U.S. patent application number 16/816814 was filed with the patent office on 2020-09-17 for manufacturing methods for producing anti-il12/il23 antibody compositions.
The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Kristopher Barnthouse, Subinay Ganguly, Maarten Groeneveld, Manuel Lopez, Michael Nedved, Kevin D. Smith.
Application Number | 20200291107 16/816814 |
Document ID | / |
Family ID | 1000004747393 |
Filed Date | 2020-09-17 |
United States Patent
Application |
20200291107 |
Kind Code |
A1 |
Barnthouse; Kristopher ; et
al. |
September 17, 2020 |
Manufacturing Methods for Producing Anti-IL12/IL23 Antibody
Compositions
Abstract
Methods of manufacture for producing anti-IL-12/IL-23p40
antibodies, e.g., the anti-IL-12/IL-23p40 antibody ustekinumab, in
CHO and specific pharmaceutical compositions of the antibody are
useful in treating various diseases.
Inventors: |
Barnthouse; Kristopher;
(Pottstown, PA) ; Ganguly; Subinay; (Newtown,
PA) ; Groeneveld; Maarten; (Oegstgeest, NL) ;
Lopez; Manuel; (Devon, PA) ; Nedved; Michael;
(Downingtown, PA) ; Smith; Kevin D.;
(Phialdelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Family ID: |
1000004747393 |
Appl. No.: |
16/816814 |
Filed: |
March 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62818359 |
Mar 14, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/14 20130101;
C07K 16/244 20130101; C12N 5/0682 20130101; C07K 1/18 20130101;
C07K 2317/31 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C07K 1/18 20060101 C07K001/18 |
Claims
1. An isolated anti-IL-12/IL-23p40 antibody comprising amino acid
sequences selected from the group consisting of: (i) a heavy chain
(HC) comprising the amino acid sequence of SEQ ID NO: 10 and a
light chain (LC) comprising the amino acid sequence of SEQ ID
NO:11; (ii) a heavy chain variable domain amino acid sequence of
SEQ ID NO:7 and a light chain variable domain amino acid sequence
of SEQ ID NO:8; and (iii) heavy chain CDR amino acid sequences of
SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, and light chain CDR
amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,
wherein the anti-IL-12/IL-23p40 antibody is expressed in a Chinese
Hamster Ovary cell (CHO cell).
2. The anti-IL-12/IL-23p40 antibody of claim 1, wherein the
oligosaccharide profile of the anti-IL-12/IL-23p40 antibody
comprises total neutral oligosaccharide species >99.0% and total
charged oligosaccharide species <1.0%
3. The anti-IL-12/IL-23p40 antibody of claim 2, wherein the
oligosaccharide profile of the anti-IL-12/IL-23p40 antibody further
comprises individual neutral oligosaccharide species G0F>70.0%,
G1F<20.0%, and G2F<5.0%.
4. The anti-IL-12/IL-23p40 antibody of claim 2, wherein the peak 3
area % of the capillary isoelectric focusing (cIEF)
electropherogram of the anti-IL-12/IL-23p40 antibody is
>70.0%.
5. The anti-IL-12/IL-23p40 antibody of claim 2, wherein the
anti-IL-12/IL-23p40 antibody has no disialylated glycan species as
determined by High Performance Liquid Chromatography (HPLC) or
Reduced Mass Analysis (RMA).
6. The anti-IL-12/IL-23p40 antibody of claim 2, wherein the
anti-IL-12/IL-23p40 antibody has a longer half-life compared to an
anti-IL-12/IL-23p40 antibody with identical amino acid heavy chain
and light chain sequences expressed in Sp2/0 cells.
7. The anti-IL-12/IL-23p40 antibody of claim 2, wherein the
anti-IL-12/IL-23p40 antibody comprises a follow-on biologic.
8. A method of manufacture for producing an anti-IL-12/IL-23p40
antibody comprising amino acid sequences selected from the group
consisting of: (i) a heavy chain (HC) comprising the amino acid
sequence of SEQ ID NO: 10 and a light chain (LC) comprising the
amino acid sequence of SEQ ID NO: 11; (ii) a heavy chain variable
domain amino acid sequence of SEQ ID NO:7 and a light chain
variable domain amino acid sequence of SEQ ID NO:8; and (iii) heavy
chain CDR amino acid sequences of SEQ ID NO: 1, SEQ ID NO:2, and
SEQ ID NO:3, and light chain CDR amino acid sequences of SEQ ID
NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein the anti-IL-12/IL-23p40
antibody is produced by the steps of: a. culturing a Chinese
Hamster Ovary cell (CHO cell) with nucleotides encoding the
anti-IL-12/IL-23p40 antibody; b. expressing the anti-IL-12/IL-23p40
antibody in the CHO cell; and c. purifying the anti-IL-12/IL-23p40
antibody.
9. The method of manufacture of claim 8, wherein the
oligosaccharide profile of the anti-IL-12/IL-23p40 antibody
comprises total neutral oligosaccharide species >99.0% and total
charged oligosaccharide species <1.0%.
10. The method of manufacture of claim 9, wherein the
oligosaccharide profile of the anti-IL-12/IL-23p40 antibody further
comprises individual neutral oligosaccharide species G0F>70.0%,
G1F<20.0%, and G2F<5.0%.
11. The method of manufacture of claim 9, wherein the peak 3 area %
of the capillary isoelectric focusing (cIEF) electropherogram of
the anti-IL-12/IL-23p40 antibody is >70.0%.
12. The method of manufacture of claim 9, wherein the
anti-IL-12/IL-23p40 antibody has no disialylated glycan species as
determined by High Performance Liquid Chromatography (HPLC) or
Reduced Mass Analysis (RMA).
13. The method of manufacture of claim 9, wherein the
anti-IL-12/IL-23p40 antibody has a longer half-life compared to an
anti-IL-12/IL-23p40 antibody with identical amino acid heavy chain
and light chain sequences expressed in Sp2/0 cells.
14. The method of manufacture of claim 9, wherein the
anti-IL-12/IL-23p40 antibody is a follow-on biologic.
15. A composition comprising an anti-IL-12/IL-23p40 antibody
comprising amino acid sequences selected from the group consisting
of: (i) a heavy chain (HC) comprising the amino acid sequence of
SEQ ID NO: 10 and a light chain (LC) comprising the amino acid
sequence of SEQ ID NO: 11; (ii) a heavy chain variable domain amino
acid sequence of SEQ ID NO:7 and a light chain variable domain
amino acid sequence of SEQ ID NO:8; and (iii) heavy chain CDR amino
acid sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, and
light chain CDR amino acid sequences of SEQ ID NO:4, SEQ ID NO:5,
and SEQ ID NO:6, wherein the anti-IL-12/IL-23p40 antibody is
expressed in a Chinese Hamster Ovary cell (CHO cell).
16. The composition of claim 15, wherein the oligosaccharide
profile of the anti-IL-12/IL-23p40 antibody comprises total neutral
oligosaccharide species >99.0% and total charged oligosaccharide
species <1.0%.
17. The composition of claim 16, wherein the oligosaccharide
profile of the anti-IL-12/IL-23p40 antibody further comprises
individual neutral oligosaccharide species G0F>70.0%,
G1F<20.0%, and G2F<5.0%.
18. The composition of claim 16, wherein the peak 3 area % of the
capillary isoelectric focusing (cIEF) electropherogram of the
anti-IL-12/IL-23p40 antibody is >70.0%.
19. The composition of claim 16, wherein the anti-IL-12/IL-23p40
antibody has no disialylated glycan species as determined by High
Performance Liquid Chromatography (HPLC).
20. The composition of claim 16, wherein the anti-IL-12/IL-23p40
antibody has a longer half-life compared to an anti-IL-12/IL-23p40
antibody with identical amino acid heavy chain and light chain
sequences expressed in Sp2/0 cells.
21. The composition of claim 16, wherein the anti-IL-12/IL-23p40
antibody is a follow-on biologic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/818,359, filed Mar. 14, 2019, the entire
contents of which are incorporated herein by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application contains a sequence listing, which is
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name "JBI6056USNP1SEQLIST.txt" creation date of
Mar. 5, 2020 and having a size of 14,000 bytes. The sequence
listing submitted via EFS-Web is part of the specification and is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of manufacture for
producing anti-IL-12/IL-23p40 antibodies, e.g., the
anti-IL-12/IL-23p40 antibody ustekinumab, and specific
pharmaceutical compositions of the antibody.
BACKGROUND OF THE INVENTION
[0004] 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.
[0005] 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).
[0006] 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). IL-12 has also
been shown to play a critical role in the pathogenesis of SLE in
two independent mouse models of systemic lupus erythematosus
(Kikawada et al. 2003; Dai et al. 2007.
[0007] Systemic lupus erythematosus (SLE) is a complex, chronic,
heterogeneous autoimmune disease of unknown etiology that can
affect almost any organ system, and which follows a waxing and
waning disease course. Systemic lupus erythematosus occurs much
more often in women than in men, up to 9 times more frequently in
some studies, and often appears during the child-bearing years
between ages 15 and 45. This disease is more prevalent in
Afro-Caribbean, Asian, and Hispanic populations. In SLE, the immune
system attacks the body's cells and tissue, resulting in
inflammation and tissue damage which can harm the heart, joints,
skin, lungs, blood vessels, liver, kidneys and nervous system.
About half of the subjects diagnosed with SLE present with
organ-threatening disease, but it can take several years to
diagnose subjects who do not present with organ involvement. Some
of the primary complaints of newly diagnosed lupus patients are
arthralgia (62%) and cutaneous symptoms (new photosensitivity;
20%), followed by persistent fever and malaise. The estimated
annual incidence of lupus varies from 1.8 to 7.6 cases per 100,000
and the worldwide prevalence ranges from 14 to 172 cases per
100,000 people. Patients with mild disease have mostly skin rashes
and joint pain and require less aggressive therapy; regimens
include nonsteroidal anti-inflammatory drugs (NSAIDs),
anti-malarials (e.g., hydroxychloroquine, chloroquine, or
quinacrine) and/or low dose corticosteroids. With more severe
disease patients may experience a variety of serious conditions
depending on the organ systems involved, including lupus nephritis
with potential renal failure, endocarditis or myocarditis,
pneumonitis, pregnancy complications, stroke, neurological
complications, vasculitis and cytopenias with associated risks of
bleeding or infection. Common treatments for more severe disease
include immunomodulatory agents, such as methotrexate (MTX),
azathioprine, cyclophosphamide, cyclosporine, high dose
corticosteroids, biologic B cell cytotoxic agents or B cell
modulators, and other immunomodulators. Patients with serious SLE
have a shortening of life expectancy by 10 to 30 years, largely due
to the complications of the disease, of standard of care therapy,
and/or accelerated atherosclerosis. In addition, SLE has a
substantial impact on quality of life, work productivity, and
healthcare expenditures. Existing therapies for SLE are generally
either cytotoxic or immunomodulatory and may have notable safety
risks. Newer treatments for SLE have provided only modest benefits
over standard of care therapy. Thus, there is a large unmet need
for new alternative treatments that can provide significant benefit
in this disease without incurring a high safety risk.
SUMMARY OF THE INVENTION
[0008] The embodiments of the invention are defined, respectively,
by the independent and dependent claims appended hereto, which for
the sake of brevity are incorporated by reference herein. Other
embodiments, features, and advantages of the various aspects of the
invention are apparent from the detailed description below taken in
conjunction with the appended drawing figures.
[0009] In certain embodiments, the present invention provides
anti-IL-12/IL-23p40 antibodies expressed in Chinese Hamster Ovary
cells (CHO cells). The "anti-IL-12/IL-23p40 Antibodies" defined by
the invention comprise antibodies having the amino acid sequences
selected from the group consisting of: (i) a heavy chain (HC)
comprising the amino acid sequence of SEQ ID NO: 10 and a light
chain (LC) comprising the amino acid sequence of SEQ ID NO: 11;
(ii) a heavy chain variable domain amino acid sequence of SEQ ID
NO:7 and a light chain variable domain amino acid sequence of SEQ
ID NO:8; and (iii) heavy chain CDR amino acid sequences of SEQ ID
NO: 1, SEQ ID NO:2, and SEQ ID NO:3, and light chain CDR amino acid
sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, expressed
in Chinese Hamster Ovary cells (CHO cells).
[0010] In certain embodiments, the oligosaccharide profile of the
anti-IL-12/IL-23p40 Antibodies comprises total neutral
oligosaccharide species >99.0% and total charged oligosaccharide
species <1.0%. In other embodiments, (i) the oligosaccharide
profile of the anti-IL-12/IL-23p40 Antibodies comprises total
neutral oligosaccharide species >99.0%, total charged
oligosaccharide species <1.0%, and individual neutral
oligosaccharide species G0F>70.0%, G1F<20.0%, and
G2F<5.0%; (ii) the oligosaccharide profile comprising total
neutral oligosaccharide species >99.0% and total charged
oligosaccharide species <1.0% and the peak 3 area % of the
capillary isoelectric focusing (cIEF) electropherogram of the
anti-IL-12/IL-23p40 Antibodies is >70.0%; (iii) the
anti-IL-12/IL-23p40 Antibodies have no disialylated glycan species
as determined by High Performance Liquid Chromatography (HPLC) or
Reduced Mass Analysis (RMA); (iv) the anti-IL-12/IL-23p40
Antibodies have a longer half-life compared to anti-IL-12/IL-23p40
antibodies expressed in Sp2/0 cells; and/or (v) the
anti-IL-12/IL-23p40 antibodies are a follow-on biologic (antibodies
relying on the regulatory approval of and/or data generated with
ustekinumab) to ustekinumab (marketed by Janssen Biotech, Inc. as
Stelara.RTM.).
[0011] In certain embodiments, the present invention provides a
method of manufacture for producing anti-IL-12/IL-23p40 Antibodies
comprising: a. culturing Chinese Hamster Ovary cells (CHO cells);
b. expressing the anti-IL-12/IL-23p40 antibodies in the CHO cells;
and, c. purifying the anti-IL-12/IL-23p40 antibodies, wherein (i)
the oligosaccharide profile of the anti-IL-12/IL-23p40 Antibodies
comprises total neutral oligosaccharide species >99.0% and total
charged oligosaccharide species <1.0%; (ii) the oligosaccharide
profile of the anti-IL-12/IL-23p40 Antibodies comprises total
neutral oligosaccharide species >99.0%, total charged
oligosaccharide species <1.0%, and individual neutral
oligosaccharide species G0F>70.0%, G1F<20.0%, and
G2F<5.0%; (iii) the oligosaccharide profile comprising total
neutral oligosaccharide species >99.0% and total charged
oligosaccharide species <1.0% and the peak 3 area % of the
capillary isoelectric focusing (cIEF) electropherogram of the
anti-IL-12/IL-23p40 Antibodies is >70.0%; (iv) the
anti-IL-12/IL-23p40 Antibodies have no disialylated glycan species
as determined by High Performance Liquid Chromatography (HPLC) or
Reduced Mass Analysis (RMA); (v) the anti-IL-12/IL-23p40 Antibodies
have a longer half-life compared to anti-IL-12/IL-23p40 antibodies
expressed in Sp2/0 cells; and/or (vi) the anti-IL-12/IL-23p40
antibodies are a follow-on biologic to ustekinumab.
[0012] In certain embodiments, the present invention provides a
composition comprising anti-IL-12/IL-23p40 Antibodies, wherein (i)
the oligosaccharide profile of the anti-IL-12/IL-23p40 Antibodies
comprises total neutral oligosaccharide species >99.0% and total
charged oligosaccharide species <1.0%; (ii) the oligosaccharide
profile of the anti-IL-12/IL-23p40 Antibodies comprises total
neutral oligosaccharide species >99.0%, total charged
oligosaccharide species <1.0%, and individual neutral
oligosaccharide species G0F>70.0%, G1F<20.0%, and
G2F<5.0%; (iii) the oligosaccharide profile comprising total
neutral oligosaccharide species >99.0% and total charged
oligosaccharide species <1.0% and the peak 3 area % of the
capillary isoelectric focusing (cIEF) electropherogram of the
anti-IL-12/IL-23p40 Antibodies is >70.0%; (iv) the
anti-IL-12/IL-23p40 Antibodies have no disialylated glycan species
as determined by High Performance Liquid Chromatography (HPLC) or
Reduced Mass Analysis (RMA); (v) the anti-IL-12/IL-23p40 Antibodies
have a longer half-life compared to anti-IL-12/IL-23p40 antibodies
expressed in Sp2/0 cells; and/or (vi) the anti-IL-12/IL-23p40
antibodies are a follow-on biologic to ustekinumab.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows an overview of the 10 stages of the ustekinumab
manufacturing process.
[0014] FIG. 2 shows a flow diagram of Stage 1 manufacturing process
for the preculture and expansion steps, including the in-process
controls and process monitoring tests.
[0015] FIG. 3 shows a flow diagram of Stage 2 manufacturing process
steps, including the in-process controls and process monitoring
tests.
[0016] FIG. 4 shows a representative HPLC chromatogram for
oligosaccharide analysis of ustekinumab produced in Sp2/0
cells.
[0017] FIG. 5 shows a representative deconvoluted mass spectrum for
IRMA analysis of ustekinumab produced in Sp2/0 cells.
[0018] FIG. 6 shows a representative cIEF electropherogram profile
of ustekinumab expressed in Sp2/0 cells. A graphic representing the
general relationship between cIEF peaks and decreasing negative
charge/degree of sialylation is also shown and Peaks A, B, 1, 2, 3,
and C are labeled.
[0019] FIG. 7 shows a diagrammatic overview of some of the primary
N-linked oligosaccharide species in ustekinumab IgG. The role of
some of the enzymes in the glycosylation maturation process and
role of some divalent cations (e.g. Mn.sup.2+ as a co-factor and
Cu.sup.2+ as an inhibitor of GalTI) are also shown (see, e.g.,
Biotechnol Bioeng. 2007 Feb. 15; 96(3):538-49; Curr Drug Targets.
