiol
and norethindrone and acetate and ferrous fumarate, levonorgestrel,
medroxyprogesterone acetate, mestranol and norethindron,
norethindrone, norethindrone acetate, norgestrel, and progesterone.
The at least one gonadroptropin can be at least one selected from
ganirelix acetate, gonadoreline acetate, histrelin acetate, and
menotropins. The at least one antidiabetic or glucaon can be at
least one selected from acarbose, chlorpropamide, glimepiride,
glipizide, glucagon, glyburide, insulins, metformin hydrochloride,
miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazone
maleate, and troglitazone. The at least one thyroid hormone can be
at least one selected from levothyroxine sodium, liothyronine
sodium, liotrix, and thyroid. The at least one thyroid hormone
antagonist can be at least one selected from methimazole, potassium
iodide, potassium iodide (saturated solution), propylthiouracil,
radioactive iodine (sodium iodide .sup.131I), and strong iodine
solution. The at least one pituitary hormone can be at least one
selected from corticotropin, cosyntropin, desmophressin acetate,
leuprolide acetate, repository corticotropin, somatrem, somatropin,
and vasopressin. The at least one parathyroid-like drug can be at
least one selected from calcifediol, calcitonin (human), calcitonin
(salmon), calcitriol, dihydrotachysterol, and etidronate disodium.
(See, e.g., pp. 696-796 of Nursing 2001 Drug Handbook.)
[0152] The at least one diuretic can be at least one selected from
acetazolamide, acetazolamide sodium, amiloride hydrochloride,
bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid,
furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,
spironolactone, torsemide, triamterene, and urea. The at least one
electrolyte or replacement solution can be at least one selected
from calcium acetate, calcium carbonate, calcium chloride, calcium
citrate, calcium glubionate, calcium gluceptate, calcium gluconate,
calcium lactate, calcium phosphate (dibasic), calcium phosphate
(tribasic), dextran (high-molecular-weight), dextran
(low-molecular-weight), hetastarch, magnesium chloride, magnesium
sulfate, potassium acetate, potassium bicarbonate, potassium
chloride, potassium gluconate, Ringer's injection, Ringer's
injection (lactated), and sodium chloride. The at least one
acidifier or alkalinizer can be at least one selected from sodium
bicarbonate, sodium lactate, and tromethamine. (See, e.g., pp.
797-833 of Nursing 2001 Drug Handbook.)
[0153] The at least one hematinic can be at least one selected from
ferrous fumarate, ferrous gluconate, ferrous sulfate, ferrous
sulfate (dried), iron dextran, iron sorbitol, polysaccharide-iron
complex, and sodium ferric gluconate complex. The at least one
anticoagulant can be at least one selected from ardeparin sodium,
dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin
calcium, heparin sodium, and warfarin sodium. The at least one
blood derivative can be at least one selected from albumin 5%,
albumin 25%, antihemophilic factor, anti-inhibitor coagulant
complex, antithrombin III (human), factor IX (human), factor IX
complex, and plasma protein fractions. The at least one
thrombolytic enzyme can be at least one selected from alteplase,
anistreplase, reteplase (recombinant), streptokinase, and
urokinase. (See, e.g., pp. 834-66 of Nursing 2001 Drug
Handbook.)
[0154] The at least one alkylating drug can be at least one
selected from busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine
hydrochloride, melphalan, melphalan hydrochloride, streptozocin,
temozolomide, and thiotepa. The at least one antimetabolite can be
at least one selected from capecitabine, cladribine, cytarabine,
floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea,
mercaptopurine, methotrexate, methotrexate sodium, and thioguanine.
The at least one antibiotic antineoplastic can be at least one
selected from bleomycin sulfate, dactinomycin, daunorubicin citrate
liposomal, daunorubicin hydrochloride, doxorubicin hydrochloride,
doxorubicin hydrochloride liposomal, epirubicin hydrochloride,
idarubicin hydrochloride, mitomycin, pentostatin, plicamycin, and
valrubicin. The at least one antineoplastic that alters hormone
balance can be at least one selected from anastrozole,
bicalutamide, estramustine phosphate sodium, exemestane, flutamide,
goserelin acetate, letrozole, leuprolide acetate, megestrol
acetate, nilutamide, tamoxifen citrate, testolactone, and
toremifene citrate. The at least one miscellaneous antineoplastic
can be at least one selected from asparaginase, bacillus
Calmette-Guerin (BCG) (live intravesical), dacarbazine, docetaxel,
etoposide, etoposide phosphate, gemcitabine hydrochloride,
irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride,
paclitaxel, pegaspargase, porfimer sodium, procarbazine
hydrochloride, rituximab, teniposide, topotecan hydrochloride,
trastuzumab, tretinoin, vinblastine sulfate, vincristine sulfate,
and vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing 2001
Drug Handbook.)
[0155] The at least one immunosuppressant can be at least one
selected from azathioprine, basiliximab, cyclosporine, daclizumab,
lymphocyte immune globulin, muromonab-CD3, mycophenolate mofetil,
mycophenolate mofetil hydrochloride, sirolimus, and tacrolimus. The
at least one vaccine or toxoid can be at least one selected from
BCG vaccine, cholera vaccine, diphtheria and tetanus toxoids
(adsorbed), diphtheria and tetanus toxoids and acellular pertussis
vaccine adsorbed, diphtheria and tetanus toxoids and whole-cell
pertussis vaccine, Haemophilus b conjugate vaccines, hepatitis A
vaccine (inactivated), hepatitis B vaccine (recombinant), influenza
virus vaccine 1999-2000 trivalent types A & B (purified surface
antigen), influenza virus vaccine 1999-2000 trivalent types A &
B (subvirion or purified subvirion), influenza virus vaccine
1999-2000 trivalent types A & B (whole virion), Japanese
encephalitis virus vaccine (inactivated), Lyme disease vaccine
(recombinant OspA), measles and mumps and rubella virus vaccine
(live), measles and mumps and rubella virus vaccine (live
attenuated), measles virus vaccine (live attenuated), meningococcal
polysaccharide vaccine, mumps virus vaccine (live), plague vaccine,
pneumococcal vaccine (polyvalent), poliovirus vaccine
(inactivated), poliovirus vaccine (live, oral, trivalent), rabies
vaccine (adsorbed), rabies vaccine (human diploid cell), rubella
and mumps virus vaccine (live), rubella virus vaccine (live,
attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid),
typhoid vaccine (oral), typhoid vaccine (parenteral), typhoid Vi
polysaccharide vaccine, varicella virus vaccine, and yellow fever
vaccine. The at least one antitoxin or antivenin can be at least
one selected from black widow spider antivenin, Crotalidae
antivenom (polyvalent), diphtheria antitoxin (equine), amd Micrurus
fulvius antivenin. The at least one immune serum can be at least
one selected from cytomegalovirus immune globulin (intraveneous),
hepatitis B immune globulin (human), immune globulin intramuscular,
immune globulin intravenous, rabies immune globulin (human),
respiratory syncytial virus immune globulin intravenous (human),
Rh.sub.0(D) immune globulin (human), Rh.sub.0(D) immune globulin
intravenous (human), tetanus immune globulin (human), and
varicella-zoster immune globulin. The at least one biological
response modifier can be at least one selected from aldesleukin,
epoetin alfa, filgrastim, glatiramer acetate for injection,
interferon alfacon-1, interferon alfa-2a (recombinant), interferon
alfa-2b (recombinant), interferon beta-1a, interferon beta-1b
(recombinant), interferon gamma-1b, levamisole hydrochloride,
oprelvekin, and sargramostim. (See, e.g., pp. 964-1040 of Nursing
2001 Drug Handbook.)
[0156] The at least one ophthalmic anti-infective can be selected
form bacitracin, chloramphenicol, ciprofloxacin hydrochloride,
erythromycin, gentamicin sulfate, ofloxacin 0.3%, polymyxin B
sulfate, sulfacetamide sodium 10%, sulfacetamide sodium 15%,
sulfacetamide sodium 30%, tobramycin, and vidarabine. The at least
one ophthalmic anti-inflammatory can be at least one selected from
dexamethasone, dexamethasone sodium phosphate, diclofenac sodium
0.1%, fluorometholone, flurbiprofen sodium, ketorolac tromethamine,
prednisolone acetate (suspension) and prednisolone sodium phosphate
(solution). The at least one miotic can be at least one selected
from acetylocholine chloride, carbachol (intraocular), carbachol
(topical), echothiophate iodide, pilocarpine, pilocarpine
hydrochloride, and pilocarpine nitrate. The at least one mydriatic
can be at least one selected from atropine sulfate, cyclopentolate
hydrochloride, epinephrine hydrochloride, epinephryl borate,
homatropine hydrobromide, phenylephrine hydrochloride, scopolamine
hydrobromide, and tropicamide. The at least one ophthalmic
vasoconstrictor can be at least one selected from naphazoline
hydrochloride, oxymetazoline hydrochloride, and tetrahydrozoline
hydrochloride. The at least one miscellaneous ophthalmic can be at
least one selected from apraclonidine hydrochloride, betaxolol
hydrochloride, brimonidine tartrate, carteolol hydrochloride,
dipivefrin hydrochloride, dorzolamide hydrochloride, emedastine
difumarate, fluorescein sodium, ketotifen fumarate, latanoprost,
levobunolol hydrochloride, metipranolol hydrochloride, sodium
chloride (hypertonic), and timolol maleate. The at least one otic
can be at least one selected from boric acid, carbamide peroxide,
chloramphenicol, and triethanolamine polypeptide oleate-condensate.
The at least one nasal drug can be at least one selected from
beclomethasone dipropionate, budesonide, ephedrine sulfate,
epinephrine hydrochloride, flunisolide, fluticasone propionate,
naphazoline hydrochloride, oxymetazoline hydrochloride,
phenylephrine hydrochloride, tetrahydrozoline hydrochloride,
triamcinolone acetonide, and xylometazoline hydrochloride. (See,
e.g., pp. 1041-97 of Nursing 2001 Drug Handbook.)
[0157] The at least one local anti-infective can be at least one
selected from acyclovir, amphotericin B, azelaic acid cream,
bacitracin, butoconazole nitrate, clindamycin phosphate,
clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate,
ketoconazole, mafenide acetate, metronidazole (topical), miconazole
nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate,
nitrofurazone, nystatin, silver sulfadiazine, terbinafine
hydrochloride, terconazole, tetracycline hydrochloride,
tioconazole, and tolnaftate. The at least one scabicide or
pediculicide can be at least one selected from crotamiton, lindane,
permethrin, and pyrethrins. The at least one topical corticosteroid
can be at least one selected from betamethasone dipropionate,
betamethasone valerate, clobetasol propionate, desonide,
desoximetasone, dexamethasone, dexamethasone sodium phosphate,
diflorasone diacetate, fluocinolone acetonide, fluocinonide,
flurandrenolide, fluticasone propionate, halcionide,
hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate,
hydrocorisone valerate, mometasone furoate, and triamcinolone
acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 Drug
Handbook.)
[0158] The at least one vitamin or mineral can be at least one
selected from vitamin A, vitamin B complex, cyanocobalamin, folic
acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide,
pyridoxine hydrochloride, riboflavin, thiamine hydrochloride,
vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D
analogue, doxercalciferol, paricalcitol, vitamin E, vitamin K
analogue, phytonadione, sodium fluoride, sodium fluoride (topical),
trace elements, chromium, copper, iodine, manganese, selenium, and
zinc. The at least one caloric can be at least one selected from
amino acid infusions (crystalline), amino acid infusions in
dextrose, amino acid infusions with electrolytes, amino acid
infusions with electrolytes in dextrose, amino acid infusions for
hepatic failure, amino acid infusions for high metabolic stress,
amino acid infusions for renal failure, dextrose, fat emulsions,
and medium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing
2001 Drug Handbook.)
[0159] Anti-IL-23p19 antibody compositions of the present invention
can further comprise at least one of any suitable and effective
amount of a composition or pharmaceutical composition comprising at
least one anti-IL-23p19 antibody contacted or administered to a
cell, tissue, organ, animal or patient in need of such modulation,
treatment or therapy, optionally further comprising at least one
selected from at least one TNF antagonist (e.g., but not limited to
a TNF chemical or protein antagonist, TNF monoclonal or polyclonal
antibody or fragment, a soluble TNF receptor (e.g., p55, p70 or
p85) or fragment, fusion polypeptides thereof, or a small molecule
TNF antagonist, e.g., TNF binding protein I or II (TBP-1 or
TBP-II), nerelimonmab, infliximab, etanercept, CDP-571, CDP-870,
afelimomab, lenercept, and the like), an antirheumatic (e.g.,
methotrexate, auranofin, aurothioglucose, azathioprine, etanercept,
gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide,
sulfasalzine), a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anethetic, a neuromuscular blocker, an
antimicrobial (e.g., aminoglycoside, an antifungal, an
antiparasitic, an antiviral, a carbapenem, cephalosporin, a
fluororquinolone, a macrolide, a penicillin, a sulfonamide, a
tetracycline, another antimicrobial), an antipsoriatic, a
corticosteriod, an anabolic steroid, a diabetes related agent, a
mineral, a nutritional, a thyroid agent, a vitamin, a calcium
related hormone, an antidiarrheal, an antitussive, an antiemetic,
an antiulcer, a laxative, an anticoagulant, an erythropoietin
(e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a
sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin,
an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab),
a growth hormone, a hormone replacement drug, an estrogen receptor
modulator, a mydriatic, a cycloplegic, an alkylating agent, an
antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an
antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a
hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an
asthma medication, a beta agonist, an inhaled steroid, a
leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine
or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine
antagonist. Non-limiting examples of such cytokines include, but
are not limted to, any of IL-1 to IL-28 (e.g., IL-1, IL-2, etc.).
Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and
Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which references are entirely
incorporated herein by reference.
[0160] Such anti-cancer or anti-infectives can also include toxin
molecules that are associated, bound, co-formulated or
co-administered with at least one antibody of the present
invention. The toxin can optionally act to selectively kill the
pathologic cell or tissue. The pathologic cell can be a cancer or
other cell. Such toxins can be, but are not limited to, purified or
recombinant toxin or toxin fragment comprising at least one
functional cytotoxic domain of toxin, e.g., selected from at least
one of ricin, diphtheria toxin, a venom toxin, or a bacterial
toxin. The term toxin also includes both endotoxins and exotoxins
produced by any naturally occurring, mutant or recombinant bacteria
or viruses which may cause any pathological condition in humans and
other mammals, including toxin shock, which can result in death.
Such toxins may include, but are not limited to, enterotoxigenic E.
coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST),
Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome
toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C
(SEC), Streptococcal enterotoxins and the like. Such bacteria
include, but are not limited to, strains of a species of
enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g.,
strains of serotype 0157:H7), Staphylococcus species (e.g.,
Staphylococcus aureus, Staphylococcus pyogenes), Shigella species
(e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii,
and Shigella sonnei), Salmonella species (e.g., Salmonella typhi,
Salmonella cholera-suis, Salmonella enteritidis), Clostridium
species (e.g., Clostridium perfringens, Clostridium dificile,
Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter
jejuni, Camphlobacter fetus), Heliobacter species, (e.g.,
Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,
Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,
Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae,
Vibrios parahemolyticus), Klebsiella species, Pseudomonas
aeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL
MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990);
Evans et al., eds., Bacterial Infections of Humans: Epidemiology
and Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York
(1991); Mandell et al, Principles and Practice of Infectious
Diseases, 3d. Ed., Churchill Livingstone, New York (1990); Berkow
et al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway,
N.J., 1992; Wood et al, FEMS Microbiology Immunology, 76:121-134
(1991); Marrack et al, Science, 248:705-711 (1990), the contents of
which references are incorporated entirely herein by reference.
[0161] Anti-IL-23p19 antibody compounds, compositions or
combinations of the present invention can further comprise at least
one of any suitable auxiliary, such as, but not limited to,
diluent, binder, stabilizer, buffers, salts, lipophilic solvents,
preservative, adjuvant or the like. Pharmaceutically acceptable
auxiliaries are preferred. Non-limiting examples of, and methods of
preparing such sterile solutions are well known in the art, such
as, but limited to, Gennaro, Ed., Remington's Pharmaceutical
Sciences, 18.sup.th Edition, Mack Publishing Co. (Easton, Pa.)
1990. Pharmaceutically acceptable carriers can be routinely
selected that are suitable for the mode of administration,
solubility and/or stability of the anti-IL-23p19 antibody, fragment
or variant composition as well known in the art or as described
herein.
[0162] Pharmaceutical excipients and additives useful in the
present composition include, but are not limited to, proteins,
peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides;
derivatized sugars, such as alditols, aldonic acids, esterified
sugars and the like; and polysaccharides or sugar polymers), which
can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein
excipients include serum albumin, such as human serum albumin
(HSA), recombinant human albumin (rHA), gelatin, casein, and the
like. Representative amino acid/antibody components, which can also
function in a buffering capacity, include alanine, glycine,
arginine, betaine, histidine, glutamic acid, aspartic acid,
cysteine, lysine, leucine, isoleucine, valine, methionine,
phenylalanine, aspartame, and the like. One preferred amino acid is
glycine.
[0163] Carbohydrate excipients suitable for use in the invention
include, for example, monosaccharides, such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol
(glucitol), myoinositol and the like. Preferred carbohydrate
excipients for use in the present invention are mannitol,
trehalose, and raffinose.
[0164] Anti-IL-23p19 antibody compositions can also include a
buffer or a pH adjusting agent; typically, the buffer is a salt
prepared from an organic acid or base. Representative buffers
include organic acid salts, such as salts of citric acid, ascorbic
acid, gluconic acid, carbonic acid, tartaric acid, succinic acid,
acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or
phosphate buffers. Preferred buffers for use in the present
compositions are organic acid salts, such as citrate.
[0165] Additionally, anti-IL-23p19 antibody compositions of the
invention can include polymeric excipients/additives, such as
polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates
(e.g., cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin),
polyethylene glycols, flavoring agents, antimicrobial agents,
sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates, such as "TWEEN 20" and "TWEEN 80"), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and
chelating agents (e.g., EDTA).
[0166] These and additional known pharmaceutical excipients and/or
additives suitable for use in the anti-IL-23p19 antibody, portion
or variant compositions according to the invention are known in the
art, e.g., as listed in "Remington: The Science & Practice of
Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in
the "Physician's Desk Reference", 52.sup.nd ed., Medical Economics,
Montvale, N.J. (1998), the disclosures of which are entirely
incorporated herein by reference. Preferred carrier or excipient
materials are carbohydrates (e.g., saccharides and alditols) and
buffers (e.g., citrate) or polymeric agents. An exemplary carrier
molecule is the mucopolysaccharide, hyaluronic acid, which may be
useful for intraarticular delivery.
[0167] Formulations
[0168] As noted above, the invention provides for stable
formulations, which preferably comprise a phosphate buffer with
saline or a chosen salt, as well as preserved solutions and
formulations containing a preservative as well as multi-use
preserved formulations suitable for pharmaceutical or veterinary
use, comprising at least one anti-IL-23p19 antibody in a
pharmaceutically acceptable formulation. Preserved formulations
contain at least one known preservative or optionally selected from
the group consisting of at least one phenol, m-cresol, p-cresol,
o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride
(e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and
the like), benzalkonium chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, polymers, or mixtures thereof in an
aqueous diluent. Any suitable concentration or mixture can be used
as known in the art, such as about 0.0015%, or any range, value, or
fraction therein. Non-limiting examples include, no preservative,
about 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), about
0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%),
about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0%
phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0%
alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005,
0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,
0.75, 0.9, 1.0%), and the like.
[0169] As noted above, the invention provides an article of
manufacture, comprising packaging material and at least one vial
comprising a solution of at least one anti-IL-23p19 antibody with
the prescribed buffers and/or preservatives, optionally in an
aqueous diluent, wherein said packaging material comprises a label
that indicates that such solution can be held over a period of 1,
2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72
hours or greater. The invention further comprises an article of
manufacture, comprising packaging material, a first vial comprising
lyophilized at least one anti-IL-23p19 antibody, and a second vial
comprising an aqueous diluent of prescribed buffer or preservative,
wherein said packaging material comprises a label that instructs a
patient to reconstitute the at least one anti-IL-23p19 antibody in
the aqueous diluent to form a solution that can be held over a
period of twenty-four hours or greater.
[0170] The at least one anti-IL-23p19 antibody used in accordance
with the present invention can be produced by recombinant means,
including from mammalian cell or transgenic preparations, or can be
purified from other biological sources, as described herein or as
known in the art.
[0171] The range of at least one anti-IL-23p19 antibody in the
product of the present invention includes amounts yielding upon
reconstitution, if in a wet/dry system, concentrations from about
1.0 .mu.g/ml to about 1000 mg/ml, although lower and higher
concentrations are operable and are dependent on the intended
delivery vehicle, e.g., solution formulations will differ from
transdermal patch, pulmonary, transmucosal, or osmotic or micro
pump methods.
[0172] Preferably, the aqueous diluent optionally further comprises
a pharmaceutically acceptable preservative. Preferred preservatives
include those selected from the group consisting of phenol,
m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
alkylparaben (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal, or mixtures thereof. The concentration of
preservative used in the formulation is a concentration sufficient
to yield an anti-microbial effect. Such concentrations are
dependent on the preservative selected and are readily determined
by the skilled artisan.
[0173] Other excipients, e.g., isotonicity agents, buffers,
antioxidants, and preservative enhancers, can be optionally and
preferably added to the diluent. An isotonicity agent, such as
glycerin, is commonly used at known concentrations. A
physiologically tolerated buffer is preferably added to provide
improved pH control. The formulations can cover a wide range of
pHs, such as from about pH 4 to about pH 10, and preferred ranges
from about pH 5 to about pH 9, and a most preferred range of about
6.0 to about 8.0. Preferably, the formulations of the present
invention have a pH between about 6.8 and about 7.8. Preferred
buffers include phosphate buffers, most preferably, sodium
phosphate, particularly, phosphate buffered saline (PBS).
[0174] Other additives, such as a pharmaceutically acceptable
solubilizers like Tween 20 (polyoxyethylene (20) sorbitan
monolaurate), Tween 40 (polyoxyethylene (20) sorbitan
monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan
monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block
copolymers), and PEG (polyethylene glycol) or non-ionic
surfactants, such as polysorbate 20 or 80 or poloxamer 184 or 188,
Pluronic.RTM. polyls, other block co-polymers, and chelators, such
as EDTA and EGTA, can optionally be added to the formulations or
compositions to reduce aggregation. These additives are
particularly useful if a pump or plastic container is used to
administer the formulation. The presence of pharmaceutically
acceptable surfactant mitigates the propensity for the protein to
aggregate.
[0175] The formulations of the present invention can be prepared by
a process which comprises mixing at least one anti-IL-23p19
antibody and a preservative selected from the group consisting of
phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
alkylparaben, (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal or mixtures thereof in an aqueous diluent. Mixing
the at least one anti-IL-23p19 antibody and preservative in an
aqueous diluent is carried out using conventional dissolution and
mixing procedures. To prepare a suitable formulation, for example,
a measured amount of at least one anti-IL-23p19 antibody in
buffered solution is combined with the desired preservative in a
buffered solution in quantities sufficient to provide the protein
and preservative at the desired concentrations. Variations of this
process would be recognized by one of ordinary skill in the art.
For example, the order the components are added, whether additional
additives are used, the temperature and pH at which the formulation
is prepared, are all factors that can be optimized for the
concentration and means of administration used.
[0176] The claimed formulations can be provided to patients as
clear solutions or as dual vials comprising a vial of lyophilized
at least one anti-IL-23p19 antibody that is reconstituted with a
second vial containing water, a preservative and/or excipients,
preferably, a phosphate buffer and/or saline and a chosen salt, in
an aqueous diluent. Either a single solution vial or dual vial
requiring reconstitution can be reused multiple times and can
suffice for a single or multiple cycles of patient treatment and
thus can provide a more convenient treatment regimen than currently
available.
[0177] The present claimed articles of manufacture are useful for
administration over a period ranging from immediate to twenty-four
hours or greater. Accordingly, the presently claimed articles of
manufacture offer significant advantages to the patient.
Formulations of the invention can optionally be safely stored at
temperatures of from about 2.degree. C. to about 40.degree. C. and
retain the biological activity of the protein for extended periods
of time, thus allowing a package label indicating that the solution
can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72,
or 96 hours or greater. If preserved diluent is used, such label
can include use up to 1-12 months, one-half, one and a half, and/or
two years.
[0178] The solutions of at least one anti-IL-23p19 antibody of the
invention can be prepared by a process that comprises mixing at
least one antibody in an aqueous diluent. Mixing is carried out
using conventional dissolution and mixing procedures. To prepare a
suitable diluent, for example, a measured amount of at least one
antibody in water or buffer is combined in quantities sufficient to
provide the protein and, optionally, a preservative or buffer at
the desired concentrations. Variations of this process would be
recognized by one of ordinary skill in the art. For example, the
order the components are added, whether additional additives are
used, the temperature and pH at which the formulation is prepared,
are all factors that can be optimized for the concentration and
means of administration used.
[0179] The claimed products can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized at
least one anti-IL-23p19 antibody that is reconstituted with a
second vial containing the aqueous diluent. Either a single
solution vial or dual vial requiring reconstitution can be reused
multiple times and can suffice for a single or multiple cycles of
patient treatment and thus provides a more convenient treatment
regimen than currently available.
[0180] The claimed products can be provided indirectly to patients
by providing to pharmacies, clinics, or other such institutions and
facilities, clear solutions or dual vials comprising a vial of
lyophilized at least one anti-IL-23p19 antibody that is
reconstituted with a second vial containing the aqueous diluent.
The clear solution in this case can be up to one liter or even
larger in size, providing a large reservoir from which smaller
portions of the at least one antibody solution can be retrieved one
or multiple times for transfer into smaller vials and provided by
the pharmacy or clinic to their customers and/or patients.
[0181] Recognized devices comprising single vial systems include
pen-injector devices for delivery of a solution, such as BD Pens,
BD Autojector.RTM., Humaject.RTM..sup., NovoPen.RTM., B-D.RTM.Pen,
AutoPen.RTM., and OptiPen.RTM., GenotropinPen.RTM., Genotronorm
Pen.RTM., Humatro Pen.RTM., Reco-Pen.RTM. Roferon Pen.RTM.,
Biojector.RTM., Iject.RTM., J-tip Needle-Free Injector.RTM.,
Intraject.RTM., Medi-Ject.RTM., e.g., as made or developed by
Becton Dickensen (Franklin Lakes, N.J., www.bectondickenson.com),
Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject,
Portland, Oreg. (www.bioject.com); National Medical Products,
Weston Medical (Peterborough, UK, www.weston-medical.com),
Medi-Ject Corp (Minneapolis, Minn., www.mediject.com), and
similarly suitable devices. Recognized devices comprising a dual
vial system include those pen-injector systems for reconstituting a
lyophilized drug in a cartridge for delivery of the reconstituted
solution, such as the HumatroPen.RTM.. Examples of other devices
suitable include pre-filled syringes, auto-injectors, needle free
injectors and needle free IV infusion sets.
[0182] The products presently claimed include packaging material.
The packaging material provides, in addition to the information
required by the regulatory agencies, the conditions under which the
product can be used. The packaging material of the present
invention provides instructions to the patient to reconstitute the
at least one anti-IL-23p19 antibody in the aqueous diluent to form
a solution and to use the solution over a period of 2-24 hours or
greater for the two vial, wet/dry, product. For the single vial,
solution product, the label indicates that such solution can be
used over a period of 2-24 hours or greater.
[0183] The presently claimed products are useful for human
pharmaceutical product use.
[0184] The formulations of the present invention can be prepared by
a process that comprises mixing at least one anti-IL-23p19 antibody
and a selected buffer, preferably, a phosphate buffer containing
saline or a chosen salt. Mixing the at least one anti-IL-23p19
antibody and buffer in an aqueous diluent is carried out using
conventional dissolution and mixing procedures. To prepare a
suitable formulation, for example, a measured amount of at least
one antibody in water or buffer is combined with the desired
buffering agent in water in quantities sufficient to provide the
protein and buffer at the desired concentrations. Variations of
this process would be recognized by one of ordinary skill in the
art. For example, the order the components are added, whether
additional additives are used, the temperature and pH at which the
formulation is prepared, are all factors that can be optimized for
the concentration and means of administration used.
[0185] The claimed stable or preserved formulations can be provided
to patients as clear solutions or as dual vials comprising a vial
of lyophilized at least one anti-IL-23p19 antibody that is
reconstituted with a second vial containing a preservative or
buffer and excipients in an aqueous diluent. Either a single
solution vial or dual vial requiring reconstitution can be reused
multiple times and can suffice for a single or multiple cycles of
patient treatment and thus provides a more convenient treatment
regimen than currently available.
[0186] Other formulations or methods of stabilizing the
anti-IL-23p19 antibody may result in other than a clear solution of
lyophilized powder comprising the antibody. Among non-clear
solutions are formulations comprising particulate suspensions, said
particulates being a composition containing the anti-IL-23p19
antibody in a structure of variable dimension and known variously
as a microsphere, microparticle, nanoparticle, nanosphere, or
liposome. Such relatively homogenous, essentially spherical,
particulate formulations containing an active agent can be formed
by contacting an aqueous phase containing the active agent and a
polymer and a nonaqueous phase followed by evaporation of the
nonaqueous phase to cause the coalescence of particles from the
aqueous phase as taught in U.S. Pat. No. 4,589,330. Porous
microparticles can be prepared using a first phase containing
active agent and a polymer dispersed in a continuous solvent and
removing said solvent from the suspension by freeze-drying or
dilution-extraction-precipitation as taught in U.S. Pat. No.