2008 April; 9(4):292-309; J Biochem Mol Biol. 2002 May 31;
35(3):330-6). Note that species with terminal sialic acid (S1 and
S2) are charged species and species lacking the terminal sialic
acid (G0F, G1F, and G2F) are neutral species, but generation of
charged species depends on the presence of the galactose in G1F and
G2F added by the GalT1 enzyme.
[0020] FIG. 8 shows a representative HPLC chromatogram for
oligosaccharide analysis of ustekinumab produced in CHO cells. Hash
marks indicate all peaks above baseline identified by the analysis
software and brackets with labels indicate groups of peaks
representing Total Neutral, Total Charged, and Monosialylated
oligosaccharide species.
[0021] FIG. 9 shows a representative cIEF electropherogram profile
of ustekinumab expressed in CHO cells. A graphic representing the
general relationship between cIEF peaks and decreasing negative
charge/degree of sialylation is also shown and Peaks 1, 2, 3, and C
are labeled.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] As used herein, an "anti-IL-12 antibody," "anti-IL-23
antibody," "anti-IL-12/23p40 antibody," "anti-IL-12/IL-23p40
antibody," "IL-12/23p40 antibody," "IL-12/IL-23p40 antibody,"
""antibody portion," or "antibody fragment" and/or "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-12
and/or 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-12/23 activity or binding, or with
IL-12/23 receptor activity or binding, in vitro, in situ and/or in
vivo. As a non-limiting example, a suitable anti-IL-12/23p40
antibody, specified portion or variant of the present invention can
bind at least one IL-12/23 molecule, or specified portions,
variants or domains thereof. A suitable anti-IL-12/23p40 antibody,
specified portion, or variant can also optionally affect at least
one of IL-12/23 activity or function, such as but not limited to,
RNA, DNA or protein synthesis, IL-12/23 release, IL-12/23 receptor
signaling, membrane IL-12/23 cleavage, IL-12/23 activity, IL-12/23
production and/or synthesis.
[0023] As used herein, the terms "antibody" or "antibodies",
include biosimilar antibody molecules approved under the Biologics
Price Competition and Innovation Act of 2009 (BPCI Act) and similar
laws and regulations globally. Under the BPCI Act, an antibody may
be demonstrated to be biosimilar if data show that it is "highly
similar" to the reference product notwithstanding minor differences
in clinically inactive components and are "expected" to produce the
same clinical result as the reference product in terms of safety,
purity and potency (Endocrine Practice: February 2018, Vol. 24, No.
2, pp. 195-204). These biosimilar antibody molecules are provided
an abbreviated approval pathway, whereby the applicant relies upon
the innovator reference product's clinical data to secure
regulatory approval. Compared to the original innovator reference
antibody that was FDA approved based on successful clinical trials,
a biosimilar antibody molecule is referred to herein as a
"follow-on biologic". As presented herein, STELARA.RTM.
(ustekinumab) is the original innovator reference anti-IL-12/23p40
antibody that was FDA approved based on successful clinical trials.
Ustekinumab has been on sale in the United States since 2009.
[0024] The term "antibody" is further intended to encompass
antibodies, digestion fragments, specified portions and variants
thereof, including antibody mimetics or comprising portions of
antibodies that mimic the structure and/or function of an antibody
or specified fragment or portion thereof, including single chain
antibodies and fragments thereof. Functional fragments include
antigen-binding fragments that bind to a mammalian IL-12/23. For
example, antibody fragments capable of binding to IL-12/23 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).
[0025] 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
C.sub.H1 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.
[0026] As used herein, the term "human antibody" refers to an
antibody in which substantially every part of the protein (e.g.,
CDR, framework, C.sub.L, C.sub.H domains (e.g., C.sub.H1, C.sub.H2,
C.sub.H3), hinge, (V.sub.L, V.sub.H)) is substantially
non-immunogenic in humans, with only minor sequence changes or
variations. A "human antibody" may also be an antibody that is
derived from or closely matches human germline immunoglobulin
sequences. Human antibodies may include amino acid residues not
encoded by germline immunoglobulin sequences (e.g., mutations
introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo). Often, this means that the human
antibody is substantially non-immunogenic in humans. Human
antibodies have been classified into groupings based on their amino
acid sequence similarities. Accordingly, using a sequence
similarity search, an antibody with a similar linear sequence can
be chosen as a template to create a human antibody. 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.
[0027] It is pointed out that a human antibody can be produced by a
non-human animal or prokaryotic or eukaryotic cell that is capable
of expressing functionally rearranged human immunoglobulin (e.g.,
heavy chain and/or light chain) genes. Further, when a human
antibody is a single chain antibody, it can comprise a linker
peptide that is not found in native human antibodies. For example,
an Fv can comprise a linker peptide, such as two to about eight
glycine or other amino acid residues, which connects the variable
region of the heavy chain and the variable region of the light
chain. Such linker peptides are considered to be of human
origin.
[0028] Anti-IL-12/23p40 antibodies (also termed IL-12/23p40
antibodies) (or antibodies to IL-23) useful in the methods and
compositions of the present invention can optionally be
characterized by high affinity binding to IL-12/23p40 (or to IL-23)
and, optionally and preferably, having low toxicity. In particular,
an antibody, specified fragment or variant of the invention, where
the individual components, such as the variable region, constant
region and framework, individually and/or collectively, optionally
and preferably possess low immunogenicity, 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 raising significant HAHA, HACA or HAMA responses
in less than about 75%, or preferably less than about 50% of the
patients treated and/or raising low titres in the patient treated
(less than about 300, preferably less than about 100 measured with
a double antigen enzyme immunoassay) (Elliott et al., Lancet
344:1125-1127 (1994)), entirely incorporated herein by reference).
"Low immunogenicity" can also be defined as the incidence of
titrable levels of antibodies to the anti-IL-12 antibody in
patients treated with anti-IL-12 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.
[0029] 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,
and CH3), hinge, (V.sub.L, V.sub.H)) is substantially
non-immunogenic in humans, with only minor sequence changes or
variations. Similarly, antibodies designated primate (monkey,
baboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pig,
hamster, and the like) and other mammals designate such species,
sub-genus, genus, sub-family, family specific antibodies. Further,
chimeric antibodies include any combination of the above. Such
changes or variations optionally and preferably retain or reduce
the immunogenicity in humans or other species relative to
non-modified antibodies. Thus, a human antibody is distinct from a
chimeric or humanized antibody. It is pointed out that a human
antibody can be produced by a non-human animal or prokaryotic or
eukaryotic cell that is capable of expressing functionally
rearranged human immunoglobulin (e.g., heavy chain and/or light
chain) genes. Further, when a human antibody is a single chain
antibody, it can comprise a linker peptide that is not found in
native human antibodies. For example, an Fv can comprise a linker
peptide, such as two to about eight glycine or other amino acid
residues, which connects the variable region of the heavy chain and
the variable region of the light chain. Such linker peptides are
considered to be of human origin.
[0030] Bispecific (e.g., DuoBody.RTM.), 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. Methods
for making bispecific antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies
is based on the co-expression of two immunoglobulin heavy
chain-light chain pairs, where the two heavy chains have different
specificities (Milstein and Cuello, Nature 305:537 (1983)). Because
of the random assortment of immunoglobulin heavy and light chains,
these hybridomas (quadromas) produce a potential mixture of 10
different antibody molecules, of which only one has the correct
bispecific structure. The purification of the correct molecule,
which is usually done by affinity chromatography steps, can be
cumbersome with low product yields and different strategies have
been developed to facilitate bispecific antibody production.
[0031] Full length bispecific antibodies can be generated for
example using Fab arm exchange (or half molecule exchange) between
two monospecific bivalent antibodies by introducing substitutions
at the heavy chain CH3 interface in each half molecule to favor
heterodimer formation of two antibody half molecules having
distinct specificity either in vitro in cell-free environment or
using co-expression. The Fab arm exchange reaction is the result of
a disulfide-bond isomerization reaction and
dissociation-association of CH3 domains. The heavy-chain disulfide
bonds in the hinge regions of the parent monospecific antibodies
are reduced. The resulting free cysteines of one of the parent
monospecific antibodies form an inter heavy-chain disulfide bond
with cysteine residues of a second parent monospecific antibody
molecule and simultaneously CH3 domains of the parent antibodies
release and reform by dissociation-association. The CH3 domains of
the Fab arms may be engineered to favor heterodimerization over
homodimerization. The resulting product is a bispecific antibody
having two Fab arms or half molecules which each can bind a
distinct epitope.
[0032] "Homodimerization" as used herein refers to an interaction
of two heavy chains having identical CH3 amino acid sequences.
"Homodimer" as used herein refers to an antibody having two heavy
chains with identical CH3 amino acid sequences.
[0033] "Heterodimerization" as used herein refers to an interaction
of two heavy chains having non-identical CH3 amino acid sequences.
"Heterodimer" as used herein refers to an antibody having two heavy
chains with non-identical CH3 amino acid sequences.
[0034] The "knob-in-hole" strategy (see, e.g., PCT Intl. Publ. No.
WO 2006/028936) can be used to generate full length bispecific
antibodies. Briefly, selected amino acids forming the interface of
the CH3 domains in human IgG can be mutated at positions affecting
CH3 domain interactions to promote heterodimer formation. An amino
acid with a small side chain (hole) is introduced into a heavy
chain of an antibody specifically binding a first antigen and an
amino acid with a large side chain (knob) is introduced into a
heavy chain of an antibody specifically binding a second antigen.
After co-expression of the two antibodies, a heterodimer is formed
as a result of the preferential interaction of the heavy chain with
a "hole" with the heavy chain with a "knob". Exemplary CH3
substitution pairs forming a knob and a hole are (expressed as
modified position in the first CH3 domain of the first heavy
chain/modified position in the second CH3 domain of the second
heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,
T394S/Y407A, T366W/T394S, F405W/T394S and
T366W/T366S_L368A_Y407V.
[0035] Other strategies such as promoting heavy chain
heterodimerization using electrostatic interactions by substituting
positively charged residues at one CH3 surface and negatively
charged residues at a second CH3 surface may be used, as described
in US Pat. Publ. No. US2010/0015133; US Pat. Publ. No.
US2009/0182127; US Pat. Publ. No. US2010/028637 or US Pat. Publ.
No. US2011/0123532. In other strategies, heterodimerization may be
promoted by following substitutions (expressed as modified position
in the first CH3 domain of the first heavy chain/modified position
in the second CH3 domain of the second heavy chain):
L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,
T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in
U.S. Pat. Publ. No. US2012/0149876 or U.S. Pat. Publ. No.
US2013/0195849.
[0036] In addition to methods described above, bispecific
antibodies can be generated in vitro in a cell-free environment by
introducing asymmetrical mutations in the CH3 regions of two
monospecific homodimeric antibodies and forming the bispecific
heterodimeric antibody from two parent monospecific homodimeric
antibodies in reducing conditions to allow disulfide bond
isomerization according to methods described in Intl. Pat. Publ.
No. WO2011/131746. In the methods, the first monospecific bivalent
antibody and the second monospecific bivalent antibody are
engineered to have certain substitutions at the CH3 domain that
promoter heterodimer stability; the antibodies are incubated
together under reducing conditions sufficient to allow the
cysteines in the hinge region to undergo disulfide bond
isomerization; thereby generating the bispecific antibody by Fab
arm exchange. The incubation conditions may optimally be restored
to non-reducing. Exemplary reducing agents that may be used are
2-mercaptoethylamine (2-MEA), dithiothreitol (DTT),
dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine
(TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing
agent selected from the group consisting of: 2-mercaptoethylamine,
dithiothreitol and tris(2-carboxyethyl)phosphine. For example,
incubation for at least 90 min at a temperature of at least
20.degree. C. in the presence of at least 25 mM 2-MEA or in the
presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for
example at pH of 7.0 or at pH of 7.4 may be used.
[0037] The terms "efficacy" and "effective" as used herein in the
context of a dose, dosage regimen, treatment or method refer to the
effectiveness of a particular dose, dosage or treatment regimen.
Efficacy can be measured based on change in the course of the
disease in response to an agent of the present invention. For
example, an anti-IL 12/23p40 or anti-IL23 antibody of the present
invention (e.g., the anti-IL12/23p40 antibody usetkinumab) is
administered to a patient in an amount and for a time sufficient to
induce an improvement, preferably a sustained improvement, in at
least one indicator that reflects the severity of the disorder that
is being treated. Various indicators that reflect the extent of the
subject's illness, disease or condition may be assessed for
determining whether the amount and time of the treatment is
sufficient. Such indicators include, for example, clinically
recognized indicators of disease severity, symptoms, or
manifestations of the disorder in question. The degree of
improvement generally is determined by a physician, who may make
this determination based on signs, symptoms, biopsies, or other
test results, and who may also employ questionnaires that are
administered to the subject, such as quality-of-life questionnaires
developed for a given disease.
[0038] The term "safe", as it relates to a dose, dosage regimen,
treatment or method with an anti-IL12/23p40 or anti-IL23 antibody
of the present invention (e.g., the anti-IL12/23p40 antibody
ustekinumab), refers to a favorable risk:benefit ratio with an
acceptable frequency and/or acceptable severity of
treatment-emergent adverse events (referred to as AEs or TEAEs)
compared to the standard of care or to another comparator. An
adverse event is an untoward medical occurrence in a patient
administered a medicinal product. In particular, safe as it relates
to a dose, dosage regimen or treatment with an anti-IL12/23p40 or
anti-IL23 antibody of the present invention refers to with an
acceptable frequency and/or acceptable severity of adverse events
associated with administration of the antibody if attribution is
considered to be possible, probable, or very likely due to the use
of the anti-IL12/23p40 or anti-IL23 antibody.
Utility
[0039] The isolated nucleic acids of the present invention can be
used for production of at least one anti-IL-12/23p40 (or
anti-IL-23) 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-12/23 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-12/23 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-12/23p40 (or anti-IL-23) 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 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.
Citations
[0041] All publications or patents cited herein, whether or not
specifically designated, are entirely incorporated herein by
reference as they show the state of the art at the time of the
present invention and/or to provide description and enablement of
the present invention. Publications refer to any scientific or
patent publications, or any other information available in any
media format, including all recorded, electronic or printed
formats. The following references are entirely incorporated herein
by reference: Ausubel, et al., ed., Current Protocols in Molecular
Biology, John Wiley & Sons, Inc., NY, N.Y.(1987-2001);
Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd
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).
Antibodies of the Present Invention--Production and Generation
[0042] At least one anti-IL-12/23p40 (or anti-IL-23) used in the
method 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), each
entirely incorporated herein by reference.
[0043] A preferred anti-IL-12/23p40 antibody is ustekinumab
(STELARA.RTM.) having the heavy chain variable region amino acid
sequence of SEQ ID NO:7 and the light chain variable region amino
acid sequence of SEQ ID NO:8 and having the heavy chain CDR amino
acid sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO: 3; and
the light chain CDR amino acid sequences of SEQ ID NO:4, SEQ ID
NO:5, and SEQ ID NO:6. A preferred anti-IL-23 antibody is
guselkumab (also referred to as CNTO1959). Other anti-IL-23
antibodies have sequences listed herein and are described in U.S.
Pat. No. 7,935,344, the entire contents of which are incorporated
herein by reference).
[0044] Human antibodies that are specific for human IL-12/23p40 or
IL-23 proteins or fragments thereof can be raised against an
appropriate immunogenic antigen, such as an isolated IL-12/23p40
protein, IL-23 protein and/or a portion thereof (including
synthetic molecules, such as synthetic peptides). Other specific or
general mammalian antibodies can be similarly raised. Preparation
of immunogenic antigens, and monoclonal antibody production can be
performed using any suitable technique.
[0045] 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.
[0046] 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).
[0047] Other suitable methods of producing or isolating antibodies
of the requisite specificity can be used, including, but not
limited to, methods that select recombinant antibody from a peptide
or protein library (e.g., but not limited to, a bacteriophage,
ribosome, oligonucleotide, RNA, cDNA, or the like, display library;
e.g., as available from Cambridge antibody Technologies,
Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation,
Aberdeen, Scotland, UK; Biolnvent, Lund, Sweden; Dyax Corp., Enzon,
Affymax/Biosite; Xoma, Berkeley, Calif.; Ixsys. See, e.g., EP
368,684, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240;
PCT/GB92/00883; PCT/GB93/00605; U.S. Ser. No. 08/350,260 (May 12,
1994); PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835; (CAT/MRC);
WO90/14443; WO90/14424; WO90/14430; PCT/US94/1234; WO92/18619;
WO96/07754; (Scripps); WO96/13583, WO97/08320 (MorphoSys);
WO95/16027 (Biolnvent); WO88/06630; WO90/3809 (Dyax); U.S. Pat. No.
4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371
998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); or
stochastically generated peptides or proteins--U.S. Pat. Nos.
5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862,
WO 86/05803, EP 590 689 (Ixsys, predecessor of Applied Molecular
Evolution (AME), each entirely incorporated herein by reference))
or that rely upon immunization of transgenic animals (e.g., SCID
mice, Nguyen et al., Microbiol. Immunol. 41:901-907 (1997); Sandhu
et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al.,
Immunol. 93:154-161 (1998), each entirely incorporated by reference
as well as related patents and applications) that are capable of
producing a repertoire of human antibodies, as known in the art
and/or as described herein. Such techniques, include, but are not
limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.
USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci.
USA, 95:14130-14135 (November 1998)); single cell antibody
producing technologies (e.g., selected lymphocyte antibody method
("SLAM") (U.S. Pat. No. 5,627,052, Wen et al., J. Immunol.
17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell
et al., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge,
Mass.; Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,
Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et
al., Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress
Biotech, Vol. 5, In Vitro Immunization in Hybridoma Technology,
Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam,
Netherlands (1988)).