4,818,542. Preferred polymers for such preparations are natural or
synthetic copolymers or polymers selected from the group consisting
of gleatin agar, starch, arabinogalactan, albumin, collagen,
polyglycolic acid, polylactic aced, glycolide-L(-) lactide
poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic
acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(8-hydroxy
butyric acid), polyethylene oxide, polyethylene,
poly(alkyl-2-cyanoacrylate), poly(hydroxyethyl methacrylate),
polyamides, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide),
poly(ester urea), poly(L-phenylalanine/ethylene
glycol/1,6-diisocyanatohexane) and poly(methyl methacrylate).
Particularly preferred polymers are polyesters, such as
polyglycolic acid, polylactic aced, glycolide-L(-) lactide
poly(episilon-caprolactone, poly(epsilon-caprolactone-CO -lactic
acid), and poly(epsilon-caprolactone-CO-glycolic acid.
[0187] Solvents useful for dissolving the polymer and/or the active
include: water, hexafluoroisopropanol, methylenechloride,
tetrahydrofuran, hexane, benzene, or hexafluoroacetone
sesquihydrate. The process of dispersing the active containing
phase with a second phase may include pressure forcing said first
phase through an orifice in a nozzle to affect droplet
formation.
[0188] Dry powder formulations may result from processes other than
lyophilization, such as by spray drying or solvent extraction by
evaporation or by precipitation of a crystalline composition
followed by one or more steps to remove aqueous or nonaqueous
solvent. Preparation of a spray-dried antibody preparation is
taught in U.S. Pat. No. 6,019,968.
[0189] The antibody-based dry powder compositions may be produced
by spray drying solutions or slurries of the antibody and,
optionally, excipients, in a solvent under conditions to provide a
respirable dry powder. Solvents may include polar compounds, such
as water and ethanol, which may be readily dried. Antibody
stability may be enhanced by performing the spray drying procedures
in the absence of oxygen, such as under a nitrogen blanket or by
using nitrogen as the drying gas. Another relatively dry
formulation is a dispersion of a plurality of perforated
microstructures dispersed in a suspension medium that typically
comprises a hydrofluoroalkane propellant as taught in WO 9,916,419.
The stabilized dispersions may be administered to the lung of a
patient using a metered dose inhaler. Equipment useful in the
commercial manufacture of spray dried medicaments are manufactured
by Buchi Ltd. or Niro Corp.
[0190] At least one anti-IL-23p19 antibody in either the stable or
preserved formulations or solutions described herein, can be
administered to a patient in accordance with the present invention
via a variety of delivery methods including SC or IM injection;
transdermal, pulmonary, transmucosal, implant, osmotic pump,
cartridge, micro pump, or other means appreciated by the skilled
artisan, as well-known in the art.
[0191] Therapeutic Applications
[0192] The present invention also provides a method for modulating
or treating at least one IL-23 related disease, in a cell, tissue,
organ, animal, or patient, as known in the art or as described
herein, using at least one IL-23p19 antibody of the present
invention, e.g., administering or contacting the cell, tissue,
organ, animal, or patient with a therapeutic effective amount of
IL-23p19 antibody. The present invention also provides a method for
modulating or treating at least one IL-23 related disease, in a
cell, tissue, organ, animal, or patient including, but not limited
to, at least one of obesity, an immune related disease, a
cardiovascular disease, an infectious disease, a malignant disease
or a neurologic disease.
[0193] The present invention also provides a method for modulating
or treating at least one IL-23 related immune related disease, in a
cell, tissue, organ, animal, or patient including, but not limited
to, at least one of rheumatoid arthritis, juvenile rheumatoid
arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic
arthritis, ankylosing spondilitis, gastric ulcer, seronegative
arthropathies, osteoarthritis, osteolysis, aseptic loosening of
orthopedic implants, inflammatory bowel disease, ulcerative
colitis, systemic lupus erythematosus, antiphospholipid syndrome,
iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary
fibrosis, systemic vasculitis/wegener's granulomatosis,
sarcoidosis, orchitis/vasectomy reversal procedures,
allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic contact dermatitis, allergic conjunctivitis,
hypersensitivity pneumonitis, transplants, organ transplant
rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis,
neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage,
burns, ionizing radiation exposure, acute pancreatitis, adult
respiratory distress syndrome, rheumatoid arthritis,
alcohol-induced hepatitis, chronic inflammatory pathologies,
sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes,
nephrosis, atopic diseases, hypersensitity reactions, allergic
rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis,
pernicious anemia, hemolytic disease, thrombocytopenia, graft
rejection of any organ or tissue, kidney transplant rejection,
heart transplant rejection, liver transplant rejection, pancreas
transplant rejection, lung transplant rejection, bone marrow
transplant (BMT) rejection, skin allograft rejection, cartilage
transplant rejection, bone graft rejection, small bowel transplant
rejection, fetal thymus implant rejection, parathyroid transplant
rejection, xenograft rejection of any organ or tissue, allograft
rejection, anti-receptor hypersensitivity reactions, Graves
disease, Raynaud's disease, type B insulin-resistant diabetes,
asthma, myasthenia gravis, antibody-meditated cytotoxicity, type
III hypersensitivity reactions, POEMS syndrome (polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, and skin
changes syndrome), polyneuropathy, organomegaly, endocrinopathy,
monoclonal gammopathy, skin changes syndrome, antiphospholipid
syndrome, pemphigus, scleroderma, mixed connective tissue disease,
idiopathic Addison's disease, diabetes mellitus, chronic active
hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,
post-MI cardiotomy syndrome, type IV hypersensitivity, contact
dermatitis, hypersensitivity pneumonitis, allograft rejection,
granulomas due to intracellular organisms, drug sensitivity,
metabolic/idiopathic, Wilson's disease, hemachromatosis,
alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto's
thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis
evaluation, primary biliary cirrhosis, thyroiditis,
encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung
disease, chronic obstructive pulmonary disease (COPD), familial
hematophagocytic lymphohistiocytosis, dermatologic conditions,
psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular
nephritis, acute renal failure, hemodialysis, uremia, toxicity,
preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy,
chemotherapy, radiation therapy (e.g., including but not limited
to, asthenia, anemia, cachexia, and the like), chronic salicylate
intoxication, and the like. See, e.g., the Merck Manual, 12th-17th
Editions, Merck & Company, Rahway, N.J. (1972, 1977, 1982,
1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., eds.,
Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),
each entirely incorporated by reference.
[0194] The present invention also provides a method for modulating
or treating at least one cardiovascular disease in a cell, tissue,
organ, animal, or patient, including, but not limited to, at least
one of cardiac stun syndrome, myocardial infarction, congestive
heart failure, stroke, ischemic stroke, hemorrhage, acute coronary
syndrome, arteriosclerosis, atherosclerosis, restenosis, diabetic
ateriosclerotic disease, hypertension, arterial hypertension,
renovascular hypertension, syncope, shock, syphilis of the
cardiovascular system, heart failure, cor pulmonale, primary
pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats,
atrial flutter, atrial fibrillation (sustained or paroxysmal), post
perfusion syndrome, cardiopulmonary bypass inflammation response,
chaotic or multifocal atrial tachycardia, regular narrow QRS
tachycardia, specific arrythmias, ventricular fibrillation, His
bundle arrythmias, atrioventricular block, bundle branch block,
myocardial ischemic disorders, coronary artery disease, angina
pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart
diseases, endocarditis, pericardial disease, cardiac tumors, aordic
and peripheral aneuryisms, aortic dissection, inflammation of the
aorta, occlusion of the abdominal aorta and its branches,
peripheral vascular disorders, occlusive arterial disorders,
peripheral atherlosclerotic disease, thromboangitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous
thrombosis, varicose veins, arteriovenous fistula, lymphederma,
lipedema, unstable angina, reperfusion injury, post pump syndrome,
ischemia-reperfusion injury, and the like. Such a method can
optionally comprise administering an effective amount of a
composition or pharmaceutical composition comprising at least one
anti-IL-23p19 antibody to a cell, tissue, organ, animal or patient
in need of such modulation, treatment or therapy.
[0195] The present invention also provides a method for modulating
or treating at least one IL-23 related infectious disease in a
cell, tissue, organ, animal or patient, including, but not limited
to, at least one of: acute or chronic bacterial infection, acute
and chronic parasitic or infectious processes, including bacterial,
viral and fungal infections, HIV infection/HIV neuropathy,
meningitis, hepatitis (e.g., A, B or C, or the like), septic
arthritis, peritonitis, pneumonia, epiglottitis, e. coli 0157:h7,
hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura,
malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic
shock syndrome, streptococcal myositis, gas gangrene, mycobacterium
tuberculosis, mycobacterium avium intracellulare, pneumocystis
carinii pneumonia, pelvic inflammatory disease,
orchitis/epidydimitis, legionella, lyme disease, influenza a,
epstein-barr virus, viral-associated hemaphagocytic syndrome, viral
encephalitis/aseptic meningitis, and the like.
[0196] The present invention also provides a method for modulating
or treating at least one IL-23 related malignant disease in a cell,
tissue, organ, animal or patient, including, but not limited to, at
least one of: leukemia, acute leukemia, acute lymphoblastic
leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB
ALL, acute myeloid leukemia (AML), acute myelogenous leukemia,
chromic myelocytic leukemia (CML), chronic lymphocytic leukemia
(CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a
lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's
lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma,
colorectal carcinoma, pancreatic carcinoma, nasopharyngeal
carcinoma, malignant histiocytosis, paraneoplastic
syndrome/hypercalcemia of malignancy, solid tumors, bladder cancer,
breast cancer, colorectal cancer, endometiral cancer, head cancer,
neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma,
liver cancer, lung cancer, non-small cell lung cancer, ovarian
cancer, pancreatic cancer, prostate cancer, renal cell carcinoma,
testicular cancer, adenocarcinomas, sarcomas, malignant melanoma,
hemangioma, metastatic disease, cancer related bone resorption,
cancer related bone pain, and the like.
[0197] The present invention also provides a method for modulating
or treating at least one IL-23 related neurologic disease in a
cell, tissue, organ, animal or patient, including, but not limited
to, at least one of: neurodegenerative diseases, multiple
sclerosis, migraine headache, AIDS dementia complex, demyelinating
diseases, such as multiple sclerosis and acute transverse myelitis;
extrapyramidal and cerebellar disorders, such as lesions of the
corticospinal system; disorders of the basal ganglia; hyperkinetic
movement disorders, such as Huntington's Chorea and senile chorea;
drug-induced movement disorders, such as those induced by drugs
which block CNS dopamine receptors; hypokinetic movement disorders,
such as Parkinson's disease; Progressive supranucleo Palsy;
structural lesions of the cerebellum; spinocerebellar
degenerations, such as spinal ataxia, Friedreich's ataxia,
cerebellar cortical degenerations, multiple systems degenerations
(Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic
disorders (Refsum's disease, abetalipoprotemia, ataxia,
telangiectasia, and mitochondrial multi-system disorder);
demyelinating core disorders, such as multiple sclerosis, acute
transverse myelitis; and disorders of the motor unit, such as
neurogenic muscular atrophies (anterior horn cell degeneration,
such as amyotrophic lateral sclerosis, infantile spinal muscular
atrophy and juvenile spinal muscular atrophy); Alzheimer's disease;
Down's Syndrome in middle age; Diffuse Lewy body disease; Senile
Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic
alcoholism; Creutzfeldt-Jakob disease; Subacute sclerosing
panencephalitis, Hallerrorden-Spatz disease; Dementia pugilistica;
neurotraumatic injury (e.g., spinal cord injury, brain injury,
concussion, repetitive concussion); pain; inflammatory pain;
autism; depression; stroke; cognitive disorders; epilepsy; and the
like. Such a method can optionally comprise administering an
effective amount of a composition or pharmaceutical composition
comprising at least one TNF antibody or specified portion or
variant to a cell, tissue, organ, animal or patient in need of such
modulation, treatment or therapy. See, e.g., the Merck Manual,
16.sup.th Edition, Merck & Company, Rahway, N.J. (1992).
[0198] The present invention also provides a method for modulating
or treating at least one IL-23 related wound, trauma or tissue
injury or related chronic condition, in a cell, tissue, organ,
animal or patient, including, but not limited to, at least one of:
bodily injury or a trauma associated with oral surgery including
periodontal surgery, tooth extraction(s), endodontic treatment,
insertion of tooth implants, application and use of tooth
prosthesis; or wherein the wound is selected from the group
consisting of aseptic wounds, contused wounds, incised wounds,
lacerated wounds, non-penetrating wounds, open wounds, penetrating
wounds, perforating wounds, puncture wounds, septic wounds,
infarctions and subcutaneous wounds; or wherein the wound is
selected from the group consisting of ischemic ulcers, pressure
sores, fistulae, severe bites, thermal burns and donor site wounds;
or wherein the wound is anaphthous wound, a traumatic wound or a
herpes associated wound.
[0199] Wounds and/or ulcers are normally found protruding from the
skin or on a mucosal surface or as a result of an infarction in an
organ ("stroke"). A wound may be a result of a soft tissue defect
or a lesion or of an underlying condition. In the present context,
the term "skin" relates to the outermost surface of the body of an
animal, including a human, and embraces intact or almost intact
skin as well as an injured skin surface. The term "mucosa" relates
to undamaged or damaged mucosa of an animal, such as a human, and
may be the oral, buccal, aural, nasal, lung, eye, gastrointestinal,
vaginal, or rectal mucosa.
[0200] In the present context the term "wound" denotes a bodily
injury with disruption of the normal integrity of tissue
structures. The term is also intended to encompass the terms
"sore," "lesion," "necrosis," and "ulcer." Normally, the term
"sore" is a popular term for almost any lesion of the skin or
mucous membranes and the term "ulcer" is a local defect, or
excavation, of the surface of an organ or tissue, which is produced
by the sloughing of necrotic tissue. Lesion generally relates to
any tissue defect. Necrosis is related to dead tissue resulting
from infection, injury, inflammation or infarctions.
[0201] The term "wound" used in the present context denotes any
wound (see below for a classification of wounds) and at any
particular stage in the healing process, including the stage before
any healing has initiated or even before a specific wound like a
surgical incision is made (prophylactic treatment). Examples of
wounds which can be prevented and/or treated in accordance with the
present invention are, e.g., aseptic wounds, contused wounds,
incised wounds, lacerated wounds, non-penetrating wounds (i.e.,
wounds in which there is no disruption of the skin but there is
injury to underlying structures), open wounds, penetrating wounds,
perforating wounds, puncture wounds, septic wounds, subcutaneous
wounds, etc. Examples of sores are bed sores, canker sores, chrome
sores, cold sores, pressure sores, etc.