[0048] Methods for engineering or humanizing non-human or human
antibodies can also be used and are well known in the art.
Generally, a humanized or engineered antibody has one or more amino
acid residues from a source that is non-human, e.g., but not
limited to, mouse, rat, rabbit, non-human primate or another
mammal. These non-human amino acid residues are replaced by
residues often referred to as "import" residues, which are
typically taken from an "import" variable, constant or other domain
of a known human sequence.
[0049] Known human Ig sequences are disclosed, e.g., [0050]
www.ncbi.nlm.nih.gov/entrez/query.fcgi; [0051]
www.ncbi.nih.gov/igblast; [0052] www.atcc.org/phage/hdb.html;
[0053] www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php; [0054]
www.kabatdatabase.com/top.html; ftp.ncbi.nih.gov/repository/kabat;
[0055] www.sciquest.com; [0056] www.abcam.com; [0057]
www.antibodyresource.com/onlinecomp.html; [0058]
www.public.iastate.edu/.about.pedro/research_tools.html; [0059]
www.whfreeman.com/immunology/CH05/kuby05.htm; [0060]
www.hhmi.org/grants/lectures/1996/vlab; [0061]
www.path.cam.ac.uk/.about.mrc7/mikeimages.html; [0062]
www.mcb.harvard.edu/BioLinks/Immunology.html; [0063]
www.immunologylink.com;
pathbox.wustl.edu/.about.hcenter/index.html; [0064]
www.appliedbiosystems.com; [0065]
www.nal.usda.gov/awic/pubs/antibody; [0066]
www.m.ehime-u.ac.jp/yasuhito/Elisa.html; [0067] www.biodesign.com;
[0068] www.cancerresearchuk.org; [0069] www.biotech.ufl.edu; [0070]
www.isac-net.org; baserv.uci.kun.nl/.about.jraats/links1.html;
[0071] www.recab.uni-hd.de/immuno.bme.nwu.edu; [0072]
www.mrc-cpe.cam.ac.uk; [0073] www.ibt.unam.mx/vir/V_mice.html;
http://www.bioinf.org.uk/abs; antibody.bath.ac.uk; [0074]
www.unizh.ch; [0075] www.cryst.bbk.ac.uk/.about.ubcg07s; [0076]
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html; [0077]
www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html; [0078]
www.ibt.unam.mx/vir/structure/stat_aim.html; [0079]
www.biosci.missouri.edu/smithgp/index.html; [0080] www.jerini.de;
[0081] Kabat et al., Sequences of Proteins of Immunological
Interest, U.S. Dept. Health (1983), each entirely incorporated
herein by reference.
[0082] 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. 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.
[0083] Antibodies can also optionally be humanized 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 products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art.
[0084] Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, 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.
[0085] In addition, the human anti-IL-12/23p40 (or anti-IL-23)
specific antibody used in the method 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.
[0086] In other embodiments, the human anti-IL-12/23p40 (or
anti-IL-23) specific antibody used in the method 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.
[0087] 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 fully human framework region.
[0088] 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,
5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539;
4816567, 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.
[0089] 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. 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
function of the Fc region of an IL-23 binding molecule. The
starting polypeptide of particular interest may be one that binds
to C1q and displays complement dependent cytotoxicity (CDC).
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 WO0042072,
which is hereby incorporated by reference.
[0090] As disclosed above, one can design an Fc region of the human
anti-IL-12/23p40 (or anti-IL-23) specific antibody of the present
invention with altered effector function, e.g., by modifying C1q
binding and/or Fc.gamma.R binding and thereby changing complement
dependent cytotoxicity (CDC) activity and/or antibody-dependent
cell-mediated cytotoxicity (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; CDC; Fc receptor binding; 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.).
[0091] For example, one can generate a variant Fc region of the
human anti-IL-12/23p40 (or anti-IL-23) 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).
[0092] 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).
[0093] Another type of amino acid substitution serves to alter the
glycosylation pattern of the Fc region of the human
anti-IL-12/23p40 (or anti-IL-23) specific 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.
[0094] 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 sites that are
not present in the polypeptide. Addition of glycosylation sites to
the Fc region of a human IL-23 specific 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.
[0095] In certain embodiments, the human anti-IL-12/23p40 (or
anti-IL-23) specific 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 human
anti-IL-12/23p40 (or anti-IL-23) antibody. Methods for producing
antibodies in such a fashion are provided in WO/9954342,
WO/03011878, patent publication 20030003097A1, and Umana et al.,
Nature Biotechnology, 17:176-180, February 1999; all of which are
herein specifically incorporated by reference in their
entireties.
[0096] The human anti-IL-12/23p40 (or anti-IL-23) antibody can also
be optionally generated by immunization of a transgenic animal
(e.g., mouse, rat, hamster, non-human primate, and the like)
capable of producing a repertoire of human antibodies, as described
herein and/or as known in the art. Cells that produce a human
anti-IL-12/23p40 (or anti-IL-23) antibody can be isolated from such
animals and immortalized using suitable methods, such as the
methods described herein.
[0097] 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.
[0098] 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.
[0099] Other systems for generating libraries of peptides have
aspects of both in vitro chemical synthesis and recombinant
methods. See, PCT Patent Publication Nos. 92/05258, 92/14843, and
96/19256. See also, U.S. Pat. Nos. 5,658,754; and 5,643,768.
Peptide display libraries, vector, and screening kits are
commercially available from such suppliers as Invitrogen (Carlsbad,
Calif.), and Cambridge antibody Technologies (Cambridgeshire, UK).
See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666, 4,946,778,
5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733,
5,767,260, 5,856,456, assigned to Enzon; U.S. Pat. Nos. 5,223,409,
5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5427908,
5580717, assigned to Affymax; 5885793, assigned to Cambridge
antibody Technologies; 5750373, assigned to Genentech, 5618920,
5595898, 5576195, 5698435, 5693493, 5698417, assigned to Xoma,
Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the
above patents and publications entirely incorporated herein by
reference.
[0100] Antibodies used in the method of the present invention can
also be prepared using at least one anti-IL-12/23p40 (or
anti-IL-23) 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.
[0101] Antibodies used in the method of the present invention can
additionally be prepared using at least one anti-IL-12/23p40 (or
anti-IL-23) 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. Each of the
above references is entirely incorporated herein by reference.
[0102] The antibodies used in the method of the invention can bind
human IL-12/IL-23p40 or IL-23 with a wide range of affinities
(K.sub.D). In a preferred embodiment, a human mAb can optionally
bind human IL-12/IL-23p40 or IL-23 with high affinity. For example,
a human mAb can bind human IL-12/IL-23p40 or IL-23 with a K.sub.D
equal to or less than about 10.sup.-7 M, such as but not limited
to, 0.1-9.9 (or any range or value therein).times.10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12,
10.sup.-13 or any range or value therein.
[0103] The affinity or avidity of an antibody for an antigen can be
determined experimentally using any suitable method. (See, for
example, Berzofsky, et al., "Antibody-Antigen Interactions," In
Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York,
N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New
York, N.Y. (1992); and methods described herein). The measured
affinity of a particular antibody-antigen interaction can vary if
measured under different conditions (e.g., salt concentration, pH).
Thus, measurements of affinity and other antigen-binding parameters
(e.g., K.sub.D, K.sub.a, K.sub.d) are preferably made with
standardized solutions of antibody and antigen, and a standardized
buffer, such as the buffer described herein.
Nucleic Acid Molecules
[0104] 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 or heavy chain variable or
CDR regions described herein, among other sequences disclosed
herein, 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 IL-12/IL-23p40 or IL-23 antibody can be
obtained using methods described herein or as known in the art.
[0105] 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.
[0106] Isolated nucleic acid molecules used in the method 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 one heavy
chain or light chain; nucleic acid molecules comprising the coding
sequence for an anti-IL-12/IL-23p40 or IL-23 antibody or variable
region; 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-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 antibodies used in the method
of the present invention. See, e.g., Ausubel, et al., supra, and
such nucleic acid variants are included in the present invention.
Non-limiting examples of isolated nucleic acid molecules include
nucleic acids encoding HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2,
and LC CDR3, respectively.
[0107] As indicated herein, nucleic acid molecules which comprise a
nucleic acid encoding an anti-IL-12/IL-23p40 or IL-23 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.
Polynucleotides Selectively Hybridizing to a Polynucleotide as
Described Herein
[0108] The method of the present invention uses 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.
[0109] 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.
[0110] Optionally, polynucleotides will encode at least a portion
of an antibody. The polynucleotides 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.
Construction of Nucleic Acids
[0111] The isolated nucleic acids can be made using (a) recombinant
methods, (b) synthetic techniques, (c) purification techniques,
and/or (d) combinations thereof, as well-known in the art.
[0112] 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.
[0113] 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)
Recombinant Methods for Constructing Nucleic Acids
[0114] The isolated nucleic acid compositions, 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)
Nucleic Acid Screening and Isolation Methods
[0115] A cDNA or genomic library can be screened using a probe
based upon the sequence of a polynucleotide used in the method 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.
[0116] 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.
[0117] Known methods of DNA or RNA amplification include, but are
not limited to, polymerase chain reaction (PCR) and related
amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; U.S. Pat. Nos.
4,795,699 and 4,921,794 to Tabor, et al; U.S. Pat. No. 5,142,033 to
Innis; U.S. Pat. No. 5,122,464 to Wilson, et al.; U.S. Pat. No.
5,091,310 to Innis; U.S. Pat. No. 5,066,584 to Gyllensten, et al;
U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat. No. 4,994,370
to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S. Pat. No.
4,656,134 to Ringold) and RNA mediated amplification that uses
anti-sense RNA to the target sequence as a template for
double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et
al, with the tradename NASBA), the entire contents of which
references are incorporated herein by reference. (See, e.g.,
Ausubel, supra; or Sambrook, supra.)
[0118] For instance, polymerase chain reaction (PCR) technology can
be used to amplify the sequences of polynucleotides used in the
method of the present invention and related genes directly from
genomic DNA or cDNA libraries. PCR and other in vitro amplification
methods can also be useful, for example, to clone nucleic acid
sequences that code for proteins to be expressed, to make nucleic
acids to use as probes for detecting the presence of the desired
mRNA in samples, for nucleic acid sequencing, or for other
purposes. Examples of techniques sufficient to direct persons of
skill through in vitro amplification methods are found in Berger,
supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et
al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR
Protocols A Guide to Methods and Applications, Eds., Academic Press
Inc., San Diego, Calif. (1990). Commercially available kits for
genomic PCR amplification are known in the art. See, e.g.,
Advantage-GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4
gene 32 protein (Boehringer Mannheim) can be used to improve yield
of long PCR products.
Synthetic Methods for Constructing Nucleic Acids
[0119] The isolated nucleic acids used in the method 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.
Recombinant Expression Cassettes
[0120] The present invention uses recombinant expression cassettes
comprising a nucleic acid. A nucleic acid sequence, for example, a
cDNA or a genomic sequence encoding an antibody used in the method
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 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.
[0121] 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. For
example, endogenous promoters can be altered in vivo or in vitro by
mutation, deletion and/or substitution.
Vectors and Host Cells
[0122] The present invention also relates to vectors that include
isolated nucleic acid molecules, host cells that are genetically
engineered with the recombinant vectors, and the production of at
least one anti-IL-23 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.
[0123] 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.
[0124] 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.
[0125] 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 affected 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.
[0126] At least one antibody used in the method 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.
[0127] Those of ordinary skill in the art are knowledgeable in the
numerous expression systems available for expression of a nucleic
acid encoding a protein used in the method of the present
invention. Alternatively, nucleic acids can be expressed in a host
cell by turning on (by manipulation) in a host cell that contains
endogenous DNA encoding an antibody. 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.
[0128] Cells useful for the production of the antibodies, specified
portions or variants thereof, include mammalian cells. Mammalian
cell systems often will be cultured in the form of monolayers of
cells, but the cells can also be adapted to grow in suspension,
e.g., in shake flasks or bioreactors. A number of suitable host
cell lines capable of expressing intact glycosylated proteins have
been developed in the art, and include, e.g., COS-1 (e.g.,
ATCC.RTM. CRL1650), COS-7 (e.g., ATCC.RTM. CRL-1651), HEK293, BHK21
(e.g., ATCC.RTM. CCL-10), BSC-1 (e.g., ATCC.RTM. CCL-26), Chinese
hamster ovary (CHO), Hep G2, P3X63Ag8.653, Sp2/0-Ag14, HeLa and the
like, which are readily available from, for example, American Type
Culture Collection, Manassas, Va. (www.atcc.org). In certain
embodiments, host cells include CHO cells and cells of lymphoid
origin, such as myeloma and lymphoma cells, e.g., CHO-K1 cells,
P3X63Ag8.653 cells (ATCC.RTM. CRL-1580) and Sp2/0-Ag14 cells
(ATCC.RTM. CRL-1581).
[0129] 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.
[0130] 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.
CHO Cell Lines
[0131] Despite the availability of several other mammalian cell
lines, a majority of recombinant therapeutic proteins produced
today are made in Chinese hamster ovary (CHO) cells (Jayapal K P,
et al. Recombinant protein therapeutics from CHO cells-20 years and
counting. Chem Eng Prog. 2007; 103:40-47; Kunert R, Reinhart D.
Advances in recombinant antibody manufacturing. Appl Microbiol
Biotechnol. 2016; 100(8):3451-61). Their strengths include, e.g.,
robust growth as adherent cells or in suspension, adaptability to
serum-free and chemically defined media, high productivity, and an
established history of regulatory approval for therapeutic
recombinant protein production. They are also very amenable to
genetic modifications and the methods for cell transfection,
recombinant protein expression, and clone selection are all well
characterized. CHO cells can also provide human-compatible
post-translational modifications. As used herein, "CHO cells"
include, but are not limited to, e.g., CHO-DG44, CHO-K1, CHO-M,
CHO-S, CHO GS knockout, and modifications and derivatives
thereof.
Cloning and Expression in CHO Cells.
[0132] One vector commonly used for expression in CHO cells is pC4.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC.RTM.
37146). The plasmid contains the mouse DHFR gene under control of
the SV40 early promoter. Chinese hamster ovary cells or other cells
lacking dihydrofolate activity that are transfected with these
plasmids can be selected by growing the cells in a selective medium
(e.g., alpha minus MEM, Life Technologies, Gaithersburg, Md.)
supplemented with the chemotherapeutic agent methotrexate. The
amplification of the DHFR genes in cells resistant to methotrexate
(MTX) has been well documented (see, e.g., F. W. Alt, et al., J.
Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem.
et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A.
Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach can be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained that contain the
amplified gene integrated into one or more chromosome(s) of the
host cell.
[0133] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early
gene of human cytomegalovirus (CMV) (Boshart, et al., Cell
41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow integration of
the genes. Behind these cloning sites the plasmid contains the 3'
intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the
expression, e.g., the human beta-actin promoter, the SV40 early or
late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On
gene expression systems and similar systems can also be used to
express proteins in a regulated way in mammalian cells (M. Gossen,
and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
For the polyadenylation of the mRNA other signals, e.g., from the
human growth hormone or globin genes can be used as well. Stable
cell lines carrying a gene of interest integrated into the
chromosomes can also be selected upon co-transfection with a
selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
Purification of an Antibody
[0134] An anti-IL-12/IL-23p40 or IL-23 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.
[0135] Antibodies used in the method 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 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.
Anti-IL-12/IL-23p40 or IL-23 Antibodies
[0136] An anti-IL-12/IL-23p40 or IL-23 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 C.sub.H1, hinge1, hinge2,
hinge3, hinge4, C.sub.H2, or C.sub.H3 or fragment thereof, further
optionally comprising at least one substitution, insertion or
deletion), or any portion thereof, that can be incorporated into an
antibody. An antibody 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.
[0137] The isolated antibodies used in the method 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-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 protein or fragment can bind the
protein or fragment and thereby inhibit activities mediated through
the binding of IL-12/IL-23p40 or IL-23 to the IL-12 and/or IL-23
receptor or through other IL-12/IL-23p40 or IL-23-dependent or
mediated mechanisms. As used herein, the term "neutralizing
antibody" refers to an antibody that can inhibit an IL-12/IL-23p40
or 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-12/IL-23p40 or IL-23 antibody to
inhibit an IL-12/IL-23p40 or IL-23-dependent activity is preferably
assessed by at least one suitable IL-12/IL-23p40 or IL-23 protein
or receptor assay, as described herein and/or as known in the art.
A human antibody 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, .gamma.4).
Antibodies of this type can be prepared by employing a transgenic
mouse or other transgenic 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-IL-23 human antibody comprises an IgG1 heavy chain and an IgG1
light chain.
[0138] An antibody binds at least one specified epitope specific to
at least one IL-12/IL-23p40 or IL-23 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.
[0139] Generally, the human antibody or antigen-binding fragment
will comprise an antigen-binding region that comprises at least one
human complementarity determining region (CDR1, CDR2 and CDR3) or
variant of at least one heavy chain variable region and at least
one human complementarity determining region (CDR1, CDR2 and CDR3)
or variant of at least one light chain variable region. 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 non-human CDRs can be
used. These CDRs may be formed by incorporation of conservative
substitutions from the original non-human sequence. In another
particular embodiment, the antibody or antigen-binding portion or
variant can have an antigen-binding region that comprises at least
a portion of at least one light chain CDR (i.e., CDR1, CDR2 and/or
CDR3) having the amino acid sequence of the corresponding CDRs 1, 2
and/or 3.
[0140] Such antibodies can be prepared by chemically joining
together the various portions (e.g., CDRs, framework) of the
antibody using conventional techniques, by preparing and expressing
a (i.e., one or more) nucleic acid molecule that encodes the
antibody using conventional techniques of recombinant DNA
technology or by using any other suitable method.
[0141] The anti-IL-12/IL-23p40 or IL-23 specific 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-12/IL-23p40 or IL-23 antibody comprises an
anti-IL-12/IL-23p40 antibody with a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO:7 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO:8.
The anti-IL-12/IL-23p40 or IL-23 specific antibody can also
comprise at least one of a heavy or light chain having a defined
amino acid sequence. In another preferred embodiment, the
anti-IL-12/IL-23p40 or IL-23 antibody comprises an
anti-IL-12/IL-23p40 antibody with a heavy chain comprising the
amino acid sequence of SEQ ID NO: 10 and a light chain comprising
the amino acid sequence of SEQ ID NO: 11. Antibodies that bind to
human IL-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 or a fragment thereof to elicit the
production of antibodies. 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.
[0142] The invention also relates to antibodies, antigen-binding
fragments, immunoglobulin chains and CDRs comprising amino acids in
a sequence that is substantially the same as an amino acid sequence
described herein. Preferably, such antibodies or antigen-binding
fragments and antibodies comprising such chains or CDRs can bind
human IL-12/IL-23p40 or IL-23 with high affinity (e.g., K.sub.D
less than or equal to about 10.sup.-9 M). Amino acid sequences that
are substantially the same as the sequences described herein
include sequences comprising conservative amino acid substitutions,
as well as amino acid deletions and/or insertions. A conservative
amino acid substitution refers to the replacement of a first amino
acid by a second amino acid that has chemical and/or physical
properties (e.g., charge, structure, polarity,
hydrophobicity/hydrophilicity) that are similar to those of the
first amino acid. Conservative substitutions include, without
limitation, replacement of one amino acid by another within the
following groups: lysine (K), arginine (R) and histidine (H);
aspartate (D) and glutamate (E); asparagine (N), glutamine (Q),
serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine
(A), valine (V), leucine (L), isoleucine (I), proline (P),
phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and
glycine (G); F, W and Y; C, S and T.
Amino Acid Codes
[0143] The amino acids that make up anti-IL-12/IL-23p40 or IL-23
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):
TABLE-US-00001 SINGLE THREE THREE LETTER LETTER NUCLEOTIDE CODE
CODE NAME CODON(S) A Ala Alanine GCA, GCC, GCG, GCU C Cys Cysteine
UGC, UGU D Asp Aspartic acid GAC, GAU E Glu Glutamic acid GAA, GAG
F Phe Phenylanine UUC, UUU G Gly Glycine GGA, GGC, GGG, GGU H His
Histidine CAC, CAU I Ile Isoleucine AUA, AUC, AUU K Lys Lysine AAA,
AAG L Leu Leucine UUA, UUG, CUA, CUC, CUG, CUU M Met Methionine AUG
N Asn Asparagine AAC, AAU P Pro Proline CCA, CCC, CCG, CCU Q Gln
Glutamine CAA, CAG R Arg Arginine AGA, AGG, CGA, CGC, CGG, CGU S
Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T Thr Threonine ACA, ACC,
ACG, ACU V Val Valine GUA, GUC, GUG, GUU W Trp Tryptophan UGG Y Tyr
Tyrosine UAC, UAU
Sequences
Example Anti-IL-12/IL-23p40 Antibody Sequences--STELARA.RTM.
(Ustekinumab)
[0144] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region heavy chain 1 (CDRH1):
TABLE-US-00002 (SEQ ID NO: 1) TYWLG
[0145] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region heavy chain 2 (CDRH2):
TABLE-US-00003 (SEQ ID NO: 2) IMSPVDSDIRYSPSFQG
[0146] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region heavy chain 3 (CDRH3):
TABLE-US-00004 (SEQ ID NO: 3) RRPGQGYFDF
[0147] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region light chain 1 (CDRL1):
TABLE-US-00005 (SEQ ID NO: 4) RASQGISSWLA
[0148] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region light chain 2 (CDRL2):
TABLE-US-00006 (SEQ ID NO: 5) AASSLQS
[0149] Amino acid sequence of anti-IL-12/IL-23p40 antibody
complementarity determining region light chain 3 (CDRL3):
TABLE-US-00007 (SEQ ID NO: 6) QQYNIYPYT
[0150] Amino acid sequence of anti-IL-12/IL-23p40 antibody variable
heavy chain region (CDRs underlined):
TABLE-US-00008 (SEQ ID NO: 7) 1 EVQLVQSGAE VKKPGESLKI SCKGSGYSFT
TYWLGWVRQM PGKGLDWIGI MSPVDSDIRY 61 SPSFQGQVTM SVDKSITTAY
LQWNSLKASD TAMYYCARRR PGQGYFDFWG QGTLVTVSS
[0151] Amino acid sequence of anti-IL-12/IL-23p40 antibody variable
light chain region (CDRs underlined):
TABLE-US-00009 (SEQ ID NO: 8) 1 DIQMTQSPSS LSASVGDRVT ITCRASQGIS
SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS 61 RFSGSGSGTD FTLTISSLQP
EDFATYYCQQ YNIYPYTFGQ GTKLEIKR
[0152] Amino acid sequence of anti-IL-12/IL-23p40 antibody heavy
chain (CDRs underlined):
TABLE-US-00010 (SEQ ID NO: 10) 1 EVQLVQSGAE VKKPGESLKI SCKGSGYSFT
TYWLGWVRQM PGKGLDWIGI MSPVDSDIRY 61 SPSFQGQVTM SVDKSITTAY
LQWNSLKASD TAMYYCARRR PGQGYFDFWG QGTLVTVSSS 121 STKGPSVFPL
APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 181
LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPELLGGP
241 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK
TKPREEQYNS 301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK
AKGQPREPQV YTLPPSRDEL 361 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE
NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 421 QGNVFSCSVM HEALHNHYTQ
KSLSLSPGK
[0153] Amino acid sequence of anti-IL-12/IL-23p40 antibody light
chain (CDRs underlined):
TABLE-US-00011 (SEQ ID NO: 11) 1 DIQMTQSPSS LSASVGDRVT ITCRASQGIS
SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS 61 RFSGSGSGTD FTLTISSLQP
EDFATYYCQQ YNIYPYTFGQ GTKLEIKRTV AAPSVFIFPP 121 SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 181
LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
Amino acid sequence IL-12
[0154] Amino acid sequence of human interleukin (IL)-12 with alpha
and beta subunits:
TABLE-US-00012 (SEQ ID NO: 9) 1 RNLPVATPDP GMFPCLHHSQ NLLRAVSNML
QKARQTLEFY PCTSEEIDHE DITKDKTSTV 61 EACLPLELTK NESCLNSRET
SFITNGSCLA SRKTSFMMAL CLSSIYEDLK MYQVEFKTMN 121 AKLLMDPKRQ
IFLDQNMLAV IDELMQALNF NSETVPQKSS LEEPDFYKTK IKLCILLHAF 181
RIRAVTIDRV MSYLNASIWE LKKDVYVVEL DWYPDAPGEM VVLTCDTPEE DGITWTLDQS
241 SEVLGSGKTL TIQVKEFGDA GQYTCHKGGE VLSHSLLLLH KKEDGIWSTD
ILKDQKEPKN 301 KTFLRCEAKN YSGRFTCWWL TTISTDLTFS VKSSRGSSDP
QGVTCGAATL SAERVRGDNK 361 EYEYSVECQE DSACPAAEES LPIEVMVDAV
HKLKYENYTS SFFIRDIIKP DPPKNLQLKP 421 LKNSRQVEVS WEYPDTWSTP
HSYFSLTFCV QVQGKSKREK KDRVFTDKTS ATVICRKNAS 481 ISVRAQDRYY
SSSWSEWASV PCS
[0155] An anti-IL-12/IL-23p40 or IL-23 antibody used in the method
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.
[0156] The number of amino acid substitutions a skilled artisan
would make depends on many factors, including those described
above. Generally speaking, the number of amino acid substitutions,
insertions or deletions for any given anti-IL-12/IL-23p40 or IL-23
antibody, fragment or variant will not be more than 40, 30, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such
as 1-30 or any range or value therein, as specified herein.
[0157] Amino acids in an anti-IL-12/IL-23p40 or IL-23 specific
antibody that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15;
Cunningham and Wells, Science 244:1081-1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity, such as, but not limited to, at least one
IL-12/IL-23p40 or 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)).
[0158] Anti-IL-12/IL-23p40 or IL-23 antibodies can include, but are
not limited to, at least one portion, sequence or combination
selected from 5 to all of the contiguous amino acids of at least
one of SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 10, or 11.
[0159] IL-12/IL-23p40 or IL-23 antibodies or specified portions or
variants can include, but are not limited to, at least one portion,
sequence or combination selected from at least 3-5 contiguous amino
acids of the SEQ ID NOs above; 5-17 contiguous amino acids of the
SEQ ID NOs above, 5-10 contiguous amino acids of the SEQ ID NOs
above, 5-11 contiguous amino acids of the SEQ ID NOs above, 5-7
contiguous amino acids of the SEQ ID NOs above; 5-9 contiguous
amino acids of the SEQ ID NOs above.
[0160] An anti-IL-12/IL-23p40 or IL-23 antibody can further
optionally comprise a polypeptide of at least one of 70-100% of 5,
17, 10, 11, 7, 9, 119, 108, 449, or 214 contiguous amino acids of
the SEQ ID NOs above. In one embodiment, the amino acid sequence of
an immunoglobulin chain, or portion thereof (e.g., variable region,
CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the
amino acid sequence of the corresponding chain of at least one of
the SEQ ID NOs above. For example, the amino acid sequence of a
light chain variable region can be compared with the sequence of
the SEQ ID NOs above, or the amino acid sequence of a heavy chain
CDR3 can be compared with the SEQ ID NOs above. Preferably, 70-100%
amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100 or any range or value therein) is determined using a suitable
computer algorithm, as known in the art.
[0161] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
"identity" also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as determined by the match
between strings of such sequences. "Identity" and "similarity" can
be readily calculated by known methods, including, but not limited
to, those described in Computational Molecular Biology, Lesk, A.
M., ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M Stockton Press, New York, 1991; and
Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988).
In addition, values for percentage identity can be obtained from
amino acid and nucleotide sequence alignments generated using the
default settings for the AlignX component of Vector NTI Suite 8.0
(Informax, Frederick, Md.).
[0162] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. Preferred computer program methods to
determine identity and similarity between two sequences include,
but are not limited to, the GCG program package (Devereux, J., et
al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and
FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990)).
The BLAST X program is publicly available from NCBI and other
sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda,
Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).
The well-known Smith Waterman algorithm may also be used to
determine identity.
[0163] Preferred parameters for polypeptide sequence comparison
include the following: [0164] (1) Algorithm: Needleman and Wunsch,
J. Mol Biol. 48:443-453 (1970) Comparison matrix: BLOSSUM62 from
Hentikoff and Hentikoff, Proc. Natl. Acad. Sci, USA. 89:10915-10919
(1992) [0165] Gap Penalty: 12 [0166] Gap Length Penalty: 4 [0167] A
program useful with these parameters is publicly available as the
"gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
sequence comparisons (along with no penalty for end gaps).
[0168] Preferred parameters for polynucleotide comparison include
the following: [0169] (1) Algorithm: Needleman and Wunsch, J. Mol
Biol. 48:443-453 (1970) [0170] Comparison matrix: matches=+10,
mismatch=0 [0171] Gap Penalty: 50 [0172] Gap Length Penalty: 3
[0173] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid sequence comparisons.
[0174] By way of example, a polynucleotide sequence may be
identical to another sequence, that is 100% identical, or it may
include up to a certain integer number of nucleotide alterations as
compared to the reference sequence. Such alterations are selected
from the group consisting of at least one nucleotide deletion,
substitution, including transition and transversion, or insertion,
and wherein the alterations may occur at the 5' or 3' terminal
positions of the reference nucleotide sequence or anywhere between
those terminal positions, interspersed either individually among
the nucleotides in the reference sequence or in one or more
contiguous groups within the reference sequence. The number of
nucleotide alterations is determined by multiplying the total
number of nucleotides in the sequence by the numerical percent of
the respective percent identity (divided by 100) and subtracting
that product from the total number of nucleotides in the sequence,
or:
n.sub.n.ltorsim.x.sub.n-(x.sub.n.y),
wherein n.sub.n is the number of nucleotide alterations, x.sub.n is
the total number of nucleotides in sequence, and y is, for
instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%,
0.95 for 95%, etc., and wherein any non-integer product of x.sub.n
and y is rounded down to the nearest integer prior to subtracting
from x.sub.n.
[0175] Alterations of a polynucleotide sequence encoding the the
SEQ ID NOs above may create nonsense, missense or frameshift
mutations in this coding sequence and thereby alter the polypeptide
encoded by the polynucleotide following such alterations.
Similarly, a polypeptide sequence may be identical to the reference
sequence of the SEQ ID NOs above, that is be 100% identical, or it
may include up to a certain integer number of amino acid
alterations as compared to the reference sequence such that the
percentage identity is less than 100%. Such alterations are
selected from the group consisting of at least one amino acid
deletion, substitution, including conservative and non-conservative
substitution, or insertion, and wherein the alterations may occur
at the amino- or carboxy-terminal positions of the reference
polypeptide sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids in the
reference sequence or in one or more contiguous groups within the
reference sequence. The number of amino acid alterations for a
given % identity is determined by multiplying the total number of
amino acids in the SEQ ID NOs above by the numerical percent of the
respective percent identity (divided by 100) and then subtracting
that product from the total number of amino acids in the SEQ ID NOs
above, or: n.sub.a.ltorsim.x.sub.a-(x.sub.a.y), wherein n.sub.a is
the number of amino acid alterations, x.sub.a is the total number
of amino acids in the SEQ ID NOs above, and y is, for instance 0.70
for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any
non-integer produce of x.sub.a and y is rounded down to the nearest
integer prior to subtracting it from x.sub.a.
[0176] Exemplary heavy chain and light chain variable regions
sequences and portions thereof are provided in the SEQ ID NOs
above. The antibodies of the present invention, or specified
variants thereof, can comprise any number of contiguous amino acid
residues from an antibody of the present invention, wherein that
number is selected from the group of integers consisting of from
10-100% of the number of contiguous residues in an
anti-IL-12/IL-23p40 or IL-23 antibody. Optionally, this subsequence
of contiguous amino acids is at least about 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220, 230, 240, 250 or more amino acids in length, or any range
or value therein. Further, the number of such subsequences can be
any integer selected from the group consisting of from 1 to 20,
such as at least 2, 3, 4, or 5.
[0177] As those of skill will appreciate, the present invention
includes at least one biologically active antibody of the present
invention. Biologically active antibodies have a specific activity
at least 20%, 30%, or 40%, and, preferably, at least 50%, 60%, or
70%, and, most preferably, at least 80%, 90%, or 95%-100% or more
(including, without limitation, up to 10 times the specific
activity) 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.
[0178] In another aspect, the invention relates to human antibodies
and antigen-binding fragments, as described herein, which are
modified by the covalent attachment of an organic moiety. Such
modification can produce an antibody or antigen-binding fragment
with improved pharmacokinetic properties (e.g., increased in vivo
serum half-life). The organic moiety can be a linear or branched
hydrophilic polymeric group, fatty acid group, or fatty acid ester
group. In particular embodiments, the hydrophilic polymeric group
can have a molecular weight of about 800 to about 120,000 Daltons
and can be a polyalkane glycol (e.g., polyethylene glycol (PEG),
polypropylene glycol (PPG)), carbohydrate polymer, amino acid
polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid
ester group can comprise from about eight to about forty carbon
atoms.
[0179] As defined herein, the term "half-life" indicates that the
plasma concentration of a drug (e.g., a therapeutic
anti-IL-12/IL-23p40 antibody ustekinumab) is halved after one
elimination half-life. Therefore, in each succeeding half-life,
less drug is eliminated. After one half-life the amount of drug
remaining in the body is 50% after two half-lives 25%, etc. The
half-life of a drug depends on its clearance and volume of
distribution. The elimination half-life is considered to be
independent of the amount of drug in the body.
[0180] The modified antibodies and antigen-binding fragments 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.
[0181] Fatty acids and fatty acid esters suitable for modifying
antibodies of the invention can be saturated or can contain one or
more units of unsaturation. Fatty acids that are suitable for
modifying antibodies of the invention include, for example,
n-dodecanoate (C.sub.12, laurate), n-tetradecanoate (C.sub.14,
myristate), n-octadecanoate (Cis, stearate), n-eicosanoate
(C.sub.20, arachidate), n-docosanoate (C.sub.22, behenate),
n-triacontanoate (C.sub.30), n-tetracontanoate (C.sub.40),
cis-A9-octadecanoate (Cis, oleate), all
cis-A5,8,11,14-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.
[0182] The modified human antibodies and antigen-binding fragments
can be prepared using suitable methods, such as by reaction with
one or more modifying agents. A "modifying agent" as the term is
used herein, refers to a suitable organic group (e.g., hydrophilic
polymer, a fatty acid, a fatty acid ester) that comprises an
activating group. An "activating group" is a chemical moiety or
functional group that can, under appropriate conditions, react with
a second chemical group thereby forming a covalent bond between the
modifying agent and the second chemical group. For example,
amine-reactive activating groups include electrophilic groups, such
as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),
N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups
that can react with thiols include, for example, maleimide,
iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic
acid thiol (TNB-thiol), and the like. An aldehyde functional group
can be coupled to amine- or hydrazide-containing molecules, and an
azide group can react with a trivalent phosphorous group to form
phosphoramidate or phosphorimide linkages. Suitable methods to
introduce activating groups into molecules are known in the art
(see for example, Hermanson, G. T., Bioconjugate Techniques,
Academic Press: San Diego, Calif. (1996)). An activating group can
be bonded directly to the organic group (e.g., hydrophilic polymer,
fatty acid, fatty acid ester), or through a linker moiety, for
example, a divalent C.sub.1-C.sub.12 group wherein one or more
carbon atoms can be replaced by a heteroatom, such as oxygen,
nitrogen or sulfur. Suitable linker moieties include, for example,
tetraethylene glycol, --(CH.sub.2).sub.3--,
--NH--(CH.sub.2).sub.6--NH--, --(CH.sub.2).sub.2--NH-- and
--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH--NH--.