[0202] Examples of ulcers are, e.g., a peptic ulcer, duodenal
ulcer, gastric ulcer, gouty ulcer, diabetic ulcer, hypertensive
ischemic ulcer, stasis ulcer, ulcus cruris (venous ulcer),
sublingual ulcer, submucous ulcer, symptomatic ulcer, trophic
ulcer, tropical ulcer, and veneral ulcer, e.g., caused by
gonorrhoea (including urethritis, endocervicitis and proctitis).
Conditions related to wounds or sores which may be successfully
treated according to the invention are burns, anthrax, tetanus, gas
gangrene, scarlatina, erysipelas, sycosis barbae, folliculitis,
impetigo contagiosa, or impetigo bullosa, etc. There is often a
certain overlap between the use of the terms "wound" and "ulcer"
and "wound" and "sore" and, furthermore, the terms are often used
at random. Therefore, as mentioned above, in the present context
the term "wound" encompasses the terms "ulcer," "lesion," "sore"
and "infarction," and the terms are indiscriminately used unless
otherwise indicated.
[0203] The kinds of wounds to be treated according to the invention
include also
(i) general wounds, such as, e.g., surgical, traumatic, infectious,
ischemic, thermal, chemical and bullous wounds; (ii) wounds
specific for the oral cavity, such as, e.g., post-extraction
wounds, endodontic wounds especially in connection with treatment
of cysts and abscesses, ulcers and lesions of bacterial, viral or
autoimmunological origin, mechanical, chemical, thermal, infectious
and lichenoid wounds; herpes ulcers, stomatitis aphthosa, acute
necrotising ulcerative gingivitis and burning mouth syndrome are
specific examples; and (iii) wounds on the skin, such as, e.g.,
neoplasm, burns (e.g. chemical, thermal), lesions (bacterial,
viral, autoimmunological), bites and surgical incisions. Another
way of classifying wounds is as (i) small tissue loss due to
surgical incisions, minor abrasions and minor bites, or as (ii)
significant tissue loss. The latter group includes ischemic ulcers,
pressure sores, fistulae, lacerations, severe bites, thermal burns
and donor site wounds (in soft and hard tissues) and
infarctions.
[0204] Other wounds that are of importance in connection with the
present invention are wounds like ischemic ulcers, pressure sores,
fistulae, severe bites, thermal burns and donor site wounds.
Ischemic ulcers and pressure sores are wounds which normally only
heal very slowly and especially in such cases, an improved and more
rapid healing process is of course of great importance for the
patient. Furthermore, the costs involved in the treatment of
patients suffering from such wounds are markedly reduced when the
healing is improved and takes place more rapidly.
[0205] Donor site wounds are wounds which, e.g., occur in
connection with removal of hard tissue from one part of the body to
another part of the body, e.g., in connection with transplantation.
The wounds resulting from such operations are very painful and an
improved healing is therefore most valuable. The term "skin" is
used in a very broad sense embracing the epidermal layer of the
skin and--in those cases where the skin surface is more or less
injured--also the dermal layer of the skin. Apart from the stratum
corneum, the epidermal layer of the skin is the outer (epithelial)
layer and the deeper connective tissue layer of the skin is called
the dermis.
[0206] The present invention also provides a method for modulating
or treating psoriasis, psoriatic arthritis, Crohn's disease,
multiple sclerosis, and optic neuritis, among the other diseases
listed above as IL-23 related, in a cell, tissue, organ, animal, or
patient including, but not limited to, at least one of immune
related disease, cardiovascular disease, infectious, malignant
and/or neurologic disease. Such a method can optionally comprise
administering an effective amount of at least one composition or
pharmaceutical composition comprising at least one anti-IL-23p19
antibody to a cell, tissue, organ, animal or patient in need of
such modulation, treatment or therapy.
[0207] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising at least one anti-IL-23p19
antibody to a cell, tissue, organ, animal or patient in need of
such modulation, treatment or therapy. Such a method can optionally
further comprise co-administration or combination therapy for
treating such diseases or disorders,
[0208] wherein the administering of said at least one anti-IL-23p19
antibody, specified portion or variant thereof, further comprises
administering, before concurrently, and/or after, at least one
selected from at least one TNF antagonist (e.g., but not limited
to, a TNF chemical or protein antagonist, TNF monoclonal or
polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55,
p70 or p85) or fragment, fusion polypeptides thereof, or a small
molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-1
or TBP-II), nerelimonmab, infliximab, etanercept (Enbrel.TM.),
adalimulab (Humira.TM.), CDP-571, CDP-870, afelimomab, lenercept,
and the like), an antirheumatic (e.g., methotrexate, auranofin,
aurothioglucose, azathioprine, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a fluororquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes
related agent, a mineral, a nutritional, a thyroid agent, a
vitamin, a calcium related hormone, an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an
anticoagulant, an erythropoietin (e.g., epoetin alpha), a
filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an immunization, an immunoglobulin, an immunosuppressive
(e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a
hormone replacement drug, an estrogen receptor modulator, a
mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a
mitotic inhibitor, a radiopharmaceutical, an antidepressant,
antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma
medication, a beta agonist, an inhaled steroid, a leukotriene
inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog,
dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist.
Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and
Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21.sup.st
edition, Springhouse Corp., Springhouse, Pa., 2001; Health
Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang,
Prentice-Hall, Inc, Upper Saddle River, N.J. each of which
references are entirely incorporated herein by reference.
[0209] TNF antagonists suitable for compositions, combination
therapy, co-administration, devices and/or methods of the present
invention (further comprising at least one antibody, specified
portion and variant thereof, of the present invention), include,
but are not limited to, anti-TNF antibodies (e.g., at least one TNF
antagonist as defined above), antigen-binding fragments thereof,
and receptor molecules which bind specifically to TNF; compounds
which prevent and/or inhibit TNF synthesis, TNF release or its
action on target cells, such as thalidomide, tenidap,
phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram),
A2b adenosine receptor agonists and A2b adenosine receptor
enhancers; compounds which prevent and/or inhibit TNF receptor
signalling, such as mitogen activated protein (MAP) kinase
inhibitors; compounds which block and/or inhibit membrane TNF
cleavage, such as metalloproteinase inhibitors; compounds which
block and/or inhibit TNF activity, such as angiotensin converting
enzyme (ACE) inhibitors (e.g., captopril); and compounds which
block and/or inhibit TNF production and/or synthesis, such as MAP
kinase inhibitors.
[0210] As used herein, a "tumor necrosis factor antibody," "TNF
antibody," "TNF.alpha. antibody," or fragment and the like
decreases, blocks, inhibits, abrogates or interferes with
TNF.alpha. activity in vitro, in situ and/or, preferably, in
vivo.
[0211] For example, a suitable TNF human antibody of the present
invention can bind TNF.alpha. and includes anti-TNF antibodies,
antigen-binding fragments thereof, and specified mutants or domains
thereof that bind specifically to TNF.alpha.. A suitable TNF
antibody or fragment can also decrease block, abrogate, interfere,
prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF
release, TNF receptor signaling, membrane TNF cleavage, TNF
activity, TNF production and/or synthesis.
[0212] An example of a TNF antibody or antagonist is the chimeric
antibody cA2. Additional examples of monoclonal anti-TNF antibodies
that can be used in the present invention are described in the art
(see, e.g., U.S. Pat. No. 5,231,024; Moller, A. et al., Cytokine
2(3):162-169 (1990); U.S. application Ser. No. 07/943,852 (filed
Sep. 11, 1992); Rathjen et al., International Publication No. WO
91/02078 (published Feb. 21, 1991); Rubin et al., EPO Patent
Publication No. 0 218 868 (published Apr. 22, 1987); Yone et al.,
EPO Patent Publication No. 0 288 088 (Oct. 26, 1988); Liang, et
al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et
al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369
(1987); Bringman, et al., Hybridoma 6:489-507 (1987); and Hirai, et
al., J. Immunol. Meth. 96:57-62 (1987).
[0213] TNF Receptor Molecules
[0214] Preferred TNF receptor molecules useful in the present
invention are those that bind TNF.alpha. with high affinity (see,
e.g., Feldmann et al., International Publication No. WO 92/07076
(published Apr. 30, 1992); Schall et al., Cell 61:361-370 (1990);
and Loetscher et al., Cell 61:351-359 (1990), which references are
entirely incorporated herein by reference) and optionally possess
low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and the
75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the
present invention. Truncated forms of these receptors, comprising
the extracellular domains (ECD) of the receptors or functional
portions thereof (see, e.g., Corcoran et al., Eur. J. Biochem.
223:831-840 (1994)), are also useful in the present invention.
Truncated forms of the TNF receptors, comprising the ECD, have been
detected in urine and serum as 30 kDa and 40 kDa TNF.alpha.
inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem.
265:1531-1536 (1990)).
[0215] TNF receptor multimeric molecules and TNF immunoreceptor
fusion molecules, and derivatives and fragments or portions
thereof, are additional examples of TNF receptor molecules which
are useful in the methods and compositions of the present
invention.
[0216] TNF receptor multimeric molecules useful in the present
invention comprise all or a functional portion of the ECD of two or
more TNF receptors linked via one or more polypeptide linkers or
other nonpeptide linkers, such as polyethylene glycol (PEG). An
example of such a TNF immunoreceptor fusion molecule is TNF
receptor/IgG fusion protein. TNF immunoreceptor fusion molecules
and methods for their production have been described in the art
(Lesslauer et al., Eur. J. Immunol. 21:2883-2886 (1991); Ashkenazi
et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Peppel et
al., J. Exp. Med. 174:1483-1489 (1991); Kolls et al., Proc. Natl.
Acad. Sci. USA 91:215-219 (1994); Butler et al., Cytokine
6(6):616-623 (1994); Baker et al., Eur. J. Immunol. 24:2040-2048
(1994); Beutler et al., U.S. Pat. No. 5,447,851; and U.S.
application Ser. No. 08/442,133 (filed May 16, 1995), each of which
references are entirely incorporated herein by reference). Methods
for producing immunoreceptor fusion molecules can also be found in
Capon et al., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No.
5,225,538; and Capon et al., Nature 337:525-531 (1989), which
references are entirely incorporated herein by reference.
[0217] Cytokines include any known cytokine. See, e.g.,
CopewithCytokines.com. Cytokine antagonists include, but are not
limited to, any antibody, fragment or mimetic, any soluble
receptor, fragment or mimetic, any small molecule antagonist, or
any combination thereof.
[0218] Therapeutic Treatments
[0219] Any method of the present invention can comprise a method
for treating an IL-23 mediated disorder, comprising administering
an effective amount of a composition or pharmaceutical composition
comprising at least one anti-IL-23p19 antibody to a cell, tissue,
organ, animal or patient in need of such modulation, treatment or
therapy. Such a method can optionally further comprise
co-administration or combination therapy for treating such diseases
or disorders, wherein the administering of said at least one
anti-IL-23p19 antibody, specified portion or variant thereof,
further comprises administering before, concurrently, and/or after,
at least one selected from an anti-infective drug, a cardiovascular
(CV) system drug, a central nervous system (CNS) drug, an autonomic
nervous system (ANS) drug, a respiratory tract drug, a
gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid
or electrolyte balance, a hematologic drug, an antineoplastic, an
immunomodulation drug, an ophthalmic, otic or nasal drug, a topical
drug, a nutritional drug or the like, at least one TNF antagonist
(e.g., but not limited to a TNF antibody or fragment, a soluble TNF
receptor or fragment, fusion proteins thereof, or a small molecule
TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin,
aurothioglucose, azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a fluororquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes
related agent, a mineral, a nutritional, a thyroid agent, a
vitamin, a calcium related hormone, an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an
anticoagulant, an erythropoietin (e.g., epoetin alpha), a
filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an immunization, an immunoglobulin, an immunosuppressive
(e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a
hormone replacement drug, an estrogen receptor modulator, a
mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a
mitotic inhibitor, a radiopharmaceutical, an antidepressant,
antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma
medication, a beta agonist, an inhaled steroid, a leukotriene
inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog,
dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist.
Such drugs are well known in the art, including formulations,
indications, dosing and administration for each presented herein
(see., e.g., Nursing 2001 Handbook of Drugs, 21.sup.st edition,
Springhouse Corp., Springhouse, Pa., 2001; Health Professional's
Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc,
Upper Saddle River, N.J.; Pharmcotherapy Handbook, Wells et al.,
ed., Appleton & Lange, Stamford, Conn., each entirely
incorporated herein by reference).
[0220] Typically, treatment of pathologic conditions is effected by
administering an effective amount or dosage of at least one
anti-IL-23p19 antibody composition that total, on average, a range
from at least about 0.01 to 500 milligrams of at least one
anti-IL-23p19 antibody per kilogram of patient per dose, and,
preferably, from at least about 0.1 to 100 milligrams
antibody/kilogram of patient per single or multiple administration,
depending upon the specific activity of the active agent contained
in the composition. Alternatively, the effective serum
concentration can comprise 0.1-5000 .mu.g/ml serum concentration
per single or multiple administration. Suitable dosages are known
to medical practitioners and will, of course, depend upon the
particular disease state, specific activity of the composition
being administered, and the particular patient undergoing
treatment. In some instances, to achieve the desired therapeutic
amount, it can be necessary to provide for repeated administration,
i.e., repeated individual administrations of a particular monitored
or metered dose, where the individual administrations are repeated
until the desired daily dose or effect is achieved.
[0221] Preferred doses can optionally include about 0.1-99 and/or
100-500 mg/kg/administration, or any range, value or fraction
thereof, or to achieve a serum concentration of about 0.1-5000
.mu.g/ml serum concentration per single or multiple administration,
or any range, value or fraction thereof. A preferred dosage range
for the anti-IL-23p19 antibody of the present invention is from
about 1 mg/kg, up to about 3, about 6 or about 12 mg/kg of body
weight of the patient.
[0222] Alternatively, the dosage administered can vary depending
upon known factors, such as the pharmacodynamic characteristics of
the particular agent, and its mode and route of administration;
age, health, and weight of the recipient; nature and extent of
symptoms, kind of concurrent treatment, frequency of treatment, and
the effect desired. Usually a dosage of active ingredient can be
about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily
0.1 to 50, and, preferably, 0.1 to 10 milligrams per kilogram per
administration or in sustained release form is effective to obtain
desired results.
[0223] As a non-limiting example, treatment of humans or animals
can be provided as a one-time or periodic dosage of at least one
antibody of the present invention about 0.1 to 100 mg/kg or any
range, value or fraction thereof per day, on at least one of day
1-40, or, alternatively or additionally, at least one of week 1-52,
or, alternatively or additionally, at least one of 1-20 years, or
any combination thereof, using single, infusion or repeated
doses.
[0224] Dosage forms (composition) suitable for internal
administration generally contain from about 0.001 milligram to
about 500 milligrams of active ingredient per unit or container. In
these pharmaceutical compositions the active ingredient will
ordinarily be present in an amount of about 0.5-99.999% by weight
based on the total weight of the composition.