Modifying agents that comprise a linker moiety can be produced, for
example, by reacting a mono-Boc-alkyldiamine (e.g.,
mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid
in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) to form an amide bond between the free amine and the fatty
acid carboxylate. The Boc protecting group can be removed from the
product by treatment with trifluoroacetic acid (TFA) to expose a
primary amine that can be coupled to another carboxylate, as
described, or can be reacted with maleic anhydride and the
resulting product cyclized to produce an activated maleimido
derivative of the fatty acid. (See, for example, Thompson, et al.,
WO 92/16221, the entire teachings of which are incorporated herein
by reference.) The modified antibodies 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).
[0183] The method of the present invention also uses an
anti-IL-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 antibody amino acid sequence selected
from the group consisting of 70-100% of the contiguous amino acids
of the SEQ ID NOs above, or specified fragments, domains or
variants thereof. Preferred anti-IL-12/IL-23p40 or IL-23 antibody
compositions include at least one or two full length, fragments,
domains or variants as at least one CDR or LBP containing portions
of the anti-IL-12/IL-23p40 or IL-23 antibody sequence described
herein, for example, 70-100% of the SEQ ID NOs above, or specified
fragments, domains or variants thereof. Further preferred
compositions comprise, for example, 40-99% of at least one of
70-100% of the SEQ ID NOs above, etc., 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.
Antibody Compositions Comprising Further Therapeutically Active
Ingredients
[0184] The antibody compositions used in the method 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, P A, 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).
[0185] By way of example of the drugs that can be combined with the
antibodies for the method of the present invention, 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 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 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.
[0186] The at least one coricosteroid 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.
[0187] 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, 6-mercaptopurine,
methotrexate, mizoribine, and tacrolimus.
[0188] 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.)
[0189] Anti-IL-12/IL-23p40 or IL-23 antibody compositions can
further comprise at least one of any suitable and effective amount
of a composition or pharmaceutical composition comprising at least
one anti-IL-12/IL-23p40 or IL-23 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, eternacept, 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), an immunization, an immunoglobulin, an
immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a
cytokine or a cytokine antagonist. Non-limiting examples of such
cytokines include, but are not limited to, any of IL-1 to IL-23 et
al. (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.
[0190] Anti-IL-12/IL-23p40 or IL-23 antibody compounds,
compositions or combinations used in the method 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-23 antibody, fragment or variant composition as well known
in the art or as described herein.
[0191] 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.
[0192] 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.
[0193] Anti-IL-12/IL-23p40 or IL-23 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.
[0194] Additionally, anti-IL-12/IL-23p40 or IL-23 antibody
compositions 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).
[0195] These and additional known pharmaceutical excipients and/or
additives suitable for use in the anti-IL-12/IL-23p40 or IL-23
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.
Formulations
[0196] 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-12/IL-23p40 or IL-23 antibody
in a pharmaceutically acceptable formulation. Preserved
formulations contain at least one known preservative or optionally
selected from the group consisting of at least one phenol,
m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
phenylmercuric nitrite, phenoxyethanol, formaldehyde,
chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium chloride, sodium dehydroacetate and thimerosal, or
mixtures thereof in an aqueous diluent. Any suitable concentration
or mixture can be used as known in the art, such as 0.001-5%, or
any range or value therein, such as, but not limited to 0.001,
0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,
3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5,
4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting
examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3.
0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1,
1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01),
0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%),
0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,
0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3,
0.5, 0.75, 0.9, 1.0%), and the like.
[0197] As noted above, the method of the invention uses an article
of manufacture, comprising packaging material and at least one vial
comprising a solution of at least one anti-IL-12/IL-23p40 or IL-23
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 uses an
article of manufacture, comprising packaging material, a first vial
comprising lyophilized anti-IL-12/IL-23p40 or IL-23 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 anti-IL-12/IL-23p40 or
IL-23 antibody in the aqueous diluent to form a solution that can
be held over a period of twenty-four hours or greater.
[0198] The anti-IL-12/IL-23p40 or IL-23 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.
[0199] The range of the anti-IL-12/IL-23p40 or IL-23 antibody
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.
[0200] 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.
[0201] 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).
[0202] 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.
[0203] The formulations can be prepared by a process which
comprises mixing at least one anti-IL-12/IL-23p40 or IL-23 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-12/IL-23p40 or IL-23 specific 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-12/IL-23p40 or IL-23 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.
[0204] The formulations can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized
anti-IL-12/IL-23p40 or IL-23 specific 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.
[0205] The present 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 biologically activity of the protein for extended
periods of time, thus allowing a package label indicating that the
solution can be held and/or used over a period of 6, 12, 18, 24,
36, 48, 72, or 96 hours or greater. If preserved diluent is used,
such label can include use up to 1-12 months, one-half, one and a
half, and/or two years.
[0206] The solutions of anti-IL-12/IL-23p40 or IL-23 specific
antibody 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.
[0207] 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-12/IL-23p40 or IL-23 specific 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.
[0208] 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-12/IL-23p40 or IL-23 specific
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.
[0209] Recognized devices comprising single vial systems include
pen-injector devices for delivery of a solution, such as BD Pens,
BD Autojector.RTM., Humaject.RTM., NovoPen.RTM., B-D.RTM.Pen,
AutoPen.RTM., and OptiPen.RTM., GenotropinPen.RTM., Genotronorm
Pen.RTM., Humatro Pen.RTM., Reco-Pen.RTM., Roferon Pen.RTM.,
Biojector.RTM., Iject.RTM., J-tip Needle-Free Injector.RTM.,
Intraject.RTM., Medi-Ject.RTM., Smartject.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 similary 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.
[0210] The products may 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, as applicable, to reconstitute the at
least one anti-IL-12/IL-23p40 or IL-23 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, pre-filled syringe or auto-injector,
the label indicates that such solution can be used over a period of
2-24 hours or greater. The products are useful for human
pharmaceutical product use.
[0211] The formulations used in the method of the present invention
can be prepared by a process that comprises mixing an
anti-IL-12/IL-23p40 or IL-23 antibody and a selected buffer,
preferably, a phosphate buffer containing saline or a chosen salt.
Mixing the anti-IL-23 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.
[0212] The method of the invention provides pharmaceutical
compositions comprising various formulations useful and acceptable
for administration to a human or animal patient. Such
pharmaceutical compositions are prepared using water at "standard
state" as the diluent and routine methods well known to those of
ordinary skill in the art. For example, buffering components such
as histidine and histidine monohydrochloride hydrate, may be
provided first followed by the addition of an appropriate,
non-final volume of water diluent, sucrose and polysorbate 80 at
"standard state." Isolated antibody may then be added. Last, the
volume of the pharmaceutical composition is adjusted to the desired
final volume under "standard state" conditions using water as the
diluent. Those skilled in the art will recognize a number of other
methods suitable for the preparation of the pharmaceutical
compositions.
[0213] The pharmaceutical compositions may be aqueous solutions or
suspensions comprising the indicated mass of each constituent per
unit of water volume or having an indicated pH at "standard state."
As used herein, the term "standard state" means a temperature of
25.degree. C.+/-2.degree. C. and a pressure of 1 atmosphere. The
term "standard state" is not used in the art to refer to a single
art recognized set of temperatures or pressure, but is instead a
reference state that specifies temperatures and pressure to be used
to describe a solution or suspension with a particular composition
under the reference "standard state" conditions. This is because
the volume of a solution is, in part, a function of temperature and
pressure. Those skilled in the art will recognize that
pharmaceutical compositions equivalent to those disclosed here can
be produced at other temperatures and pressures. Whether such
pharmaceutical compositions are equivalent to those disclosed here
should be determined under the "standard state" conditions defined
above (e.g. 25.degree. C.+/-2.degree. C. and a pressure of 1
atmosphere).
[0214] Importantly, such pharmaceutical compositions may contain
component masses "about" a certain value (e.g. "about 0.53 mg
L-histidine") per unit volume of the pharmaceutical composition or
have pH values about a certain value. A component mass present in a
pharmaceutical composition or pH value is "about" a given numerical
value if the isolated antibody present in the pharmaceutical
composition is able to bind a peptide chain while the isolated
antibody is present in the pharmaceutical composition or after the
isolated antibody has been removed from the pharmaceutical
composition (e.g., by dilution). Stated differently, a value, such
as a component mass value or pH value, is "about" a given numerical
value when the binding activity of the isolated antibody is
maintained and detectable after placing the isolated antibody in
the pharmaceutical composition.
[0215] Competition binding analysis is performed to determine if
the IL-12/IL-23p40 or IL-23 specific mAbs bind to similar or
different epitopes and/or compete with each other. Abs are
individually coated on ELISA plates. Competing mAbs are added,
followed by the addition of biotinylated hrIL-12 or IL-23. For
positive control, the same mAb for coating may be used as the
competing mAb ("self-competition"). IL-12/IL-23p40 or IL-23 binding
is detected using streptavidin. These results demonstrate whether
the mAbs recognize similar or partially overlapping epitopes on
IL-12/IL-23p40 or IL-23.
[0216] One aspect of the method of the invention administers to a
patient a pharmaceutical composition comprising
[0217] In one embodiment of the pharmaceutical compositions, the
isolated antibody concentration is from about 77 to about 104 mg
per ml of the pharmaceutical composition. In another embodiment of
the pharmaceutical compositions the pH is from about 5.5 to about
6.5.
[0218] The 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-23 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.
[0219] Other formulations or methods of stabilizing the anti-IL-23
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-23 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(.beta.-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. 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.
[0220] 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. 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
9916419. 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.
[0221] An anti-IL-23 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.
Therapeutic Applications
[0222] The present invention also provides a method for modulating
or treating lupus, in a cell, tissue, organ, animal, or patient, as
known in the art or as described herein, using at least one IL-23
antibody of the present invention, e.g., administering or
contacting the cell, tissue, organ, animal, or patient with a
therapeutic effective amount of IL-12/IL-23p40 or IL-23 specific
antibody.
[0223] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising an anti-IL-23 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-23 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, eternacept (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 flurorquinolone, 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.
Therapeutic Treatments
[0224] Typically, treatment of lupus is affected by administering
an effective amount or dosage of an anti-IL-12/23p40 or anti-IL-23
antibody composition that total, on average, a range from at least
about 0.01 to 500 milligrams of an anti-IL-12/23p40 or anti-IL-23
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
administrations. 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.
[0225] Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
and/or 100-500 mg/kg/administration, or any range, value or
fraction thereof, or to achieve a serum concentration of 0.1, 0.5,
0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0,
4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5,
8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9,
13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9,
7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,
11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5,
15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5,
19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000,
4500, and/or 5000 .mu.g/ml serum concentration per single or
multiple administration, or any range, value or fraction
thereof.
[0226] 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.
[0227] As a non-limiting example, treatment of humans or animals
can be provided as a one-time or periodic dosage of at least one
antibody of the present invention 0.1 to 100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or, alternatively or additionally, at least one of
week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or
52, or, alternatively or additionally, at least one of 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years,
or any combination thereof, using single, infusion or repeated
doses.
[0228] 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.
[0229] 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 1-10% human serum albumin.
[0230] 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.
[0231] Suitable pharmaceutical carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
Alternative Administration
[0232] Many known and developed modes can be used according to the
present invention for administering pharmaceutically effective
amounts of an anti-IL-23 antibody. 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-12/IL-23p40 or IL-23 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.
Parenteral Formulations and Administration
[0233] 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.
Alternative Delivery
[0234] The invention further relates to the administration of an
anti-IL-12/IL-23p40 or IL-23 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. An anti-IL-12/IL-23p40 or IL-23 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).
[0235] 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. Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
Example: Manufacturing Processes to Produce STELARA.RTM.
(ustekinumab)
Background
[0236] STELARA.RTM. (ustekinumab) is a fully human G1 kappa
monoclonal antibody that binds with high affinity and specificity
to the shared p40 subunit of human interleukin (IL)-12 and IL-23
cytokines. Ustekinumab comprises a heavy chain of the amino acid
sequence of SEQ ID NO: 10 and a light chain of the amino acid
sequence of SEQ ID NO: 11; a heavy chain variable domain amino acid
sequence of SEQ ID NO:7 and a light chain variable domain amino
acid sequence of SEQ ID NO:8; the heavy chain CDR amino acid
sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3; and the
light chain CDR amino acid sequences of SEQ ID NO:4, SEQ ID NO:5,
and SEQ ID NO:6. The binding of ustekinumab to the IL-12/23p40
subunit blocks the binding of IL-12 or IL-23 to the IL-12R.beta.1
receptor on the surface of natural killer and CD4.sup.+ T cells,
inhibiting IL-12- and IL-23-specific intracellular signaling and
subsequent activation and cytokine production. Abnormal regulation
of IL-12 and IL-23 has been associated with multiple
immune-mediated diseases.
[0237] To date, ustekinumab has received marketing approval
globally, including countries in North America, Europe, South
America, and the Asia-Pacific region, for the treatment of adult
patients including those with chronic moderate to severe plaque
psoriasis and/or active psoriatic arthritis, Crohn's disease (CD)
and ulcerative colitis (UC). Ustekinumab is also being evaluated in
a Phase 3 study for the treatment of active Systemic Lupus
Erythematosus (SLE).
Manufacturing Process Overview
[0238] STELARA.RTM. (ustekinumab) is manufactured in a 10-stage
process that includes continuous perfusion cell culture followed by
purification. An overview of the manufacturing process is provided
in FIG. 1.
[0239] As used herein, the terms "culture", "culturing",
"cultured", and "cell culture" refer to a cell population that is
suspended in a medium under conditions suitable to survival and/or
growth of the cell population. As will be clear from context to
those of ordinary skill in the art, these terms as used herein also
refer to the combination comprising the cell population and the
medium in which the population is suspended. Cell culture includes,
e.g., cells grown by batch, fed-batch or perfusion cell culture
methods and the like. In certain embodiments, the cell culture is a
mammalian cell culture.
[0240] Cell lines for use in the present invention include
mammalian cell lines including, but not limited to, Chinese Hamster
Ovary cells (CHO cells), human embryonic kidney cells (HEK cells),
baby hamster kidney cells (BHK cells), mouse myeloma cells (e.g.,
NS0 cells and Sp2/0 cells), and human retinal cells (e.g., PER.C6
cells).
[0241] As used herein, the terms "chemically defined medium",
"chemically defined media", "chemically defined hybridoma medium",
or "chemically defined hybridoma media" refer to a synthetic growth
medium in which the identity and concentration of all the
components are known. Chemically defined media do not contain
bacterial, yeast, animal, or plant extracts, animal serum or plasma
although they may or may not include individual plant or
animal-derived components (e.g., proteins, polypeptides, etc).
Chemically defined media may contain inorganic salts such as
phosphates, sulfates, and the like needed to support growth. The
carbon source is defined, and is usually a sugar such as glucose,
lactose, galactose, and the like, or other compounds such as
glycerol, lactate, acetate, and the like. While certain chemically
defined media also use phosphate salts as a buffer, other buffers
may be employed such as citrate, triethanolamine, and the like.
Examples of commercially available chemically defined media
include, but are not limited to, ThermoFisher's CD Hybridoma Medium
and CD Hybridoma AGT.TM. Medium, various Dulbecco's Modified
Eagle's (DME) mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc),
Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc),
combinations thereof, and the like. Methods of preparing chemically
defined mediums are known in the art, for example in U.S. Pat. Nos.
6,171,825 and 6,936,441, WO 2007/077217, and U.S. Patent
Application Publication Nos. 2008/0009040 and 2007/0212770.
[0242] The term "bioreactor" as used herein refers to any vessel
useful for the growth of a cell culture. The bioreactor can be of
any size so long as it is useful for the culturing of cells. In
certain embodiments, such cells are mammalian cells. Typically, the
bioreactor will be at least 1 liter and may be 10, 100, 250, 500,
1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any
volume in between. The internal conditions of the bioreactor,
including, but not limited to pH and temperature, are optionally
controlled during the culturing period. The bioreactor can be
composed of any material that is suitable for holding mammalian
cell cultures suspended in media under the culture conditions of
the present invention, including glass, plastic or metal. The term
"production bioreactor" as used herein refers to the final
bioreactor used in the production of the polypeptide or
glycoprotein of interest. The volume of the production bioreactor
is typically at least 500 liters and may be 1,000, 2,500, 5,000,
8,000, 10,000, 12,000 liters or more, or any volume in between. One
of ordinary skill in the art will be aware of and will be able to
choose suitable bioreactors for use in practicing the present
invention.
[0243] Preculture, expansion, and production of ustekinumab are
performed in Stage 1 and Stage 2. In Stage 1, preculture is
initiated from one or more working cell bank vials of transfected
Sp2/0 cells expressing the HC and LC sequences of ustekinumab and
expanded in culture flasks, disposable culture bags, and a 100 L
seed bioreactor. The cells are cultured until the cell density and
volume required for inoculation of a 500 L production bioreactor
are obtained. In Stage 2, the cell culture is perfused in a 500 L
production bioreactor using an alternating tangential flow (ATF)
hollow fiber filter cell retention system. Cell culture permeate
(harvest) is collected from the ATF system while cells are retained
within the bioreactor and the culture is replenished with fresh
medium. Harvest from one or more 500 L production bioreactors may
be combined in Stage 3. The harvests are purified using MabSelect
Protein A resin affinity chromatography. The resultant direct
product capture (DPC) eluate is frozen until further
processing.
[0244] Purification of ustekinumab from DPC is performed in Stage 4
through Stage 8 by ion exchange chromatography steps and other
steps to inactivate or remove potential virus contamination
(solvent/detergent [S/D] treatment and virus removal filtration).