[0225] For parenteral administration, the antibody can be
formulated as a solution, suspension, emulsion, particle, powder,
or lyophilized powder in association, or separately provided, with
a pharmaceutically acceptable parenteral vehicle. Examples of such
vehicles are water, saline, Ringer's solution, dextrose solution,
and about 1-10% human serum albumin. Liposomes and nonaqueous
vehicles, such as fixed oils, can also be used. The vehicle or
lyophilized powder can contain additives that maintain isotonicity
(e.g., sodium chloride, mannitol) and chemical stability (e.g.,
buffers and preservatives). The formulation is sterilized by known
or suitable techniques.
[0226] Suitable pharmaceutical carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
[0227] Alternative Administration
[0228] Many known and developed modes can be used according to the
present invention for administering pharmaceutically effective
amounts of at least one anti-IL-23p19 antibody according to the
present invention. While pulmonary administration is used in the
following description, other modes of administration can be used
according to the present invention with suitable results. IL-23p19
antibodies of the present invention can be delivered in a carrier,
as a solution, emulsion, colloid, or suspension, or as a dry
powder, using any of a variety of devices and methods suitable for
administration by inhalation or other modes described here within
or known in the art.
[0229] Parenteral Formulations and Administration
[0230] Formulations for parenteral administration can contain as
common excipients sterile water or saline, polyalkylene glycols,
such as polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes and the like. Aqueous or oily suspensions for
injection can be prepared by using an appropriate emulsifier or
humidifier and a suspending agent, according to known methods.
Agents for injection can be a non-toxic, non-orally administrable
diluting agent, such as aqueous solution, a sterile injectable
solution or suspension in a solvent. As the usable vehicle or
solvent, water, Ringer's solution, isotonic saline, etc. are
allowed; as an ordinary solvent or suspending solvent, sterile
involatile oil can be used. For these purposes, any kind of
involatile oil and fatty acid can be used, including natural or
synthetic or semisynthetic fatty oils or fatty acids; natural or
synthetic or semisynthtetic mono- or di- or tri-glycerides.
Parental administration is known in the art and includes, but is
not limited to, conventional means of injections, a gas pressured
needle-less injection device as described in U.S. Pat. No.
5,851,198, and a laser perforator device as described in U.S. Pat.
No. 5,839,446 entirely incorporated herein by reference.
[0231] Alternative Delivery
[0232] The invention further relates to the administration of at
least one
anti-IL-23p19 antibody by parenteral, subcutaneous, intramuscular,
intravenous, intrarticular, 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, intralesional, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal
means.
[0233] At least one anti-IL-23p19 antibody composition can be
prepared for use for parenteral (subcutaneous, intramuscular or
intravenous) or any other administration particularly in the form
of liquid solutions or suspensions; for use in vaginal or rectal
administration particularly in semisolid forms, such as, but not
limited to, creams and suppositories; for buccal, or sublingual
administration, such as, but not limited to, in the form of tablets
or capsules; or intranasally, such as, but not limited to, the form
of powders, nasal drops or aerosols or certain agents; or
transdermally, such as not limited to a gel, ointment, lotion,
suspension or patch delivery system with chemical enhancers such as
dimethyl sulfoxide to either modify the skin structure or to
increase the drug concentration in the transdermal patch
(Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D. S.,
Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely
incorporated herein by reference), or with oxidizing agents that
enable the application of formulations containing proteins and
peptides onto the skin (WO 98/53847), or applications of electric
fields to create transient transport pathways, such as
electroporation, or to increase the mobility of charged drugs
through the skin, such as iontophoresis, or application of
ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and
4,767,402) (the above publications and patents being entirely
incorporated herein by reference).
[0234] Pulmonary/Nasal Administration
[0235] For pulmonary administration, preferably, at least one
anti-IL-23p19 antibody composition is delivered in a particle size
effective for reaching the lower airways of the lung or sinuses.
According to the invention, at least one anti-IL-23p19 antibody can
be delivered by any of a variety of inhalation or nasal devices
known in the art for administration of a therapeutic agent by
inhalation. These devices capable of depositing aerosolized
formulations in the sinus cavity or alveoli of a patient include
metered dose inhalers, nebulizers, dry powder generators, sprayers,
and the like. Other devices suitable for directing the pulmonary or
nasal administration of antibodies are also known in the art. All
such devices can use formulations suitable for the administration
for the dispensing of antibody in an aerosol.
[0236] Such aerosols can be comprised of either solutions (both
aqueous and non aqueous) or solid particles.
[0237] Metered dose inhalers like the Ventolin.RTM. metered dose
inhaler, typically use a propellent gas and require actuation
during inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry
powder inhalers like Turbuhaler.TM. (Astra), Rotahaler.RTM.
(Glaxo), Diskus.RTM. (Glaxo), Spiros.TM. inhaler (Dura), devices
marketed by Inhale Therapeutics, and the Spinhaler.RTM. powder
inhaler (Fisons), use breath-actuation of a mixed powder (U.S. Pat.
No. 4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO
94/08552 Dura, U.S. Pat. No. 5,458,135 Inhale, WO 94/06498 Fisons,
entirely incorporated herein by reference). Nebulizers like
AERx.TM. Aradigm, the Ultravent.RTM. nebulizer (Mallinckrodt), and
the Acorn II.RTM. nebulizer (Marquest Medical Products) (U.S. Pat.
No. 5,404,871 Aradigm, WO 97/22376), the above references entirely
incorporated herein by reference, produce aerosols from solutions,
while metered dose inhalers, dry powder inhalers, etc. generate
small particle aerosols. These specific examples of commercially
available inhalation devices are intended to be a representative of
specific devices suitable for the practice of this invention, and
are not intended as limiting the scope of the invention.
[0238] Preferably, a composition comprising at least one
anti-IL-23p19 antibody is delivered by a dry powder inhaler or a
sprayer. There are several desirable features of an inhalation
device for administering at least one antibody of the present
invention. For example, delivery by the inhalation device is
advantageously reliable, reproducible, and accurate. The inhalation
device can optionally deliver small dry particles, e.g., less than
about 10 .mu.m, preferably about 1-5 .mu.m, for good
respirability.
[0239] Administration of IL-23p19 Antibody Compositions as a
Spray
[0240] A spray including IL-23p19 antibody composition can be
produced by forcing a suspension or solution of at least one
anti-IL-23p19 antibody through a nozzle under pressure. The nozzle
size and configuration, the applied pressure, and the liquid feed
rate can be chosen to achieve the desired output and particle size.
An electrospray can be produced, for example, by an electric field
in connection with a capillary or nozzle feed. Advantageously,
particles of at least one anti-IL-23p19 antibody composition
delivered by a sprayer have a particle size less than about 10
.mu.m, preferably, in the range of about 1 .mu.m to about 5 .mu.m,
and, most preferably, about 2 .mu.m to about 3 .mu.m.
[0241] Formulations of at least one anti-IL-23p19 antibody
composition suitable for use with a sprayer typically include
antibody composition in an aqueous solution at a concentration of
about 0.1 mg to about 100 mg of at least one anti-IL-23p19 antibody
composition per ml of solution or mg/gm, or any range, value, or
fraction therein. The formulation can include agents, such as an
excipient, a buffer, an isotonicity agent, a preservative, a
surfactant, and, preferably, zinc. The formulation can also include
an excipient or agent for stabilization of the antibody
composition, such as a buffer, a reducing agent, a bulk protein, or
a carbohydrate. Bulk proteins useful in formulating antibody
compositions include albumin, protamine, or the like. Typical
carbohydrates useful in formulating antibody compositions include
sucrose, mannitol, lactose, trehalose, glucose, or the like. The
antibody composition formulation can also include a surfactant,
which can reduce or prevent surface-induced aggregation of the
antibody composition caused by atomization of the solution in
forming an aerosol. Various conventional surfactants can be
employed, such as polyoxyethylene fatty acid esters and alcohols,
and polyoxyethylene sorbitol fatty acid esters. Amounts will
generally range between 0.001 and 14% by weight of the formulation.
Especially preferred surfactants for purposes of this invention are
polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate
20, or the like. Additional agents known in the art for formulation
of a protein, such as IL-23p19 antibodies, or specified portions or
variants, can also be included in the formulation.
[0242] Administration of IL-23p19 Antibody Compositions by a
Nebulizer
[0243] Antibody compositions of the invention can be administered
by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
Typically, in a jet nebulizer, a compressed air source is used to
create a high-velocity air jet through an orifice. As the gas
expands beyond the nozzle, a low-pressure region is created, which
draws a solution of antibody composition through a capillary tube
connected to a liquid reservoir. The liquid stream from the
capillary tube is sheared into unstable filaments and droplets as
it exits the tube, creating the aerosol. A range of configurations,
flow rates, and baffle types can be employed to achieve the desired
performance characteristics from a given jet nebulizer. In an
ultrasonic nebulizer, high-frequency electrical energy is used to
create vibrational, mechanical energy, typically employing a
piezoelectric transducer. This energy is transmitted to the
formulation of antibody composition either directly or through a
coupling fluid, creating an aerosol including the antibody
composition. Advantageously, particles of antibody composition
delivered by a nebulizer have a particle size less than about 10
.mu.m, preferably, in the range of about 1 .mu.m to about 5 .mu.m,
and, most preferably, about 2 .mu.m to about 3 .mu.m.
[0244] Formulations of at least one anti-IL-23p19 antibody suitable
for use with a nebulizer, either jet or ultrasonic, typically
include a concentration of about 0.1 mg to about 100 mg of at least
one anti-IL-23p19 antibody protein per ml of solution. The
formulation can include agents, such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for
stabilization of the at least one anti-IL-23p19 antibody
composition, such as a buffer, a reducing agent, a bulk protein, or
a carbohydrate.
[0245] Bulk proteins useful in formulating at least one
anti-IL-23p19 antibody compositions include albumin, protamine, or
the like. Typical carbohydrates useful in formulating at least one
anti-IL-23p19 antibody include sucrose, mannitol, lactose,
trehalose, glucose, or the like. The at least one anti-IL-23p19
antibody formulation can also include a surfactant, which can
reduce or prevent surface-induced aggregation of the at least one
anti-IL-23p19 antibody caused by atomization of the solution in
forming an aerosol. Various conventional surfactants can be
employed, such as polyoxyethylene fatty acid esters and alcohols,
and polyoxyethylene sorbital fatty acid esters. Amounts will
generally range between about 0.001 and 4% by weight of the
formulation. Especially preferred surfactants for purposes of this
invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80,
polysorbate 20, or the like. Additional agents known in the art for
formulation of a protein, such as antibody protein, can also be
included in the formulation.
[0246] Administration of IL-23p19 Antibody Compositions by a
Metered Dose Inhaler
[0247] In a metered dose inhaler (MDI), a propellant, at least one
anti-IL-23p19 antibody, and any excipients or other additives are
contained in a canister as a mixture including a liquefied
compressed gas. Actuation of the metering valve releases the
mixture as an aerosol, preferably containing particles in the size
range of less than about 10 .mu.m, preferably, about 1 .mu.m to
about 5 .mu.m, and, most preferably, about 2 .mu.m to about 3
.mu.m. The desired aerosol particle size can be obtained by
employing a formulation of antibody composition produced by various
methods known to those of skill in the art, including jet-milling,
spray drying, critical point condensation, or the like. Preferred
metered dose inhalers include those manufactured by 3M or Glaxo and
employing a hydrofluorocarbon propellant. Formulations of at least
one anti-IL-23p19 antibody for use with a metered-dose inhaler
device will generally include a finely divided powder containing at
least one anti-IL-23p19 antibody as a suspension in a non-aqueous
medium, for example, suspended in a propellant with the aid of a
surfactant. The propellant can be any conventional material
employed for this purpose, such as chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a
(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the
like. Preferably, the propellant is a hydrofluorocarbon. The
surfactant can be chosen to stabilize the at least one
anti-IL-23p19 antibody as a suspension in the propellant, to
protect the active agent against chemical degradation, and the
like. Suitable surfactants include sorbitan trioleate, soya
lecithin, oleic acid, or the like. In some cases, solution aerosols
are preferred using solvents, such as ethanol. Additional agents
known in the art for formulation of a protein can also be included
in the formulation. One of ordinary skill in the art will recognize
that the methods of the current invention can be achieved by
pulmonary administration of at least one anti-IL-23p19 antibody
composition via devices not described herein.
[0248] Oral Formulations and Administration
[0249] Formulations for oral administration rely on the
co-administration of adjuvants (e.g., resorcinols and nonionic
surfactants, such as polyoxyethylene oleyl ether and
n-hexadecylpolyethylene ether) to increase artificially the
permeability of the intestinal walls, as well as the
co-administration of enzymatic inhibitors (e.g., pancreatic trypsin
inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to
inhibit enzymatic degradation. Formulations for delivery of
hydrophilic agents including proteins and antibodies and a
combination of at least two surfactants intended for oral, buccal,
mucosal, nasal, pulmonary, vaginal transmembrane, or rectal
administration are taught in U.S. Pat. No. 6,309,663. The active
constituent compound of the solid-type dosage form for oral
administration can be mixed with at least one additive, including
sucrose, lactose, cellulose, mannitol, trehalose, raffinose,
maltitol, dextran, starches, agar, arginates, chitins, chitosans,
pectins, gum tragacanth, gum arabic, gelatin, collagen, casein,
albumin, synthetic or semisynthetic polymer, and glyceride. These
dosage forms can also contain other type(s) of additives, e.g.,
inactive diluting agent, lubricant, such as magnesium stearate,
paraben, preserving agent, such as sorbic acid, ascorbic acid,
.alpha.-tocopherol, antioxidant such as cysteine, disintegrator,
binder, thickener, buffering agent, sweetening agent, flavoring
agent, perfuming agent, etc.
[0250] Tablets and pills can be further processed into
enteric-coated preparations. The liquid preparations for oral
administration include emulsion, syrup, elixir, suspension and
solution preparations allowable for medical use. These preparations
can contain inactive diluting agents ordinarily used in said field,
e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More
recently, microspheres of artificial polymers of mixed amino acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat.
No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 and used to
deliver biologically active agents orally are known in the art.
[0251] Mucosal Formulations and Administration
[0252] A formulation for orally administering a bioactive agent
encapsulated in one or more biocompatible polymer or copolymer
excipients, preferably, a biodegradable polymer or copolymer,
affording microcapsules which due to the proper size of the
resultant microcapsules results in the agent reaching and being
taken up by the folliculi lymphatic aggregati, otherwise known as
the "Peyer's patch," or "GALT" of the animal without loss of
effectiveness due to the agent having passed through the
gastrointestinal tract. Similar folliculi lymphatic aggregati can
be found in the bronchei tubes (BALT) and the large intestine. The
above-described tissues are referred to in general as mucosally
associated lymphoreticular tissues (MALT). For absorption through
mucosal surfaces, compositions and methods of administering at
least one anti-IL-23p19 antibody include an emulsion comprising a
plurality of submicron particles, a mucoadhesive macromolecule, a
bioactive peptide, and an aqueous continuous phase, which promotes
absorption through mucosal surfaces by achieving mucoadhesion of
the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces
suitable for application of the emulsions of the present invention
can include corneal, conjunctival, buccal, sublingual, nasal,
vaginal, pulmonary, stomachic, intestinal, and rectal routes of
administration. Formulations for vaginal or rectal administration,
e.g., suppositories, can contain as excipients, for example,
polyalkyleneglycols, vaseline, cocoa butter, and the like.
Formulations for intranasal administration can be solid and contain
as excipients, for example, lactose or can be aqueous or oily
solutions of nasal drops. For buccal administration, excipients
include sugars, calcium stearate, magnesium stearate,
pregelinatined starch, and the like (U.S. Pat. No. 5,849,695).
[0253] Transdermal Formulations and Administration
[0254] For transdermal administration, the at least one
anti-IL-23p19 antibody is encapsulated in a delivery device, such
as a liposome or polymeric nanoparticles, microparticle,
microcapsule, or microspheres (referred to collectively as
microparticles unless otherwise stated). A number of suitable
devices are known, including microparticles made of synthetic
polymers, such as polyhydroxy acids, such as polylactic acid,
polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and polyphosphazenes, and natural polymers, such as
collagen, polyamino acids, albumin and other proteins, alginate and
other polysaccharides, and combinations thereof (U.S. Pat. No.
5,814,599).
[0255] Prolonged Administration and Formulations
[0256] It can be desirable to deliver the compounds of the present
invention to the subject over prolonged periods of time, for
example, for periods of one week to one year from a single
administration. Various slow release, depot or implant dosage forms
can be utilized. For example, a dosage form can contain a
pharmaceutically acceptable non-toxic salt of the compounds that
has a low degree of solubility in body fluids, for example, (a) an
acid addition salt with a polybasic acid, such as phosphoric acid,
sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic
acid, alginic acid, polyglutamic acid, naphthalene mono- or
di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt
with a polyvalent metal cation, such as zinc, calcium, bismuth,
barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and
the like, or with an organic cation formed from e.g.,
N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c)
combinations of (a) and (b), e.g., a zinc tannate salt.
Additionally, the compounds of the present invention or,
preferably, a relatively insoluble salt, such as those just
described, can be formulated in a gel, for example, an aluminum
monostearate gel with, e.g., sesame oil, suitable for injection.
Particularly preferred salts are zinc salts, zinc tannate salts,
pamoate salts, and the like. Another type of slow release depot
formulation for injection would contain the compound or salt
dispersed for encapsulation in a slow degrading, non-toxic,
non-antigenic polymer, such as a polylactic acid/polyglycolic acid
polymer for example as described in U.S. Pat. No. 3,773,919. The
compounds or, preferably, relatively insoluble salts, such as those
described above, can also be formulated in cholesterol matrix
silastic pellets, particularly for use in animals. Additional slow
release, depot or implant formulations, e.g., gas or liquid
liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and
"Sustained and Controlled Release Drug Delivery Systems", J. R.
Robinson ed., Marcel Dekker, Inc., N.Y., 1978).
[0257] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Subunit Specificity of IL-23p19 Monoclonal Antibodies
[0258] Purified mouse anti-human IL-23 mAbs were evaluated in
cytokine capture ELISA to determine their antigen subunit
specificity. Briefly, IL-23 mAbs were coated onto plates and
incubated with 100 ng/ml (hr=human recombinant) hrIL-23, hrIL-12,
and hrp40, respectively. Following incubation with biotinylated
anti-p40 mAb, the binding was detected using HRP-conjugated
streptavidin. An anti-p40 mAb and an anti-IL-12 mAb (20C2, Catalog
No. 555065, BD Pharmingen, San Diego, Calif.) with known
specificity were used as controls.
[0259] FIG. 1 demonstrates that four mAbs bind specifically hrIL-23
and not hrIL-12 or hrp40 monomer. Because the IL-23p19 subunit must
covalently associate with p40 to be secreted from mammalian cells,
IL-23 mAbs that do not recognize p40 monomer must bind either the
IL-23p19 subunit alone or a joint epitope of the p19-p40
heterodimer. Therefore, these IL-23 mAbs are referred to as
IL-23p19 mAbs.
[0260] All four anti-human IL-23p19 mAbs demonstrate similar
binding curves to hrIL-23 (FIG. 2) and subsequent BIAcore analysis
demonstrated affinities ranging from 43-338 pM. It was further
determined that these IL-23 mAbs do not cross-react with murine
IL-23 (data not shown).
Example 2
Inhibition of IL-23 Receptor Binding by IL-23p19 mAbs
[0261] To demonstrate that the IL-23p19 mAbs are neutralizing
antibodies against the p19 subunit, the mAbs were tested for their
inhibition of IL-23 and IL-23R binding. In this experiment, IL-23R
was immobilized on a plate. Biotinylated hrIL-23 was added to the
plate either alone or after preincubation with individual IL-23p19
mAbs. Soluble IL-23R (IL-23R-Fc) was used as a positive control.
IL-23 binding was detected with HRP-conjugated streptavidin. As
shown in FIG. 3, all four IL-23p19 mAbs were able to prevent
IL-23/IL-23R binding with comparable potency to soluble IL-23R-Fc.
In contrast, when IL-12R.beta.1 was immobilized on a plate, none of
the IL-23p19 mAbs were able to inhibit IL-23/IL-12R.beta.1 binding
(data not shown). Similarly, the IL-23p19 mAbs do not block
IL-12/IL-12R.beta.1 binding (data not shown). The selective
inhibition of IL-23/IL-23R binding and the lack of interference
with IL-12 or IL-23 binding to IL-12R.beta.1 further demonstrates
that these IL-23p19 mAbs do not bind the p40 subunit and thus are
neutralizing anti-human IL-23p19 antibodies.
Example 3
Neutralization of IL-23 Biological Function by IL-23p19 mAbs
[0262] IL-23 is known to induce intracellular STAT3 phosphorylation
and IL-17 production by T cells. Therefore, the IL-23p19 mAbs were
tested for their ability to inhibit these biological functions of
human IL-23.
[0263] In one experiment, natural killer (NKL) cells were
stimulated with hrIL-23 either alone or after preincubation with
individual IL-23p19 mAbs. Treated cells were stained with
fluorochrome-conjugated anti-phospho-STAT3 antibodies and analyzed
by intracellular flow cytometry. It was shown that all four
IL-23p19 mAbs inhibit STAT3 phosphorylation with comparable potency
to a neutralizing anti-p40 mAb.
[0264] In another experiment, freshly isolated murine splenocytes
were treated with hrIL-23 preincubated with titrated IL-23p19 mAbs
or control mAbs. hrIL-23 with no antibody preincubation was used as
the positive control. After 3 days in culture, cell supernatants
were collected and assayed by ELISA using IL-17 ELISA duo set
(R&D Systems). As shown in FIG. 4, IL-23p19 mAbs inhibit
hrIL-23 mediated IL-17 production. These mAbs were also shown to
inhibit native human IL-23 (produced by human PBMC) mediated IL-17
production.
[0265] In comparison, IL-23p19 mAbs were also tested for their
ability to inhibit IL-12 induced IFN.gamma. production. Briefly,
NK92MI cells were treated with IL-12 preincubated with titrated
IL-23p19 mAbs or control mAbs. IL-12 with no antibody preincubation
was used as the positive control. ELISA analysis performed 24 hours
post-stimulation showed no effect of IL-23p19 mAbs on IL-12 induced
IFN.gamma. production demonstrating that the antibodies do not bind
to the p40 subunit shared by IL-12 and IL-23.
Example 4
Epitope Identification of IL-23p19 mAbs and Competitive Binding
[0266] Competition binding analysis was performed to determine if
the four neutralizing IL-23p19 mAbs bind to similar or different
IL-23p19 epitopes. IL-23 mAbs were individually coated on ELISA
plates. Competing mAbs were added, followed by the addition of
biotinylated hrIL-23. For positive control, the same mAb for
coating was used as the competing mAb ("self-competition"). IL-23
binding was detected using streptavidin. C1269, C1273 and C1275 all
show cross-competition indicating binding to a spatially similar
site. C1249 shows little or no competition with C1269 or C1273 and
partial inhibition of C1275. These results indicate that the mAbs
recognize similar or partially overlapping epitopes on IL-23.
[0267] Competition ELISA of labeled C1269 antibody bound to IL-23
was performed as shown in FIG. 7. 5 .mu.L of a 20 .mu.g/ml hIL-23
coated on plate, mixed with 10 nM of labeled C1269 and serially
diluted from 3000 nM to 0 unlabeled competitors, C1269 and C1249,
in 25 .mu.L. Relative binding was calculated by setting the signal
in the absence of competitor to 100%. The results indicate that
antibodies C1249 and C1269 do not compete for the same binding
epitope.
[0268] To directly map their binding epitopes, the IL-23p19 mAbs,
75 .mu.g of C1249 or mouse IgG, was mixed with about 65 .mu.g of
IL-23 and incubated at 4.degree. C. overnight. The antigen-antibody
complex was isolated by gel chromatography and transferred into
digestion buffer (0.1M Tris-HCL pH 8.5) using a 100,000 NMWL filter
unit. Then, 4 .mu.g of trypsin (0.16 units) in digestion buffer was
added and incubated for 2 hours at 37.degree. C. After incubation,
the complexes were captured by Protein G beads, washed twice with
PBS and once with ammonium bicarbonate, and the captured peptides
eluted in 30 .mu.L of elution buffer (10% Acetonitrile, 0.5% TFA).
Complex formation and trypsin digestion of C1269 was carried out in
the same manner.
[0269] The eluted peptides were analyzed by MALDI-TOF mass
spectrometry as described below. Eluents of the trypsin-digest
complex were desalted by pipetting the sample through a C18 Zip
Tip, the zip tip was wetted with 100% Acetonitrile followed by
equilibration with 0.1% TFA. The eluent was then bound to the
media, washed with 0.1% TFA and eluted in a volume of 2 .mu.L
.alpha.-cyano matrix (10 mg/mL .alpha.-cyano, 0.1% TFA, 50%
Acetonitrile in water) and directly spotted on the MALDI-TOF
target. The MALDI-TOF (Voyager-DE STR, ABI) was calibrated with
Calibration mixture 2 (ABI). Spectra were obtained at laser
intensity between 1500-1800 units and acquisition mass range from
800 to 5000 m/z.
[0270] One p19 peptide at m/z=1248 (.sub.74IHQGLIFYEK.sub.83) was
captured by both the C1249 and C1269 mAbs. However, with C1269,
this peptide was less intense and may be non-specifically bound.
These results indicate that the region .sub.74IHQGLIFYEK.sub.83
contributes to the binding epitope for C1249 and C1269.
[0271] Epitope-Analysis by Swap Mutagenesis of
huIL-23p19/muIL-23p19 Proteins
[0272] It has been observed that C1269 and C1249 do not bind to
mouse IL-23, however, C1269, but not C1249, neutralizes native
IL-23 from cynomolgus monkey. Therefore, sequence differences among
human, cynomolgus monkey, and mouse IL-23p19 were analyzed to
identify putative binding regions for these monoclonal antibodies.
The p19 subunit of cynomolgus monkey was cloned and sequenced at
Centocor. Alignment of the p19 sequences from human, cynomolgus and
mouse is shown from the region identified as comprising at least a
portion of the epitope region for the antibodies of the present
invention--.sub.74IHQGLIFYEK.sub.83. In this 10-residue amino acid
region, there is only a single difference at H75 between human and
cynomolgus monkey IL-23p19. This indicates that H75 is critical for
the binding of C1249, which does not neutralize cynomolgus
IL-23p19, but does not appear to be critical for binding of
C1269.
[0273] Based on the sequence alignment, species swap mutagenesis
was performed. A mutant human IL-23 protein (designated "3220") was
generated in which the tryptic peptide region was mutated to the
mouse sequence, .sub.74IRQGLAFYKH.sub.83,with the four mutations
highlighted in bold (four single point mutations in 3220--Human
.sub.75His.fwdarw.Mouse .sub.75Arg, Human .sub.79 Ile.fwdarw.Mouse
.sub.79Ala, Human .sub.82Glu.fwdarw.Mouse .sub.82Lys and Human
.sub.83Lys.fwdarw.Mouse .sub.83 His). A second mutant protein
(designated "3397") was constructed that incorporates the D and K
substitutions immediately C-terminal of this
peptide-.sub.74IRQGLAFYKHLLDSDIFK.sub.91 (from wild-type
.sub.74IHQGLIFYEKLLGSDOFT.sub.91) with the six mutations
highlighted in bold (the four mutations of 3220 along with two
additional single point mutations--Human .sub.75His.fwdarw.Mouse
.sub.75Arg, Human .sub.79 Ile.fwdarw.Mouse .sub.79Ala, Human
.sub.82Glu.fwdarw.Mouse .sub.82Lys and Human
.sub.83Lys.fwdarw.Mouse .sub.83 His, Human .sub.86 Gly.fwdarw.Mouse
.sub.86 Asp and Human .sub.91 Thr.fwdarw.Mouse .sub.91 Lys).
[0274] To assess binding specificity for the mutant proteins, ELISA
binding was carried out for C1249, C1269, as well as the control
surrogate antibody CNTO 209 (rat anti-mouse IL-23p19). The results
are shown in FIGS. 8A, 8B, and 9. The ELISA results show that the
mutations in IL-23 p19 (3220) reduced the binding activity to C1249
and C1269 by more than 80% (FIG. 5 and FIGS. 8A and 8B). The
additional substitutions in mutant 3397 further reduced the binding
by 95% in C1249 and by 90% in C1269.
[0275] About 65 .mu.g of IL-23 was mixed with 75 .mu.g of C1249,
C1269, mouse IgG, respectively, and incubated for overnight at
4.degree. C. overnight. The antigen-antibody complex was
transferred into digestion buffer using a 100,000 NMWL filter unit.
Then, 4 .mu.g of trypsin (0.16 units) in digestion buffer was added
and incubated for 2 hours at 37.degree. C. After digestion, Protein
G beads were added to capture antibody and fragments bound by
antibody. Then, the protein G beads were washed once with PBS and
twice with 50 mM Ammonium Bicarbonate, and the captured complexes
were eluted with 30 .mu.l of elution buffer (10% Acetonitrile, 0.5%
Trifluoracetic Acid).
[0276] Eluents of the trypsin-digest complex were desalted by
pipetting the sample through ZipTip C18, washed with 0.1% TFA in
water, then eluted in 2 .mu.l of .alpha.-cyano matrix (10 mg/ml
.alpha.-cyano, 0.1% TFA, 50% Acetonitrile in water) and spotted on
a MALDI-TOF target. The MALDI-TOF (Voyager-DE.TM. STR, ABI) was
calibrated with Calibration mixture 2 (ABI). Spectra were obtained
at laser intensity between 1,500-1,800 units and acquisition mass
range 800-5,000 m/z.