DPC eluates are thawed, pooled and filtered in Stage 4 and
incubated with Tri-n-butyl Phosphate (TNBP) and polysorbate 80 S/D
treatment in Stage 5 to inactivate any lipid-enveloped viruses
present. TNBP and polysorbate 80 reagents, aggregates, and
impurities are removed from ustekinumab in Stage 6, using SPXL.RTM.
sepharose cation exchange resin chromatography. Ustekinumab is
further purified using QXL.RTM. sepharose anion exchange resin
chromatography in Stage 7 to remove DNA, viruses, and impurities.
In Stage 8, the purified ustekinumab is diluted and filtered
through a virus retentive filter (NFP.RTM.).
[0245] Preparation of the ustekinumab pre-formulated bulk (PFB) and
formulated bulk (FB) is performed in Stages 9 and 10, respectively.
In Stage 9, the ultrafiltration step concentrates the ustekinumab
and the diafiltration step adds the formulation excipients and
removes the in-process buffer salts. Polysorbate 80 is added to the
ustekinumab PFB in Stage 10 to obtain the FB. The FB is filtered
into polycarbonate containers for frozen storage. The frozen FB is
packaged in insulated containers with dry ice for transport to the
drug product manufacturing site.
Detailed Description of Cell Culture in Large-Scale Manufacturing
Process
Stage 1
Preculture and Expansion
[0246] The first stage in the production of ustekinumab is the
initiation of preculture from a Working Cell Bank (WCB) vial of
transfected Sp2/0 cells expressing the HC and LC sequences of
ustekinumab and expanded in culture flasks, disposable culture
bags, and a 100 L seed bioreactor. The cells are cultured until the
cell density and volume required for inoculation of a 500 L
production bioreactor are obtained. A flow diagram depicting the
preculture and expansion process is provided in FIG. 2.
Manufacturing Procedure
[0247] One or more cryopreserved vials of WCB are thawed and
diluted with CD (chemically defined) hybridoma medium supplemented
with 6 mM L-glutamine, 0.5 mg/L mycophenolic acid, 2.5 mg/L
hypoxanthine, and 50 mg/L xanthine (CDH-A). The culture viability
must be .gtoreq.45%. The cells are further diluted with CDH-A in a
culture flask to a seeding density of 0.2 to 0.5.times.10.sup.6
viable cells (VC)/mL. The preculture is maintained in a humidified
CO2 incubator, with temperature, CO.sub.2 concentration, and
agitation controlled within ranges defined in the batch record. The
preculture is incubated for .ltoreq.3 days until a minimum cell
density of .gtoreq.0.6.times.10.sup.6 VC/mL and a culture viability
of .gtoreq.50% are obtained. The preculture is expanded
sequentially in a series of culture flasks and then culture bags as
a mechanism to scale up for inoculation of the 100 L seed
bioreactor. During the culture expansion phase, each incubation
step takes .ltoreq.3 days to achieve passage conditions, which
require a cell density of .gtoreq.0.6.times.10.sup.6 VC/mL and a
culture viability of .gtoreq.80%. The seeding density for each
passage is 0.2 to 0.5.times.10.sup.6 VC/mL in culture flasks, and
0.2 to 0.6.times.10.sup.6 VC/mL in culture bags. Each passage is
sampled for viable cell density (VCD), culture viability, and
microscopic examination. Prior to inoculation of the 100 L seed
bioreactor, the preculture is sampled for bioburden.
[0248] Preculture expansions may be maintained for a maximum of 30
days post-thaw. Precultures not used within 30 days are discarded.
Back-up precultures, expanded as described above and subject to the
same in-process monitoring, control tests, and process parameters
as the primary precultures, may be maintained and used to inoculate
another 100 L seed bioreactor as needed
[0249] When the preculture meets inoculum criteria, the contents of
the culture bag(s) are transferred to the 100 L seed bioreactor
containing CDH-A to target a seeding density of
.gtoreq.0.3.times.10.sup.6 VC/mL. The seed bioreactor culture pH,
temperature, and dissolved oxygen concentration are controlled
within ranges defined in the batch record. The culture is expanded
until a cell density of .gtoreq.1.5.times.10.sup.6 VC/mL and a
culture viability of .gtoreq.80% are obtained. The culture is
sampled for VCD, culture viability, and microscopic examination
throughout the seed bioreactor process. Prior to inoculation of the
500 L production bioreactor, the culture is sampled for
bioburden.
[0250] When the VCD of the seed bioreactor culture reaches
.gtoreq.1.5.times.10.sup.6 VC/mL, the culture may be used to
inoculate the 500 L production bioreactor. Alternatively, a portion
of the culture can be drawn from the 100 L seed bioreactor and the
remaining culture diluted with fresh medium. Following this "draw
and fill" process, the culture is allowed to expand to sufficient
cell density to inoculate the 500 L production bioreactor. The
maximum duration of the 100 L seed bioreactor culture is 9 days
post-inoculation.
Stage 2
Bioreactor Production
[0251] In Stage 2, cell culture is continuously perfused in a 500 L
production bioreactor using an alternating tangential flow hollow
fiber filter cell retention system (ATF system). Cell culture
permeate (harvest) is collected from the ATF system while cells are
returned to the bioreactor, and the culture is replenished with
fresh medium. A flow diagram depicting the bioreactor production
process is provided in FIG. 3.
Manufacturing Procedure
[0252] The inoculation of the 500 L production bioreactor is
performed by transferring the contents of the 100 L seed bioreactor
into the 500 L production bioreactor containing CD (chemically
defined) hybridoma medium supplemented with 6 mM L-glutamine, 0.5
mg/L mycophenolic acid, 2.5 mg/L hypoxanthine, and 50 mg/L xanthine
(CDH-A). The volume transferred must be sufficient to target a
seeding density of .gtoreq.0.3.times.10.sup.6 viable cells (VC)/mL.
The culture is maintained at a temperature of 34 to 38.degree. C.,
a pH of 6.8 to 7.6, and a dissolved oxygen (DO) concentration of 1
to 100%.
[0253] Continuous perfusion is initiated, and culture is drawn from
the 500 L bioreactor into the ATF system to separate the cells from
the permeate. The permeate is filtered through the 0.2 m ATF filter
and collected as harvest in bioprocess containers (BPCs). The cells
are returned to the bioreactor, and fresh CDH-A is supplied to
maintain a constant culture volume. Viable cell density (VCD),
culture viability, pH, DO, temperature and immunoglobulin G (IgG)
content are monitored during the production run. The perfusion rate
is gradually increased in proportion to VCD until a target rate of
approximately one bioreactor volume per day is reached. The
perfusion rate is controlled, not to exceed 1.20 bioreactor volumes
per day. Retention of the ATF system is monitored to facilitate
shutdown of an ATF filter prior to the IgG retention across the
filter exceeding 50%.
[0254] When the VCD within the 500 L bioreactor reaches
8.0.times.10.sup.6 VC/mL or on day 10, whichever comes first, the
pH target is lowered from 7.2 to 7.1. Biomass removal is initiated
at either day 20 or when a VCD of 12.0.times.10.sup.6 VC/mL is
reached, whichever comes first. Biomass is removed from the 500 L
production bioreactor into BPCs at a rate of up to 20% bioreactor
volumes per day. Each harvest is sampled for bioburden.
[0255] The continuous perfusion cell culture operation in the 500 L
production bioreactor continues for up to 46 days post-inoculation.
At the end of production, the culture is sampled for mycoplasma and
adventitious virus testing. Harvest may be stored for .ltoreq.30
days at 2 to 8.degree. C. after disconnection from the
bioreactor.
Description for Small-Scale Production of Ustekinumab Expressed in
CHO Cells
Generation of CHO Cells Expressing Ustekinumab
[0256] The CHO cell line was originally created by T. T. Puck from
the ovary of an adult Chinese hamster. CHO-K1 (ATCC.RTM. CCL-61) is
a subclone of the parental CHO cell line that lacks the proline
synthesis gene. CHO-K1 was also deposited at the European
Collection of Cell Cultures, CHO-K1 (ECACC 85051005). A master cell
bank (MCB) of CHO-K1, 024 M, was established at Celltech Biologics
(now Lonza Biologics) and used for adaptation of CHO-K1 to
suspension culture and serum-free medium. The adapted cell line was
named CHOKiSV. The CHOKiSV cell line was further adapted in
protein-free medium to create a MCB of cells referred to as 269-M.
Cells derived from the 269-M MCB were transfected as described
below to create the CHO cell lines expressing ustekinumab.
[0257] Cell lines were generated, expanded, and maintained in a
humidified incubator at 37.degree. C. and 5% CO.sub.2 using cell
culture plates and shake flasks. Routine seeding density in shake
flasks was 3.times.10.sup.5 viable cells per mL (vc/mL). All shake
flask cultures were maintained at 130 revolutions per minute (rpm)
with a 25 mm orbit and 96-deepwell (DW, Thermo Scientific, Waltham,
Mass., Cat. #278743) cultures were maintained at 800 rpm with a 3
mm orbit.
[0258] CHO clones expressing ustekinumab were created using media
identified as MACH-1, an in-house developed, chemically-defined
medium for CHO cell culture. The basal medium for the routine
passage of the CHO host cell line was MACH-1 supplemented with 6 mM
L-glutamine (Invitrogen, Carlsbad, Calif., Cat. #25030-081). CHO
cells transfected with the glutamine synthetase (GS) gene were
grown in MACH-1+MSX unless otherwise noted, which is MACH-1
supplemented with 25 .mu.M L-methionine sulfoximine (MSX, Sigma,
St. Louis, Mo., Cat. # M5379-1G) to inhibit glutamine synthetase
function. For bolus fed-batch shake flask and bioreactor
experiments, cells were cultured in MACH-1+F8, which is MACH-1
supplemented with 8 g/kg F8 (a supplement of proprietary growth
enhancers) to further support cell growth and antibody production.
Proprietary feed media were used in shake flask and bioreactor
experiments.
[0259] The DNA encoding the genes of interest were cloned into a
glutamine-synthetase (GS) double gene expression plasmid (Lonza
Biologics). Expression of the heavy chain (HC) and light chain (LC)
genes were driven by separate human cytomegalovirus (hCMV-MIE)
promoters. The GS gene selection marker, driven by the Simian Virus
SV40 promoter, allows for the selection of transfected cells in
glutamine-free media in the presence of MSX.
[0260] Prior to each transfection, 1 aliquot of plasmid DNA,
containing both the HC and LC coding regions of ustekinumab, was
linearized by restriction enzyme digestion. A linearized 15 .mu.g
DNA aliquot was transfected into a 1.times.10.sup.7 cell aliquot
using the BTX ECM 830 Electro Cell Manipulator (Harvard Apparatus,
Holliston, Mass.). Cells were electroporated 3 times at 250 volts
with 15 millisecond pulse lengths and 5 second pulse intervals in a
4 mm gap cuvette. Transfected cells were transferred to
MACH-1+L-glutamine in a shake flask and incubated for 1 day.
Transfections were centrifuged, then resuspended in MACH-1+25 uM
MSX for selection and transferred to shake flasks to incubate for 6
days.
[0261] Following chemical selection, cells were plated in a single
cell suspension in custom glutamine-free Methocult medium
containing 2.5% (w/v) methylcellulose in a Dulbecco's Modified
Eagle's Medium (DMEM) base media (Methocult, StemCell Technologies,
Inc., Vancouver, BC, Cat. #03899). The working solution also
contained 30% (v/v) gamma-irradiated dialyzed fetal bovine serum
(dFBS.IR, Hyclone, Logan, Utah, Cat. # SH30079.03), ix GS
Supplement (SAFC, St. Louis, Mo., Cat. #58672-100M), 1.5 mg animal
component-free Protein G Alexa Fluor 488 conjugate (Protein G,
Invitrogen, Carlsbad, Calif., Cat. # C47010), 25 .mu.M MSX,
Dulbecco's Modified Eagle's Medium with F12 (DMEM/F12,
Gibco/Invitrogen, Carlsbad, Calif., Cat. #21331-020), and cell
suspension.
[0262] Protein G recognizes human monoclonal antibodies and binds
to the IgG that is secreted by the cells. The Protein G is
conjugated to the fluorescent label Alexa Fluor 488, so that cell
colonies secreting the most antibodies will show the highest levels
of fluorescence. After incubation for 12 to 18 days, colonies with
the highest fluorescence levels were picked into 100 .mu.L phenol
red-containing MACH-1+MSX in 96-well plates using a ClonePix FL
colony picking instrument (Molecular Devices, Sunnyvale, Calif.)
and incubated without shaking for 5-7 days. After 5-7 days, cells
from the 96-well plate were expanded by adding to 50-100 .mu.L
phenol red-containing MACH-1+MSX in a 96DW plate (Thermo
Scientific, Waltham, Mass., Cat. #278743) and shaken at 800 rpm
with a 3 mm orbit. The 96DW plates were fed and at 7 days post 96DW
seeding were titered via Octet (ForteBio, Menlo Park, Calif.). The
top 10 cultures corresponding to the highest batch 96DW overgrow
titers were expanded to shake flasks in MACH-1+MSX, and frozen cell
banks were created with cells suspended in in MACH-1+MSX medium
containing 10% DMSO.
Cell Culture for Small-Scale Production
[0263] As in large-scale production of ustekinumab expressed in
Sp2/0 cells, preculture, cell expansion, and cell production are
performed in Stages 1 and 2 for small-scale production of
ustekinumab expressed in Chinese Hamster Ovary cells (CHO cells).
In Stage 1, preculture is initiated from a single cell bank vial of
transfected CHO cells expressing the HC and LC sequences of
ustekinumab and the cells are expanded in culture flasks. The cells
are cultured until the cell density and volume required for
inoculation of a 10-L production bioreactor are obtained. In Stage
2, the cell culture is run in fed-batch mode in a 10-L production
bioreactor. For the duration of the 15-day bioreactor run the
culture is fed as required with concentrated glucose-based and
amino acid-based feeds. At the completion of the production
bioreactor run cell culture harvest is clarified to remove biomass
and filtered for further processing.
Purification for Small-Scale Production
[0264] The purification steps for small-scale production of
ustekinumab were identical to the large-scale manufacturing
process, except the Stage 8 virus filtration step was omitted for
small-scale production. In brief, for small-scale production,
purification of ustekinumab from the cell culture harvest is
performed in Stages 3 through 7 by a combination of affinity and
ion exchange chromatography steps and steps to inactivate or remove
potential virus contamination (solvent/detergent treatment and
virus removal). In Stage 3, harvest and/or pooled harvest is
clarified and purified using Protein A affinity chromatography. The
resultant direct product capture (DPC) eluate is frozen until
further processing. DPC eluates are filtered and pooled in Stage 4
following thaw, and subsequently treated in Stage 5 with
tri-n-butyl phosphate (TNBP) and polysorbate 80 (PS 80) to
inactivate any lipid-enveloped viruses potentially present.
[0265] In Stage 6, TNBP and PS 80 reagents and impurities are
removed from the ustekinumab product using cation exchange
chromatography. The ustekinumab product is further purified using
anion exchange chromatography in Stage 7 to remove DNA, potentially
present viruses, and impurities. As noted above, Stage 8 filtering
through a virus retentive filter was omitted from the CHO derived
ustekinumab product purification process.
[0266] Final preparation of ustekinumab pre-formulated bulk (PFB)
and formulated bulk (FB) is performed in Stages 9 and 10,
respectively (references to large-scale stages). In Stage 9, the
ultrafiltration step concentrates the ustekinumab product, and the
diafiltration step adds the formulation excipients and removes the
in-process buffer salts. Polysorbate 80 is added to the ustekinumab
PFB in Stage 10 to obtain the FB and the FB is filtered into
polycarbonate containers for frozen storage.
Methods
Methods for Determining Viable Cell Density (VCD) and %
Viability
[0267] Total cells per/ml, viable cells/ml (VCD), and % viability
are typically determined with a Beckman Coulter Vi-CELL-XR cell
viability analyzer using manufacturer provided protocols, software
and reagents. Alternatively, a CEDEX automated cell counting system
has also been used. It should also be noted, however, that other
methods for determining VCD and % viability are well known by those
skilled in the art, e.g., using a hemocytometer and trypan blue
exclusion.
Bioactivity Assay
[0268] The bioactivity of ustekinumab is determined by
neutralization of IL-12 induced interferon-gamma (IFN-.gamma.)
production by an IL-12-responsive human natural killer cell line,
NK-92MI (ATCC.RTM. CRL-2408). Ustekinumab binds the p40 subunit of
IL-12 and impedes the interaction with the IL-12R.beta.1 on the
cell surface of NK cells. This results in the blockade of IL-12
mediated production of IFN-.gamma. (Aggeletopoulou I, et al.
Interleukin 12/interleukin 23 pathway: Biological basis and
therapeutic effect in patients with Crohn's disease. World J
Gastroenterol. 2018; 24(36):4093-4103). In brief, the assay method
involves incubating NK-92MI cells with recombinant human IL-12
(rhIL-12) and comparing the levels of IFN-.gamma. secreted by the
cells in the presence and absence of ustekinumab. The levels of
IFN-.gamma. are quantified with an enzyme-linked immunosorbent
assay (ELISA) using an anti-IFN-.gamma. antibody (see, e.g.,
Jayanthi S, et al. Modulation of Interleukin-12 activity in the
presence of heparin. Sci Rep. 2017; 7(1):5360).
Methods for Determining Oligosaccharide Composition
Oligosaccharide Composition by HPLC
[0269] The N-linked oligosaccharide composition of ustekinumab is
determined with a normal phase anion exchange HPLC method with
fluorescent detection using an Agilent 1100/1200 Series HPLC System
with Chemstation/Chemstore software. To quantitate the relative
amounts of glycans, the N-linked oligosaccharides are first cleaved
from the reduced and denatured test article with N-glycanase
(PNGase F). The released glycans are labeled using anthranilic
acid, purified by filtration using 0.45-.mu.m nylon filters, and
analyzed by HPLC with fluorescence detection. The HPLC chromatogram
serves as a map that can be used to identify and quantitate the
relative amounts of N-linked oligosaccharides present in the
sample. Glycans are identified by co-elution with oligosaccharide
standards and by retention time in accordance with historical
results from extensive characterizations. A representative HPLC
chromatogram for ustekinumab is shown in FIG. 4.