[0277] MSD high bind plates (Meso Scale Diagnostics, Gaithersburg,
Md.) were coated, at 4.degree. C. overnight, with 5 11 of serially
diluted samples, from 100 to 0 .mu.g/ml, of different protein
reagents, including human IL-23, murine IL-23 and two
segment-swapped mutant proteins, 3220 and 3397. One-hundred and
fifty .mu.l of 5% MSD Blocker A buffer was added to each well and
incubated for 1 hr at room temperature. Plates were washed three
times with 0.1 M HEPES buffer, pH 7.4. These protein-charged ELISA
micro-wells were incubated with 25 .mu.l of 2 g/mL MSD Sulfo-TAG
labeled C1249, C1269, or CNTO 209 mAbs. After incubation for 2
hours with shaking at room temperature, plates were washed 3 times
with 0.1 M HEPES buffer (pH 7.4). MSD Read Buffer T was diluted
with distilled water (4 fold) and dispensed at a volume of 150
.mu.l/well and analyzed with a SECTOR imager 6000.
[0278] IL-23 Structural Analysis
[0279] FIG. 6 shows the structural model for human IL-23 based upon
the crystal structure of human IL-12 (pdb code 1f45). It is clear
that residues (H75, 179, E82, K83 and G86) are surface exposed and
clustered together. This arrangement is typical of conformational
epitopes for antibody binding. Therefore, this segment
(.sub.74IHQGLIFYEKLLG.sub.86) may constitute part of the binding
epitope for C1249 as well as species specificity determinants of
the epitope. Typical Ab/Ag binding buries approximately 1000
.ANG..sup.2 of surface area. This suggests that additional exposed
residues of the IL-23p19 in the vicinity of the above segment would
also be required for a typical binding site. Inspection of the
molecular model suggests that residues from the neighboring C helix
(residues L109, L110, 5113, Q114, L116, and Q117) are exposed and
would form an extended surface cluster together with the residues
in the 74-86 segment. Residues identified and proposed to be part
of the epitope for C1249 are labeled and shown in stick models in
FIG. 6. The p40 subunit is labeled 2 and the p19 subunit labeled
1.
[0280] The sequence for this part of the C helix is identical
between human and murine p19. It is plausible that as part of the
epitope these residues would contribute to the residual binding of
the human-to-murine swap mutants described above. Therefore, the
epitope may be comprised of two segments of p19 (74-85 and 108-118)
with the exposed residues (H75, 179, E82, K83, G86, L109, L110,
S113, Q114, L116, and Q117) to be most likely involved in direct
antibody interactions.
[0281] Mutagenesis analysis is performed with the IL-23p19 segment
spanning residues 108-118 in which the residues are selectively
changed individually and/or in groups (i.e., multiple changes).
IL-23 activity is monitored after the mutations are made as in the
experiments above using the IL-23p19 segment spanning amino acid
residues 74-86. The epitope is confirmed based on the change in
activity correlated with the various combinations of mutations.
[0282] For the purposes of this invention, 70-100% amino acid or
nucleotide sequence identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100 or any range or value therein) is determined using a
suitable computer algorithm, as known in the art.
[0283] It will be clear that the invention can be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
TABLE-US-00001 Sequence Tables SEQ ID NO: 1 (human IL-23p19
subunit) Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro
Trp Thr 1 5 10 15 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro
Ala Trp Thr Gln 20 25 30 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr
Leu Ala Trp Ser Ala His 35 40 45 Pro Leu Val Gly His Met Asp Leu
Arg Glu Glu Gly Asp Glu Glu Thr 50 55 60 Thr Asn Asp Val Pro His
Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln 65 70 75 80 Gly Leu Arg Asp
Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly 85 90 95 Leu Ile
Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 100 105 110
Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 115
120 125 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu
Thr 130 135 140 Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln
Arg Leu Leu 145 150 155 160 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln
Ala Phe Val Ala Val Ala 165 170 175 Ala Arg Val Phe Ala His Gly Ala
Ala Thr Leu Ser Pro 180 185 SEQ ID NO: 2-C1273 Heavy Chain
Nucleotide Sequence
Atgagcagtgaacacagacccctcaccatgaacttcgggctcagattgattttccttgtccttacttt
aaaaggtgtccagtgtgacgtgaacttggtggagtctgggggaggcttagtgaagcctggagggtccc
tgaaactctcctgtgcagcctctggattcactttcagtagctataccatgtcttgggttcgccagact
ccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttacacctactatccagacag
tgtgaagggccgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtc
tgaagtctgaggacacagccatgttttactgtacaagagataaccatgcttacgacaggggccctttc
tttgactactggggccaaggcgccactctcacagtctcctca SEQ ID NO: 3-C1273 Heavy
Chain Amino Acid Sequence (CDR sequences in boldface and underline)
MSSEHRPLTMNFGLRLIFLVLTLKGVQCDVNLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQT
PEKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMFYCTRDNHAYDRGPF
FDYWGQGATLTVSS SEQ ID NO: 4-C1273 Heavy Chain CDR1 Amino Acid
Sequence GFTFSSYTMS SEQ ID NO: 5-C1273 Heavy Chain CDR2 Amino Acid
Sequence TISSGGTYTYYPDSVKG SEQ ID NO: 6-C1273 Heavy Chain CDR3
Amino Acid Sequence DNHAYDRGPFFDY SEQ ID NO: 7-C1273 Light Chain
Nucleotide Sequence
Atggattcacaggcccaggttcttttgttactgctgctatgggtttctggtacctgtggggacattgt
gatgtcacagtccccatcctccctagttgtgtcagttggagagaaggttactatgagctgcaagtcca
gtcagaacctcttttataggagtaatcaaaagaaccacttggcctggtaccagcagaaaccagggcag
tctcctacactgctgatttactggacgtccactagggaatctggggtccctgatcgcttcacaggcag
tggatctgggacagatttcactctcaccatcagccgtgtgaaggctgaagacctggcagtttattact
gtcagcaatattatagctatcctccgacgttcggtggaggcaccaagctggaaatcaaa SEQ ID
NO: 8-C1273 Light Chain Amino Acid Sequence
MDSQAQVLLLLLLWVSGTCGDIVMSQSPSSLVVSVGEKVTMSCKSSQNLFYRSNQKNHLAWYQQKPGQ
SPTLLIYWTSTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYSYPPTFGGGTKLEIK
SEQ ID NO: 9-C1273 Light Chain CDR1 Amino Acid Sequence
KSSQNLFYRSNQKNHLA SEQ ID NO: 10-C1273 Light Chain CDR2 Amino Acid
Sequence WTSTRES SEQ ID NO: 11-C1273 Light Chain CDR3 Amino Acid
Sequence QQYYSYPPT SEQ ID NO: 12-C1269 Heavy Chain Nucleotide
Sequence
Atgagcagtgaacacagacccctcaccatgaacttcgggctcagattgattttccttgtcctgacttt
aaaaggtgtccagtgtgacgtgaacttggtggagtctgggggaggcttagtgaagcctggagggtccc
tgaaactctcctgtgcagcctctggattcactttcagtagctataccatgtcttgggttcgccagact
ccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttacacctactatccagacag
tgtgaagggccgattcaccatttccagagacaatgccaagaatacattgtatctgcaaatgagcagtc
tgaagtctgaggacacagccatcttttattgtacaagagataaccatgcttacgacaggggccctttc
tttgactcctggggccaaggcgccactctcacagtctcctca SEQ ID NO: 13-C1269
Heavy Chain Amino Acid Sequence
MSSEHRPLTMNFGLRLIFLVLTLKGVQCDVNLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQT
PEKRLEWVATISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAIFYCTRDNHAYDRGPF
FDSWGQGATLTVSS SEQ ID NO: 14-C1269 Heavy Chain CDR1 Amino Acid
Sequence GFTFSSYTMS SEQ ID NO: 15-C1269 Heavy Chain CDR2 Amino Acid
Sequence ISSGGTYTYYPDSVKG SEQ ID NO: 16-C1269 Heavy Chain CDR3
Amino Acid Sequence DNHAYDRGPFFDS SEQ ID NO: 17-C1269 Light Chain
Nucleotide Sequence
Atggattcacaggcccaggttcttatgttactgctgctatgggtttctggtacctgtggggacattgt
gatgtcacagtctccatcctccctagctgtgtcagttggagagaaggttactatgagctgcaagtcca
gtcagaacctcttttataggaataatcaaaagaactacttggcctggtaccagcagaaaccagggcag
tctcctacactgctgatttactggacgtccactagggagtctggggtccctgatcgcttcacaggcag
tggatctgggacagatttcactctcaccatcagccgtgtgaaggctgaagacctggcagtttattact
gtcagcaatattatagctatcctccgacgttcggtggaggcaccaagctggaaatcaaa SEQ ID
NO: 18-C1269 Light Chain Amino Acid Sequence
MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLAVSVGEKVTMSCKSSQNLFYRNNQKNYLAWYQQKPGQ
SPTLLIYWTSTRESGVPDRFTGSGSGTDFTLTISRVKAEDLAVYYCQQYYSYPPTFGGGTKLEIK
SEQ ID NO: 19-C1269 Light Chain CDR1 Amino Acid Sequence
KSSQNLFYRNNQKNYLA SEQ ID NO: 20-C1269 Light Chain CDR2 Amino Acid
Sequence YWTSTRES SEQ ID NO: 21-C1269 Light Chain CDR3 Amino Acid
Sequence QQYYSYPPT SEQ ID NO: 22-C1275 Heavy Chain Nucleotide
Sequence
Atgtacttgggactgaactgtgtattcatagtttttctcttaaaaggtgtccagagtgaagtgaacct
tgaggagtctggaggaggcttggtgcaacctggaagatccatgaaactctcctgtgttgcctctggat
tcactttcagtaactactggatgacctgggtccgccagtctccagagaaggggcttgagtgggttgct
gaaattagattgaaatctaataattatgcaacacattatgcggagtctgtgaaagggaggttcaccat
ctcaagagatgattccaaaagtagtgtctacctgcaaatgaacaacttaagagctgaagacactgcca
tttattactgtaccaggggggggggttacgacgtaggagcctggtttgcttactggggccaagggact
ctggtcactgtctctgca SEQ ID NO: 23-C1275 Heavy Chain Amino Acid
Sequence
MYLGLNCVFIVFLLKGVQSEVNLEESGGGLVQPGRSMKLSCVASGFTFSNYWMTWVRQSPEKGLEWVA
EIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTAIYYCTRGGGYDVGAWFAYWGQGT
LVTVSA SEQ ID NO: 24-C1275 Heavy Chain CDR1 Amino Acid Sequence
GFTFSNYWMT SEQ ID NO: 25-C1275 Heavy Chain CDR2 Amino Acid Sequence
EIRLKSNNYATHYAESVKG SEQ ID NO: 26-C1275 Heavy Chain CDR3 Amino Acid
Sequence GGGYDVGAWFAY SEQ ID NO: 27-C1275 Light Chain Nucleotide
Sequence
Atggagtcagacacactcctgctatgggtgctgctgctctgggttccaggctccactggtgacattgt
gctcacccaatctccagcttctttggctgtgtctctagggcagagagccaccatctcctgcagagcca
gtgaaaatgttgaatattatggcacaggtttaattcagtggtaccaacagaaaccaggacagccaccc
aaactcctcatctatgcttcatccaacgtagaatctggggtccctgccaggtttagtggcagtgggtc
tgggacagacttcagcctctacatccatcctgtggaggaggatgatattgcaatgtatttctgtcagc
aaagtaggaaggttccttcgacgttcggtggaggcaccaagctggaaatcaaa SEQ ID NO:
28-C1275 Light Chain Amino Acid Sequence
MESDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASENVEYYGTGLIQWYQQKPGQPP
KLLIYASSNVESGVPARFSGSGSGTDFSLYIHPVEEDDIAMYFCQQSRKVPSTFGGGTKLEIK SEQ
ID NO: 29-C1275 Light Chain CDR1 Amino Acid Sequence
RASENVEYYGTGLIQ SEQ ID NO: 30-C1275 Light Chain CDR2 Amino Acid
Sequence ASSNVES SEQ ID NO: 31-C1275 Light Chain CDR3 Amino Acid
Sequence QQSRKVPST SEQ ID NO: 32-C1249 Heavy Chain Nucleotide
Sequence
Atggtgttggggctgaagtgggttttctttgttgttttttatcaaggtgtgcattgtgaggtgcaact
tgttgagtctggtggaggattggtgcagcctaaaggatcattgaaactctcatgtgccgcctctggtt
tcaacttcaatacctatgccatgcactgggtctgccaggctccaggaaagggtttggaatggattggt
cgcataagaagtaaaagtcataattatgcaacagactatgccgatccagtgaaagacagattcaccat
ctccagagatgattcacaaggcttgctctatctgctaatgaacaacctgaaaactgaggacacagcca
tgtattactgtatgagggagggaatctatggtagttttgcttactggggccaagggactctggtcact
gtctctgca SEQ ID NO: 33-C1249 Heavy Chain Amino Acid Sequence
MVLGLKWVFFVVFYQGVHCEVQLVESGGGLVQPKGSLKLSCAASGFNFNTYAMHWVCQAPGKGLEWIG
RIRSKSHNYATDYADPVKDRFTISRDDSQGLLYLLMNNLKTEDTAMYYCMREGIYGSFAYWGQGTLVT
VSA SEQ ID NO: 34-C1249 Heavy Chain CDR1 Amino Acid Sequence
GFNFNTYAMH SEQ ID NO: 35-C1249 Heavy Chain CDR2 Amino Acid Sequence
IRSKSHNYATDYADPVKD SEQ ID NO: 36-C1249 Heavy Chain CDR3 Amino Acid
Sequence EGIYGSFAY SEQ ID NO: 37-C1249 Light Chain Nucleotide
Sequence
Atggagacagacacactcctgttatgggtactgctgctctgggttccaggttccactggtgacattgt
gctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggcca
gcaaaagtgtcagttcatctgcctatagttttttccactggtaccaacagaagccaggacagccaccc
aaactcctcatctatcttgcatccaacctacaatctggggtccctgccaggttcagtggcagtgggtc
tgggacagacttcaccctcaacatccatcctgtggaggcggaggatgctgcaacctattactgtcaac
acagtggggagcttccattcacgttcggctcggggacaaagttggaaataaaa SEQ ID NO:
38-C1249 Light Chain Amino Acid Sequence
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSSSAYSFFHWYQQKPGQPPKL
LIYLASNLQSGVPARFSGSGSGTDFTLNIHPVEAEDAATYYCQHSGELPFTFGSGTKLEIK SEQ
ID NO: 39-C1249 Light Chain CDR1 Amino Acid Sequence
CRASKSVSSSAYSFFH SEQ ID NO: 40-C1249 Light Chain Amino Acid
Sequence LASNLQS SEQ ID NO: 41-C1249 Light Chain Amino Acid
Sequence CQHSGELPFT
Sequence CWU 1
1
411189PRTHomo sapiens 1Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu
Leu Leu Pro Trp Thr1 5 10 15Ala Gln Gly Arg Ala Val Pro Gly Gly Ser
Ser Pro Ala Trp Thr Gln 20 25 30Cys Gln Gln Leu Ser Gln Lys Leu Cys