[0270] The amount of each glycan is quantitated by peak area
integration and expressed as a percentage of total glycan peak area
(peak area %). Results are reported for G0F, G1F, G2F, total
neutrals, and total charged glycans. Other neutrals are the sum of
all integrated peaks between 17 and 35 minutes, excluding the peaks
corresponding to G0F, G1F and G2F. Total neutral glycans is the sum
of G0F, G1F, G2F and the other neutrals. Total charged glycans is
the sum of all mono-sialylated glycan peaks eluting between 42 and
55 minutes and all di-sialylated glycan peaks eluting between 78
and 90 minutes.
[0271] A mixture of oligosaccharide standards (G0F, G2F,
G2F+N-acetylneuraminic acid (NANA) and G2F+2NANA) is analyzed in
parallel as a positive control for the labeling reaction, as
standards for peak identification, and as a measure of system
suitability. Reconstituted oligosaccharides from Prozyme, G0F (Cat.
No. GKC-004301), G2F (Cat. No. GKC-024301), SA1F (Cat. No.
GKC-124301), and SA2F (Cat. No. GKC-224301), or equivalent, are
used as reference standards. A method blank negative control and
pre-labeled G0F standard are also run for system suitability
purposes. The following system suitability and assay (test article)
acceptance criteria are applied during the performance of the
oligosaccharide mapping procedure in order to yield a valid
result:
System Suitability Criteria:
[0272] Resolution (USP) between the G0F and G2F peaks in the
oligosaccharide standard must be .gtoreq.3.0. [0273] Theoretical
plate count (tangent method) of the G0F peak in the oligosaccharide
standards must be .gtoreq.5000. [0274] The total glycan peak area
for the ustekinumab reference standard must be .gtoreq.1.5 times of
the major glycan peak area of the pre-labeled G0F. [0275] If any
reference standard glycan peak is off-scale, the reference standard
is re-injected with less injection volume [0276] The retention time
of G0F peak in the ustekinumab reference standard must be within
0.4 min of the G0F retention time in the oligosaccharide
standards.
Assay Acceptance Criteria:
[0276] [0277] The method blank must have no detectable peaks that
co-elute with assigned oligosaccharide peaks in ustekinumab. [0278]
The total glycan peak area of each test article must be .gtoreq.1.5
times the major glycan peak area of the pre-labeled G0F standard.
[0279] If any sample glycan peak is off-scale, that sample is
re-injected with less injection volume, together with pre-labeled
G0F, the oligosaccharide standards, Method Blank and reference
standard with normal volume. [0280] The retention time for the G0F
peak in each test article must be within 0.4 min of the retention
time for the G0F peak in the oligosaccharide standards. [0281] If
the assay fails to meet any acceptance criteria, the assay is
invalidated
Oligosaccharide Composition by IRMA
[0282] The IdeS-RMA (IRMA) method allows differentiation between
major glycoforms by Reduced Mass Analysis (RMA) after the enzymatic
treatment of immunoglobulin G (IgG) with FabRICATOR.RTM., an IgG
degrading enzyme of Streptococcus pyogenes (IdeS) available from
Genovis AB (SKU: A0-FR1-050). See also, for example, U.S. Pat. No.
7,666,582. Reduced Mass Analysis (RMA) involves disulfide bond
reduction of antibodies followed by the intact mass analysis of the
heavy chain of the antibody and its attached glycan moieties. Some
antibodies show a large degree of heterogeneity due to the presence
of N-terminal modifications such as pyroglutamate formation and
carboxylation. Consequently, disulfide reduction and heavy chain
mass measurement results in a complex pattern of deconvoluted
peaks. Therefore, in some applications, proteolytic generation of
antibody fragments is desired over generation of light and heavy
chains using reduction agents such as dithiothreitol (DTT).
Traditionally papain and pepsin are used to generate antibody
fragments all of which are laborious processes. Cleavage of IgG
with pepsin requires extensive optimization and it is done at low
acidic pH. Papain needs an activator and both F(ab').sub.2 and Fab
can be obtained depending on the reaction conditions resulting in a
heterogeneous pool of fragments. These drawbacks can be
circumvented by using the novel enzyme, FabRICATOR.RTM.. The
cleavage procedure is very fast, simple, and importantly no
optimization is needed. It is performed at neutral pH generating
precise F(ab').sub.2 and Fc fragments. No further degradation or
over-digestion is observed as is commonly associated with other
proteolytic enzymes like pepsin or papain. Importantly, as
FabRICATOR.RTM. cleaves just C-terminally of the disulfide bridges
in the heavy chain, no reduction step is required and an intact
F(ab').sub.2 and two residual Fc fragments are obtained.
Definitions
[0283] H: hexose (mannose, glucose, and galactose) [0284] Man5:
mannose 5 [0285] N: N-acetylhexosamine (N-acetylglucosamine and
N-acetylgalactosamine) [0286] F: fucose [0287] S: sialic acid
(N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid
(NGNA)) [0288] G0: asialo-agalacto-afucosylated biantennary
oligosaccharide [0289] G0F: asialo-agalacto-fucosylated biantennary
oligosaccharide [0290] G1: asialo-monogalactosylated-afucosylated
biantennary oligosaccharide [0291] G1F:
asialo-monogalactosylated-fucosylated biantennary oligosaccharide
[0292] G2: asialo-digalactosylated-afucosylated biantennary
oligosaccharide [0293] G2F: asialo-digalactosylated-fucosylated
biantennary oligosaccharide [0294] GlcNAc: N-Acetyl-D-Glucosamine
[0295] Lys: Lysine [0296] -Lys: Truncated heavy chain (no
C-terminal Lysine residue present) [0297] +Lys: Heavy chain
containing C-terminal Lysine [0298] ppm: parts per million
Equipment
[0298] [0299] Thermo Scientific Q Exactive (Plus) mass spectrometer
[0300] Agilent 1200 HPLC system [0301] Applied Biosystems POROS
R2/10 2.1 mmD.times.100 mmL column [0302] Thermo Scientific Q
Exactive Tune software [0303] Thermo Scientific Protein
Deconvolution software [0304] Analytical balance capable of
weighing 0.01 mg [0305] Vortex mixer, any suitable model [0306]
Water bath or heating block, any suitable model [0307] Calibrated
Thermometer--10 to 110.degree. C., any suitable model [0308]
Calibrated Pipettes [0309] Microcentrifuge, any suitable model
Procedure
IdeS Digestion of Samples
[0309] [0310] samples (equal to 50 .mu.g IgG). [0311] add 1 .mu.l
(50 units) of IdeS enzyme to 50 .mu.g of IgG, vortex briefly, spin
down, and incubate at 37.degree. C. for 30 minutes (stock enzyme @
5000 units per 100 al. 1 unit of enzyme fully digests 1 .mu.g of
IgG in 30 minutes at 37.degree. C.) [0312] spin down samples and
transfer to LC-MS vials, and load sample vials into Agilent 1200
autosampler
LC-MS Method
Solution Preparation
[0312] [0313] Mobile phase A (0.1% Formic Acid (FA) in ultrapure
water)--Add 999 mL of ultrapure water to a 1 L HPLC Mobile phase
bottle, add 1 mL FA and stir. This solution can be stored at RT for
2 months. [0314] Mobile phase B (0.1% FA, 99.9% acetonitrile)--Add
999 mL of acetonitrile to a 1 L HPLC Mobile phase bottle, add 1 mL
FA and stir. This solution can be stored at RT for 2 months.
LC Method
[0314] [0315] Column: Applied Biosystems POROS R2/10 2.1
mmD.times.100 mmL [0316] Column temperature: 60.degree. C. [0317]
Auto sampler temperature: 4.degree. C. [0318] Flow rate: 300
.mu.L/min [0319] Injection volume: 5 .mu.L [0320] Mobile phase A:
0.1% FA in ultrapure water [0321] Mobile phase B: 0.1% FA in
acetonitrile
TABLE-US-00013 [0321] TABLE 1 LC Gradient Table Time (min) % Mobile
phase B 0.0 10 6.0 30 11.9 42 12.0 95 15.9 95 16.0 10 21.0 10
MS Method
[0322] Scan Parameters: [0323] Scan type: Full MS [0324] Scan
range: 700 to 3500 m/z [0325] Fragmentation: In-source CID 35.0 eV
[0326] Resolution: 17500 [0327] Polarity: Positive [0328] Lock
masses: On, m/z 445.12002 [0329] AGC target: 3e6 [0330] Maximum
injection time: 250
[0331] HESI Source: [0332] Sheath gas flow rate: 32 [0333] Aux gas
flow rate: 7 [0334] Sweep gas flow rate: 0 [0335] Spray voltage
(IkVJ): 4.20 [0336] Capillary temp. (.degree. C.): 280 [0337]
S-lens RF level: 55.0 [0338] Heater temp. (.degree. C.): 80
Data Analysis
[0339] The relative content of each detected glycan species is
recorded based on analysis of deconvoluted mass spectra. FIG. 5
shows a representative deconvoluted mass spectrum for IRMA analysis
of ustekinumab produced in Sp2/0 cells. The major structures
determined by IRMA analysis include, e.g., G0 (H3N4), G0F (H3N4F1),
G1F-GlcNAc (H4N3F1), H5N3, G1 (H4N4), H5N3F1, G1F (H4N4F1), G2
(H5N4), G2F (H5N4F1), G1FS (H4N4F1S1), H6N4F1, G2FS (H5N4F1S1),
H7N4F1, H6N4F1S1, G2FS2 (H5N4F1S2). The percentage of each of these
structures is monitored. The measured peak intensity represents the
percentage of each structure after normalization (% of Total
Assigned). Glycans of which the observed mass is outside the 100
ppm mass deviation threshold are not included in the calculations,
e.g., (*G1F-GlcNAc-Lys, *H5N3-Lys, *G1-Lys, *H5N3F1-Lys, and
*G2-Lys). As noted, these are indicated with an asterisk ("*").
Also, Man5-Lys is not always detected in the spectra since it has a
very low intensity, nevertheless it is considered and included into
the calculations when present. The percentage of a glycan is
calculated as detected on both isoforms of the Fc fragment with and
without terminal Lysine, e.g., percentage G0F is (% G0F-Lys+%
G0F+Lys). Structures detected on only one of the heavy chain
isoforms are indicated with a double asterisk ("**"), e.g.,
**G1F-GlcNAc-Lys, **H5N3-Lys, **H5N4-Lys, and **H5N3F1+Lys. Most of
these structures are low abundant and cannot be resolved from
adjacent peaks with higher intensities or are below the detection
capabilities of the method. *Note: Differences between the HPLC and
IRMA methods (e.g., see Table 2 below) may result from co-elution
of species in HPLC and possibly underestimation of some sialylated
species by IRMA because some of the intensities are very close to
the limit of detection capabilities of the IRMA method.
TABLE-US-00014 TABLE 2 Glycan abundance comparison for IRMA and
HPLC for representative ustekinumab sample produced in Sp2/0 cells
Glycan Group IRMA % HPLC % G0F 21.6 25.0 G1F 28.5 33.2 G2F 9.2 7.8
Other neutral oligosaccharides 11.4 5.9 Total neutral
oligosaccharides 70.7 71.9 Monosialylated 25.0 25.9 Disialylated
4.3 2.2 Total charged oligosaccharides 29.3 28.1
Capillary Isoelectric Focusing
[0340] Capillary isoelectric focusing (cIEF) separates proteins on
the basis of overall charge or isoelectric point (p1). The method
is used to monitor the distribution of charge-based isoforms in
ustekinumab. Unlike the gel-based IEF procedures, cIEF provides a
quantitative measure of the charged species present. In addition,
cIEF shows increased resolution, sensitivity, and reproducibility
compared to the gel-based method. The assay is performed on a
commercially available imaging cIEF analyzer equipped with an
autosampler able to maintain sample temperature
.ltoreq.10.5.degree. C. in an ambient environment of
.ltoreq.30.degree. C., such as the Alcott autosampler (GP
Instruments, Inc.). The analysis employs an inner wall-coated
silica capillary without an outer wall polyimide coating to allow
for whole column detection. In addition, an anolyte solution of
dilute phosphoric acid and methylcellulose, a catholyte solution of
sodium hydroxide and methylcellulose, and a defined mixture of
broad range (pH 3-10) and narrow range (pH 8-10.5) ampholytes are
used. The assay employs a pre-treatment of both test articles and
Reference Standard (RS) with carboxypeptidase B (CPB) which removes
the heavy chain C-terminal lysine and eliminates ambiguities
introduced by the presence of multiple C-terminal variants. A
representative cIEF electropherogram of ustekinumab expressed in
Sp2/0 cells is shown in FIG. 6 and representative cIEF
electropherogram of ustekinumab expressed in CHO cells is shown in
FIG. 9.
[0341] Before each analysis, the autosampler temperature set-point
is set to 4.degree. C. and the autosampler is pre-cooled for at
least 30 minutes and the ambient room temperature of the lab is
maintained .ltoreq.30.degree. C. The pre-treated test article and
RS, sample vials, vial inserts, the reagents used in the assay
including purified water, the parent solution containing
N,N,N',N'-Tetramethylethylenediamine (TEMED) (which optimizes
focusing within the capillary), ampholytes, pI 7.6 and 9.5 markers
for internal standards and methylcellulose (MC) are kept on ice for
at least 30 minutes before starting sample preparation. The samples
are prepared on ice and the time of addition of the parent solution
is recorded and exposure to TEMED is controlled. The assay must be
completed within 180 minutes after this addition. System
suitability controls are injected once, and test articles and RS
are injected twice following the sequence table below (Table
3):
TABLE-US-00015 TABLE 3 Sample Running Sequence Sample Vial Number
of Sample Name Position Injections System Suitability 1 1 Blank 2 1
CPB Control 3 1 CPB Treated RS 4 2 CPB Treated Sample 1 5 2 CPB
Treated RS 6 2
[0342] After the samples are injected into the capillary by a
syringe pump, an electric field (3 kV) is applied across the
capillary for 8 min, forming a pH gradient, and charge-based
isoforms of ustekinumab are separated according to their
isoelectric point (pI). The protein isoforms in the capillary are
detected by imaging the entire capillary at 280 nm, and the data
are presented in the form of an electropherogram as a function of
pI value vs A280. Values for pI are assigned by comparison to the
internal pI standards (pI 7.6 and 9.5) using the instrument
software, and peak areas are determined from the electropherogram
using standard data acquisition software. The average pI and
average peak area percentage from duplicate injections of all peaks
.gtoreq.LOD, the .DELTA.pI value compared to Reference Standard,
and percent area peaks are reported.
Introduction to Manufacturing Control Strategies
[0343] During large-scale commercial production, manufacturing
control strategies are developed to maintain consistent drug
substance (DS) and drug product (DP) characteristics of therapeutic
proteins (e.g., therapeutic antibodies like ustekinumab), with
regard to oligosaccharide profile, bioactivity (potency), and/or
other characteristics of the DS and DP (e.g., See characteristics
identified in Table 4 and Table 5). For example, ustekinumab
glycosylation is monitored as an in-process control for formulated
bulk (FB) at Stage 10 of the manufacturing process, with upper and
lower specifications in place for mean % total neutral
oligosaccharides, % total charged oligosaccharides, and %
individual neutral oligosaccharide species, G0F, G1F, and G2F. As
used herein, the terms "drug substance" (abbreviated as "DS") and
"drug product" (abbreviated as "DP") refer to compositions for use
as commercial drugs, for example in clinical trials or as marketed
drugs. A DS is an active ingredient that is intended to furnish
pharmacological activity or other direct effect in the diagnosis,
cure, mitigation, treatment, or prevention of disease or to affect
the structure or any function of the human body. The formulated
bulk (FB) produced in the manufacturing process is the drug
substance (DS). A DP (also referred to as a medicinal product,
medicine, medication, or medicament) is a drug used in the
diagnosis, cure, mitigation, treatment, or prevention of disease or
to affect the structure or any function of the human body. The DP
is the DS that has been prepared as the medicinal product for sale
and/or administration to the patient.
[0344] As shown in Table 4, there are only very small differences
in % Monomer, % Purity, and % Bioactivity for ustekinumab produced
in Sp2/0 cells and CHO cells. However, there are substantial
differences in the cIEF profiles that are caused primarily by
differences in the oligosaccharide profile of ustekinumab produced
in Sp2/0 cells and CHO cells. For a comparison of cIEF profiles for
ustekinumab produced in Sp2/0 cells and CHO cells, see also, e.g.,
FIG. 6 and FIG. 9.
TABLE-US-00016 TABLE 4 Representative comparison of selected
ustekinumab characteristics expressed in Sp2/0 cells and CHO cells
Test Parameter Sp2/0 Cells CHO Cells DW-SE-HPLC % Monomer 99.75%
99.40% % Aggregate 0.23% 0.57% % Fragment <LOD <LOD cSDS
Reduced % Purity 98.9% 98.2% cSDS Non-Reduced % Purity 98.9% 97.3%
Bioactivity % Bioactivity NA .sup. 96%.sup.a cIEF peak A 0.6%
<LOD peak B 3.9% <LOD peak 1 12.6% 5.5% peak 2 28.7% 15.8%
peak 3 53.6% 76.9% peak C 0.8% 1.7% <LOD--below limit of
detection NA--Not Applicable for reference material produced in
Sp2/0 cells .sup.aCompared to reference material expressed in Sp2/0
cells
Oligosaccharide Profile of Ustekinumab
[0345] Ustekinumab is N-glycosylated at a single site on each heavy
chain, on asparagine 299. These N-linked oligosaccharide structures
can be any in a group of biantennary oligosaccharide structures
linked to the protein through the primary amine of the asparagine
residue, but on ustekinumab they consist primarily of biantennal
core-fucosylated species, with galactose and sialic acid
heterogeneity. Major individual oligosaccharide species include,
e.g., "G0F", an asialo, agalacto core-fucosylated biantennary
glycan, "G1F", an asialo, mono-galacto core-fucosylated biantennary
glycan, and "G2F", an asialo, di-galacto core-fucosylated
biantennary glycan. A diagrammatic overview of some of the primary
N-linked oligosaccharide species in ustekinumab IgG is shown in
FIG. 7. The role of some of the enzymes in the glycosylation
maturation process, including roles of some divalent cations (e.g.,
Mn.sup.2+ and Cu.sup.2+) in these enzymatic processes are also
shown.