Thr Leu Ala Trp Ser Ala His 35 40 45Pro Leu Val Gly His Met Asp Leu
Arg Glu Glu Gly Asp Glu Glu Thr 50 55 60Thr Asn Asp Val Pro His Ile
Gln Cys Gly Asp Gly Cys Asp Pro Gln65 70 75 80Gly Leu Arg Asp Asn
Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly 85 90 95Leu Ile Phe Tyr
Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 100 105 110Pro Ser
Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 115 120
125Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Gln Thr
130 135 140Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg
Leu Leu145 150 155 160Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala
Phe Val Ala Val Ala 165 170 175Ala Arg Val Phe Ala His Gly Ala Ala
Thr Leu Ser Pro 180 1852450DNAHomo sapiens 2atgagcagtg aacacagacc
cctcaccatg aacttcgggc tcagattgat tttccttgtc 60cttactttaa aaggtgtcca
gtgtgacgtg aacttggtgg agtctggggg aggcttagtg 120aagcctggag
ggtccctgaa actctcctgt gcagcctctg gattcacttt cagtagctat
180accatgtctt gggttcgcca gactccggag aagaggctgg agtgggtcgc
aaccattagt 240agtggtggta cttacaccta ctatccagac agtgtgaagg
gccgattcac catctccaga 300gacaatgcca agaacaccct gtacctgcaa
atgagcagtc tgaagtctga ggacacagcc 360atgttttact gtacaagaga
taaccatgct tacgacaggg gccctttctt tgactactgg 420ggccaaggcg
ccactctcac agtctcctca 4503150PRTHomo sapiens 3Met Ser Ser Glu His
Arg Pro Leu Thr Met Asn Phe Gly Leu Arg Leu1 5 10 15Ile Phe Leu Val
Leu Thr Leu Lys Gly Val Gln Cys Asp Val Asn Leu 20 25 30Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu 35 40 45Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp 50 55 60Val
Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Ser65 70 75
80Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe
85 90 95Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met
Ser 100 105 110Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys Thr
Arg Asp Asn 115 120 125His Ala Tyr Asp Arg Gly Pro Phe Phe Asp Tyr
Trp Gly Gln Gly Ala 130 135 140Thr Leu Thr Val Ser Ser145
150410PRTHomo sapiens 4Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser1 5
10517PRTHomo sapiens 5Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr
Pro Asp Ser Val Lys1 5 10 15Gly613PRTHomo sapiens 6Asp Asn His Ala
Tyr Asp Arg Gly Pro Phe Phe Asp Tyr1 5 107399DNAHomo sapiens
7atggattcac aggcccaggt tcttttgtta ctgctgctat gggtttctgg tacctgtggg
60gacattgtga tgtcacagtc cccatcctcc ctagttgtgt cagttggaga gaaggttact
120atgagctgca agtccagtca gaacctcttt tataggagta atcaaaagaa
ccacttggcc 180tggtaccagc agaaaccagg gcagtctcct acactgctga
tttactggac gtccactagg 240gaatctgggg tccctgatcg cttcacaggc
agtggatctg ggacagattt cactctcacc 300atcagccgtg tgaaggctga
agacctggca gtttattact gtcagcaata ttatagctat 360cctccgacgt
tcggtggagg caccaagctg gaaatcaaa 3998133PRTHomo sapiens 8Met Asp Ser
Gln Ala Gln Val Leu Leu Leu Leu Leu Leu Trp Val Ser1 5 10 15Gly Thr
Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Val 20 25 30Val
Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Asn 35 40
45Leu Phe Tyr Arg Ser Asn Gln Lys Asn His Leu Ala Trp Tyr Gln Gln
50 55 60Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile Tyr Trp Thr Ser Thr
Arg65 70 75 80Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
Gly Thr Asp 85 90 95Phe Thr Leu Thr Ile Ser Arg Val Lys Ala Glu Asp
Leu Ala Val Tyr 100 105 110Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Pro
Thr Phe Gly Gly Gly Thr 115 120 125Lys Leu Glu Ile Lys
130917PRTHomo sapiens 9Lys Ser Ser Gln Asn Leu Phe Tyr Arg Ser Asn
Gln Lys Asn His Leu1 5 10 15Ala107PRTHomo sapiens 10Trp Thr Ser Thr
Arg Glu Ser1 5119PRTHomo sapiens 11Gln Gln Tyr Tyr Ser Tyr Pro Pro
Thr1 512450DNAHomo sapiens 12atgagcagtg aacacagacc cctcaccatg
aacttcgggc tcagattgat tttccttgtc 60ctgactttaa aaggtgtcca gtgtgacgtg
aacttggtgg agtctggggg aggcttagtg 120aagcctggag ggtccctgaa
actctcctgt gcagcctctg gattcacttt cagtagctat 180accatgtctt
gggttcgcca gactccggag aagaggctgg agtgggtcgc aaccattagt
240agtggtggta cttacaccta ctatccagac agtgtgaagg gccgattcac
catttccaga 300gacaatgcca agaatacatt gtatctgcaa atgagcagtc
tgaagtctga ggacacagcc 360atcttttatt gtacaagaga taaccatgct
tacgacaggg gccctttctt tgactcctgg 420ggccaaggcg ccactctcac
agtctcctca 45013150PRTHomo sapiens 13Met Ser Ser Glu His Arg Pro
Leu Thr Met Asn Phe Gly Leu Arg Leu1 5 10 15Ile Phe Leu Val Leu Thr
Leu Lys Gly Val Gln Cys Asp Val Asn Leu 20 25 30Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu 35 40 45Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp 50 55 60Val Arg Gln
Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Thr Ile Ser65 70 75 80Ser
Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe 85 90
95Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser
100 105 110Ser Leu Lys Ser Glu Asp Thr Ala Ile Phe Tyr Cys Thr Arg
Asp Asn 115 120 125His Ala Tyr Asp Arg Gly Pro Phe Phe Asp Ser Trp
Gly Gln Gly Ala 130 135 140Thr Leu Thr Val Ser Ser145
1501410PRTHomo sapiens 14Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser1 5
101516PRTHomo sapiens 15Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro
Asp Ser Val Lys Gly1 5 10 151613PRTHomo sapiens 16Asp Asn His Ala
Tyr Asp Arg Gly Pro Phe Phe Asp Ser1 5 1017399DNAHomo sapiens
17atggattcac aggcccaggt tcttatgtta ctgctgctat gggtttctgg tacctgtggg
60gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact
120atgagctgca agtccagtca gaacctcttt tataggaata atcaaaagaa
ctacttggcc 180tggtaccagc agaaaccagg gcagtctcct acactgctga
tttactggac gtccactagg 240gagtctgggg tccctgatcg cttcacaggc
agtggatctg ggacagattt cactctcacc 300atcagccgtg tgaaggctga
agacctggca gtttattact gtcagcaata ttatagctat 360cctccgacgt
tcggtggagg caccaagctg gaaatcaaa 39918133PRTHomo sapiens 18Met Asp
Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Trp Val Ser1 5 10 15Gly
Thr Cys Gly Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala 20 25
30Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Asn
35 40 45Leu Phe Tyr Arg Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
Gln 50 55 60Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile Tyr Trp Thr Ser
Thr Arg65 70 75 80Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp 85 90 95Phe Thr Leu Thr Ile Ser Arg Val Lys Ala Glu
Asp Leu Ala Val Tyr 100 105 110Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro
Pro Thr Phe Gly Gly Gly Thr 115 120 125Lys Leu Glu Ile Lys
1301917PRTHomo sapiens 19Lys Ser Ser Gln Asn Leu Phe Tyr Arg Asn
Asn Gln Lys Asn Tyr Leu1 5 10 15Ala208PRTHomo sapiens 20Tyr Trp Thr
Ser Thr Arg Glu Ser1 5219PRTHomo sapiens 21Gln Gln Tyr Tyr Ser Tyr
Pro Pro Thr1 522426DNAHomo sapiens 22atgtacttgg gactgaactg
tgtattcata gtttttctct taaaaggtgt ccagagtgaa 60gtgaaccttg aggagtctgg
aggaggcttg gtgcaacctg gaagatccat gaaactctcc 120tgtgttgcct
ctggattcac tttcagtaac tactggatga cctgggtccg ccagtctcca
180gagaaggggc ttgagtgggt tgctgaaatt agattgaaat ctaataatta
tgcaacacat 240tatgcggagt ctgtgaaagg gaggttcacc atctcaagag
atgattccaa aagtagtgtc 300tacctgcaaa tgaacaactt aagagctgaa
gacactgcca tttattactg taccaggggg 360gggggttacg acgtaggagc
ctggtttgct tactggggcc aagggactct ggtcactgtc 420tctgca
42623142PRTHomo sapiens 23Met Tyr Leu Gly Leu Asn Cys Val Phe Ile
Val Phe Leu Leu Lys Gly1 5 10 15Val Gln Ser Glu Val Asn Leu Glu Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Arg Ser Met Lys Leu Ser
Cys Val Ala Ser Gly Phe Thr Phe 35 40 45Ser Asn Tyr Trp Met Thr Trp
Val Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60Glu Trp Val Ala Glu Ile
Arg Leu Lys Ser Asn Asn Tyr Ala Thr His65 70 75 80Tyr Ala Glu Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95Lys Ser Ser
Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr 100 105 110Ala
Ile Tyr Tyr Cys Thr Arg Gly Gly Gly Tyr Asp Val Gly Ala Trp 115 120
125Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 130 135
1402410PRTHomo sapiens 24Gly Phe Thr Phe Ser Asn Tyr Trp Met Thr1 5
102519PRTHomo sapiens 25Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr
His Tyr Ala Glu Ser1 5 10 15Val Lys Gly2612PRTHomo sapiens 26Gly
Gly Gly Tyr Asp Val Gly Ala Trp Phe Ala Tyr1 5 1027393DNAHomo
sapiens 27atggagtcag acacactcct gctatgggtg ctgctgctct gggttccagg
ctccactggt 60gacattgtgc tcacccaatc tccagcttct ttggctgtgt ctctagggca
gagagccacc 120atctcctgca gagccagtga aaatgttgaa tattatggca
caggtttaat tcagtggtac 180caacagaaac caggacagcc acccaaactc
ctcatctatg cttcatccaa cgtagaatct 240ggggtccctg ccaggtttag
tggcagtggg tctgggacag acttcagcct ctacatccat 300cctgtggagg
aggatgatat tgcaatgtat ttctgtcagc aaagtaggaa ggttccttcg
360acgttcggtg gaggcaccaa gctggaaatc aaa 39328131PRTHomo sapiens
28Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala 20 25 30Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Glu Asn 35 40 45Val Glu Tyr Tyr Gly Thr Gly Leu Ile Gln Trp Tyr Gln
Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ala Ser Ser
Asn Val Glu Ser65 70 75 80Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Ser 85 90 95Leu Tyr Ile His Pro Val Glu Glu Asp
Asp Ile Ala Met Tyr Phe Cys 100 105 110Gln Gln Ser Arg Lys Val Pro
Ser Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125Glu Ile Lys
1302915PRTHomo sapiens 29Arg Ala Ser Glu Asn Val Glu Tyr Tyr Gly
Thr Gly Leu Ile Gln1 5 10 15307PRTHomo sapiens 30Ala Ser Ser Asn
Val Glu Ser1 5319PRTHomo sapiens 31Gln Gln Ser Arg Lys Val Pro Ser
Thr1 532417DNAHomo sapiens 32atggtgttgg ggctgaagtg ggttttcttt
gttgtttttt atcaaggtgt gcattgtgag 60gtgcaacttg ttgagtctgg tggaggattg
gtgcagccta aaggatcatt gaaactctca 120tgtgccgcct ctggtttcaa
cttcaatacc tatgccatgc actgggtctg ccaggctcca 180ggaaagggtt
tggaatggat tggtcgcata agaagtaaaa gtcataatta tgcaacagac
240tatgccgatc cagtgaaaga cagattcacc atctccagag atgattcaca
aggcttgctc 300tatctgctaa tgaacaacct gaaaactgag gacacagcca
tgtattactg tatgagggag 360ggaatctatg gtagttttgc ttactggggc
caagggactc tggtcactgt ctctgca 41733139PRTHomo sapiens 33Met Val Leu
Gly Leu Lys Trp Val Phe Phe Val Val Phe Tyr Gln Gly1 5 10 15Val His
Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro
Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe 35 40
45Asn Thr Tyr Ala Met His Trp Val Cys Gln Ala Pro Gly Lys Gly Leu
50 55 60Glu Trp Ile Gly Arg Ile Arg Ser Lys Ser His Asn Tyr Ala Thr
Asp65 70 75 80Tyr Ala Asp Pro Val Lys Asp Arg Phe Thr Ile Ser Arg
Asp Asp Ser 85 90 95Gln Gly Leu Leu Tyr Leu Leu Met Asn Asn Leu Lys
Thr Glu Asp Thr 100 105 110Ala Met Tyr Tyr Cys Met Arg Glu Gly Ile
Tyr Gly Ser Phe Ala Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ala 130 1353410PRTHomo sapiens 34Gly Phe Asn Phe Asn Thr
Tyr Ala Met His1 5 103518PRTHomo sapiens 35Ile Arg Ser Lys Ser His
Asn Tyr Ala Thr Asp Tyr Ala Asp Pro Val1 5 10 15Lys Asp369PRTHomo
sapiens 36Glu Gly Ile Tyr Gly Ser Phe Ala Tyr1 537393DNAHomo
sapiens 37atggagacag acacactcct gttatgggta ctgctgctct gggttccagg
ttccactggt 60gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca
gagggccacc 120atctcatgca gggccagcaa aagtgtcagt tcatctgcct
atagtttttt ccactggtac 180caacagaagc caggacagcc acccaaactc
ctcatctatc ttgcatccaa cctacaatct 240ggggtccctg ccaggttcag
tggcagtggg tctgggacag acttcaccct caacatccat 300cctgtggagg
cggaggatgc tgcaacctat tactgtcaac acagtgggga gcttccattc
360acgttcggct cggggacaaa gttggaaata aaa 39338131PRTHomo sapiens
38Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala 20 25 30Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser
Lys Ser 35 40 45Val Ser Ser Ser Ala Tyr Ser Phe Phe His Trp Tyr Gln
Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser
Asn Leu Gln Ser65 70 75 80Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr 85 90 95Leu Asn Ile His Pro Val Glu Ala Glu
Asp Ala Ala Thr Tyr Tyr Cys 100 105 110Gln His Ser Gly Glu Leu Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125Glu Ile Lys
1303916PRTHomo sapiens 39Cys Arg Ala Ser Lys Ser Val Ser Ser Ser
Ala Tyr Ser Phe Phe His1 5 10 15407PRTHomo sapiens 40Leu Ala Ser
Asn Leu Gln Ser1 54110PRTHomo sapiens 41Cys Gln His Ser Gly Glu Leu
Pro Phe Thr1 5 10
* * * * *
References