[0346] HPLC is an analytical procedure that is deployed to analyze
glycosylation of ustekinumab during the manufacturing method. For
analysis by HPLC, the glycans are first enzymatically cleaved from
the heavy chain and then labeled with a fluorescent label to allow
detection. In the method, uncharged peaks for G0F, G1F and G2F can
be distinguished, as well as a subset of smaller neutral peaks.
Furthermore, peaks for differentially sialylated material can also
be observed (FIG. 4). Another method for oligosaccharide analysis
is IRMA, a reduced mass analysis (RMA) method using Immunoglobulin
G (IgG) degrading enzyme of Streptococcus pyogenes (IdeS) that
allows differentiation between major glycoforms after the enzymatic
treatment of IgGs. FIG. 5 shows a representative deconvoluted mass
spectrum for IRMA analysis of ustekinumab produced in Sp2/0 cells.
For ustekinumab, there is also a direct relationship between the
degree of sialylation on the oligosaccharide structures and the
charge heterogeneity as determined by cIEF, IRMA, or HPLC (see,
e.g., FIG. 4, FIG. 5, FIG. 6, FIG. 8, and FIG. 9).
Controlling Oligosaccharide Profile
[0347] Controlling the oligosaccharide profile is critical because
changes in the oligosaccharide profile of a recombinant monoclonal
antibody can significantly affect antibody biological functions.
For example, biological studies have shown that the distribution of
different glycoforms on the Fc region can significantly impact
antibody efficacy, stability, and effector function (J. Biosci.
Bioeng. 2014 117(5):639-644; Bio-Process Int. 2011, 9(6):48-53;
Nat. Rev. Immunol. 2010, 10(5):345-352). In particular,
afucosylation (J. Mol. Biol. 368:767-779) and galactosylation
(Biotechnol. Prog. 21:1644-1652) can play a huge role in the
antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC), two important mechanisms
by which antibodies mediate killing target cells through the immune
function. In addition, high mannose levels have been shown to
adversely affect efficacy by increasing clearance of the antibody
(Glycobiology. 2011, 21(7):949-959) and sialic acid content can
affect anti-inflammatory activity (Antibodies. 2013 2(3):392-414).
As a result of these biological consequences from changes in the
oligosaccharide profile, regulatory agencies require control of the
antibody glycosylation pattern to ensure adherence to lot release
specifications for a consistent, safe and effective product.
Oligosaccharide Profile--Effects from Expression in Different
Cells
[0348] Two commonly used rodent host cell lines for the recombinant
expression of antibodies are Chinese Hamster Ovary cells (CHO) and
mouse myeloma cells (e.g., Sp2/0 cells). CHO cells express
recombinant antibodies which can be virtually devoid of sialic acid
glycan and the glycans can be up to 99% fucosylated. In contrast,
mouse myeloma cells express recombinant antibodies that can contain
up to 50% sialic acid and generally have less fucose. These
differences can have significant effects on antibody activity in
vivo, e.g., it has been shown that such differences can affect the
structure of the Fc-portion of the molecule and thereby alter
antibody effector functions such as antibody-dependent cellular
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
(see, e.g., U.S. Pat. No. 8,975,040). For example, reduced ADCC
activity has been noted with increased sialylated (charged) Fc
glycans (Scallon et al., Mol Immunol 2007; 44:1524-34) and
increased ADCC activity has been reported for antibodies that were
deficient in fucose (Shields et al., J Biol Chem. 2002;
277:26733-26740; Shinkawa et al., J Biol Chem. 2003;
278:3466-3473).
[0349] In addition, antibodies produced in CHO and Sp2/0 cells can
have significant differences in the levels of two glycan epitopes,
galactose-.alpha.-1,3-galactose (.alpha.-gal) and the sialylated
N-glycan Neu5Gc-.alpha.-2-6-galactose (Neu5Gc). For example, it has
been shown that CHO cells can express antibodies with undetectable
or only trace levels of .alpha.-Gal and Neu5Gc, while Sp2/0 cells
can express much higher levels of the two glycan structures (Yu et
al., Sci Rep. 2016 Jan. 29; 7:20029). In contrast, humans are
genetically deficient in the gene for biosynthesizing .alpha.-gal
and the gene responsible for production of Neu5Gc is irreversibly
mutated in all humans. As a result, .alpha.-Gal and Neu5Gc are not
produced in humans. Furthermore, the presence of these non-human
glycan epitopes on therapeutic antibodies can cause undesirable
immune reactions in certain human populations because of higher
levels of pre-existing antibodies to .alpha.-Gal and Neu5Gc. For
example, anti-.alpha.-gal IgE mediated anaphylactic responses have
been reported for Cetuximab (Chung, C. H. et al., N Engl J Med.
2008 Mar. 13; 358(11): 1109-17) and the presence of circulating
anti-Neu5Gc antibodies has been reported to promote clearance of
Cetuximab (Ghaderi et al., Nat Biotechnol. 2010 August;
28(8):863-7).
[0350] It has also been reported that ustekinumab expressed in
Sp2/0 cells contains higher levels of Neu5Gc compared to a number
of other antibodies. Western blot analysis showed that anti-Neu5Gc
antibody preparations highly mono-specific for Neu5Gc bound to
ustekinumab, but not to ustekinumab treated with PNGase F, which
removes nearly 100% of the N-glycan (Yu et al., Sci Rep. 2016 Jan.
29; 7:20029). Further analysis also showed that anti-Neu5Gc
antibody preparations could not bind ustekinumab with only one
Neu5Gc (mono-sialylated on one Fc region) but could bind antibodies
with two to four Neu5Gc. It was not determined if the anti-Neu5Gc
antibody could bind two Neu5Gc located on two different Fc regions
of the same antibody (monosialylated on both Fc regions) or only to
a disialylated N-glycan on one Fc region of an antibody, but
regardless of their distribution it was determined that at least
two Fc Neu5Gc residues are required for binding to the anti-Neu5Gc
antibody.
Oligosaccharide Profile of Ustekinumab Expressed in Sp2/0 Cells and
CHO Cells
[0351] Compiled HPLC data from multiple commercial production runs
of ustekinumab showed that DS or DP produced in Sp2/0 cells
comprises total neutral oligosaccharide species .gtoreq.64.8% to
.ltoreq.85.4%, total charged oligosaccharide species .gtoreq.14.4%
to .ltoreq.35.6%, and individual neutral oligosaccharide species
G0F.gtoreq.11.5% to .ltoreq.40.2%, G1F.gtoreq.29.9% to
.ltoreq.40.6%, and G2F.gtoreq.4.1% to .ltoreq.11.3%. Furthermore,
the peak 3 area % of the capillary isoelectric focusing (cIEF)
electropherogram of ustekinumab produced in Sp2/0 cells is
.gtoreq.39.8% to .ltoreq.64.4%. As shown in Table 5 and Table 6,
based on IRMA or HPLC analysis, ustekinumab produced in CHO cells
has a very different oligosaccharide profile compared to
ustekinumab produced in Sp2/0 cells for total neutral, total
charged, and individual neutral oligosaccharide species G0F, G1F,
and G2F. These differences are apparent in representative HPLC
chromatograms for ustekinumab produced in Sp2/0 cells and CHO
cells, as shown in FIG. 4 and FIG. 8, respectively. Compared to
ustekinumab produced in Sp2/0 cells, the oligosaccharide profile
for ustekinumab produced in CHO cells is shifted toward very low
levels of charged glycans and higher levels of neutral glycans,
that are predominantly G0F. The oligosaccharide profile for
ustekinumab produced in CHO cells comprises total neutral
oligosaccharide species >99.0%, total charged oligosaccharide
species <1.0%, and individual neutral oligosaccharide species
G0F>70.0%, G1F<20.0%, and G2F<5.0%. The peak 3 area % of
the capillary isoelectric focusing (cIEF) electropherogram of
ustekinumab produced in CHO cells is >70.0%. Furthermore, no
disialylated glycan species were detected by IRMA or by HPLC for
ustekinumab produced in CHO cells and monosialylated glycan species
were at very low levels based on HPLC analysis and undetectable by
IRMA analysis (see, e.g., Table 5 and FIG. 8).
TABLE-US-00017 TABLE 5 Representative results for IRMA and HPLC
analysis of total neutral, total charged, and other selected
oligosaccharide species for ustekinumab produced in Sp2/0 cells and
CHO cells IRMA HPLC Glycans Sp2/0 CHO Sp2/0 CHO G0F 26.7 71.0 25.0
78.0 G1F 29.2 13.3 33.2 15.2 G2F 8.6 1.5 7.8 2.2 Other neutral 11.0
14.2 5.9 4.2 Total neutral 75.5 100.0 71.9 99.6 Monosialylated 20.9
0.0 25.9 0.4 Disialylated 3.5 0.0 2.2 <LOD Total charged 24.5
0.0 28.1 0.4 <LOD--below limit of detection Numbers are % of
totals
TABLE-US-00018 TABLE 6 Representative results for IRMA analysis of
individual oligosaccharide species for ustekinumab produced in
Sp2/0 cells and CHO cells Sp2/0 Cells CHO Cells % of % of Glycan
Total Assigned Total Assigned G0F 26.7 71.0 G1F 29.2 13.3 G2F 8.6
1.5 G0 2.9 10.1 G1FS 9.7 0.0 H6N4F1 1.6 0.0 G2FS 7.4 0.0 H7N4F1 0.9
0.0 H6N4F1S1 3.9 0.0 G2FS2 3.5 0.0 *Man5 + Lys 0.7 0.9 *G1F-GlcNAc
+ Lys 1.1 0.6 *H5N3 + Lys 0.8 0.8 *G1 + Lys 1.5 1.2 *G2 + Lys 0.4
0.2 **G1F-GlcNAc - Lys 0.0 0.0 **H5N3 - Lys 0.0 0.0 **G1 - Lys 0.0
0.0 **H5N4 - Lys 0.0 0.5 **H5N3F1 + Lys 1.0 0.0
CONCLUSION
[0352] Thus, as described supra, manufacturing control strategies
are developed to maintain consistent drug substance (DS) and drug
product (DP) characteristics of therapeutic proteins with regard to
oligosaccharide profile and/or other characteristics of the DS or
DP (e.g., DS and/or DP comprising the therapeutic antibody
ustekinumab). In particular, controlling the oligosaccharide
profile of therapeutic antibodies is critical because changes in
the oligosaccharide profile can significantly affect antibody
biological functions. A point of control for the oligosaccharide
profile of therapeutic antibodies is the selection of the cellular
host for expression of the therapeutic antibodies. As presented
herein, ustekinumab expressed in Sp2/0 cells comprises
anti-IL-12/IL-23p40 antibodies having a heavy chain (HC) comprising
amino acid sequence of SEQ ID NO: 10 and a light chain (LC)
comprising amino acid sequence of SEQ ID NO: 11; a heavy chain
variable domain amino acid sequence of SEQ ID NO:7 and a light
chain variable domain amino acid sequence of SEQ ID NO:8; the heavy
chain CDR amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ
ID NO:3; and the light chain CDR amino acid sequences of SEQ ID
NO:4, SEQ ID NO:5, and SEQ ID NO:6; wherein the oligosaccharide
profile of the anti-IL-12/IL-23p40 antibodies comprises total
neutral oligosaccharide species .gtoreq.64.8% to .ltoreq.85.4%,
total charged oligosaccharide species .gtoreq.14.4% to
.ltoreq.35.6%, and individual neutral oligosaccharide species
G0F.gtoreq.11.5% to .ltoreq.40.2%, G1F.gtoreq.29.9% to
.ltoreq.40.6%, and G2F.gtoreq.4.1% to .ltoreq.11.3%. Furthermore,
the peak 3 area % of the capillary isoelectric focusing (cIEF)
electropherogram of the anti-IL-12/IL-23p40 antibodies produced in
Sp2/0 cells is .gtoreq.39.8% to .ltoreq.64.4%.
[0353] In contrast, for ustekinumab produced in CHO cells, the
oligosaccharide profile is shifted toward very low levels of
charged glycans and higher levels of neutral glycans that are
predominantly G0F. The oligosaccharide profile for ustekinumab
produced in CHO cells comprises anti-IL-12/IL-23p40 antibodies
having a heavy chain (HC) comprising amino acid sequence of SEQ ID
NO: 10 and a light chain (LC) comprising amino acid sequence of SEQ
ID NO: 11; a heavy chain variable domain amino acid sequence of SEQ
ID NO:7 and a light chain variable domain amino acid sequence of
SEQ ID NO:8; the heavy chain CDR amino acid sequences of SEQ ID NO:
1, SEQ ID NO:2, and SEQ ID NO:3, and the light chain CDR amino acid
sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; wherein the
oligosaccharide profile of the anti-IL-12/IL-23p40 antibodies
comprises total neutral oligosaccharide species .gtoreq.99.0%,
total charged oligosaccharide species .ltoreq.1.0%, and individual
neutral oligosaccharide species G0F.gtoreq.70.0%, G1F.ltoreq.20.0%,
and G2F.ltoreq.5.0%. The peak 3 area % of the capillary isoelectric
focusing (cIEF) electropherogram of ustekinumab produced in CHO
cells is .gtoreq.70.0%. Furthermore, no disialylated glycan species
were detected by IRMA or by HPLC for ustekinumab produced in CHO
cells and monosialylated glycan species were at very low levels
based on HPLC analysis and undetectable by IRMA analysis. The
reduction in sialylated species generally and the reduction of
Neu5Gc specifically for ustekinumab produced in CHO cells may
provide a benefit by reducing undesirable immunogenic responses
when administered to humans. For example, reduced levels of Neu5Gc
could reduce clearance so that anti-IL-12/23p40 antibodies produced
in CHO cells would have a longer half-life compared to
anti-IL-12/23p40 antibodies expressed in Sp2/0 cells, especially
for patient populations with higher levels of anti-Neu5Gc
antibodies.
Sequence CWU 1
1
1115PRTHomo sapiens 1Thr Tyr Trp Leu Gly1 5217PRTHomo sapiens 2Ile
Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln1 5 10
15Gly310PRTHomo sapiens 3Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe1 5
10411PRTHomo sapiens 4Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala1
5 1057PRTHomo sapiens 5Ala Ala Ser Ser Leu Gln Ser1 569PRTHomo
sapiens 6Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr1 57119PRTHomo sapiens
7Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5
10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr
Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp
Trp Ile 35 40 45Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser
Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile
Thr Thr Ala Tyr65 70 75 80Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Arg Pro Gly Gln Gly Tyr
Phe Asp Phe Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1158108PRTHomo sapiens 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu
Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr 85 90 95Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg 100 1059503PRTHomo sapiens 9Arg Asn
Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu1 5 10 15His
His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys 20 25
30Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp
35 40 45His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys
Leu 50 55 60Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg
Glu Thr65 70 75 80Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg
Lys Thr Ser Phe 85 90 95Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu
Asp Leu Lys Met Tyr 100 105 110Gln Val Glu Phe Lys Thr Met Asn Ala
Lys Leu Leu Met Asp Pro Lys 115 120 125Arg Gln Ile Phe Leu Asp Gln
Asn Met Leu Ala Val Ile Asp Glu Leu 130 135 140Met Gln Ala Leu Asn
Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser145 150 155 160Leu Glu
Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu 165 170
175Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser
180 185 190Tyr Leu Asn Ala Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr
Val Val 195 200 205Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met
Val Val Leu Thr 210 215 220Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr
Trp Thr Leu Asp Gln Ser225 230 235 240Ser Glu Val Leu Gly Ser Gly
Lys Thr Leu Thr Ile Gln Val Lys Glu 245 250 255Phe Gly Asp Ala Gly
Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu 260 265 270Ser His Ser
Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser 275 280 285Thr
Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu 290 295
300Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
Leu305 310 315 320Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys
Ser Ser Arg Gly 325 330 335Ser Ser Asp Pro Gln Gly Val Thr Cys Gly
Ala Ala Thr Leu Ser Ala 340 345 350Glu Arg Val Arg Gly Asp Asn Lys
Glu Tyr Glu Tyr Ser Val Glu Cys 355 360 365Gln Glu Asp Ser Ala Cys
Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu 370 375 380Val Met Val Asp
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser385 390 395 400Ser
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu 405 410
415Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu
420 425 430Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu
Thr Phe 435 440 445Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys
Lys Asp Arg Val 450 455 460Phe Thr Asp Lys Thr Ser Ala Thr Val Ile
Cys Arg Lys Asn Ala Ser465 470 475 480Ile Ser Val Arg Ala Gln Asp
Arg Tyr Tyr Ser Ser Ser Trp Ser Glu 485 490 495Trp Ala Ser Val Pro
Cys Ser 50010449PRTHomo sapiens 10Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30Trp Leu Gly Trp Val Arg
Gln Met Pro Gly Lys Gly Leu Asp Trp Ile 35 40 45Gly Ile Met Ser Pro
Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val
Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr65 70 75 80Leu Gln
Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala
Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys11214PRTHomo sapiens 11Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys
Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr 85 90 95Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 210
* * * * *
References