U.S. patent application number 16/070385 was filed with the patent office on 2019-03-28 for anti-tnf antibodies, compositions, methods and use for the treatment or prevention of type 1 diabetes.
This patent application is currently assigned to Janssen Biotech, Inc.. The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Joseph Hedrick, Elizabeth C. Hsia, Paul Imm, Jocelyn Leu, Bethany Paxson, Mark Rigby, Songmao Zheng, Ramineh Zoka.
Application Number | 20190092849 16/070385 |
Document ID | / |
Family ID | 59500155 |
Filed Date | 2019-03-28 |
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United States Patent
Application |
20190092849 |
Kind Code |
A1 |
Hedrick; Joseph ; et
al. |
March 28, 2019 |
ANTI-TNF ANTIBODIES, COMPOSITIONS, METHODS AND USE FOR THE
TREATMENT OR PREVENTION OF TYPE 1 DIABETES
Abstract
The present invention relates to compositions and methods
utilizing anti-TNF antibodies having a heavy chain (HC) comprising
SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37 for use
in the treatment or prevention of Type I Diabetes (T1D).
Inventors: |
Hedrick; Joseph; (New Hope,
PA) ; Hsia; Elizabeth C.; (Kenneth Square, PA)
; Imm; Paul; (Newtown, PA) ; Leu; Jocelyn;
(Ambler, PA) ; Paxson; Bethany; (Philadelphia,
PA) ; Rigby; Mark; (Abington, PA) ; Zheng;
Songmao; (Chongqing, CH) ; Zoka; Ramineh;
(Lower Gwynedd, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Assignee: |
Janssen Biotech, Inc.
Horsham
PA
|
Family ID: |
59500155 |
Appl. No.: |
16/070385 |
Filed: |
February 2, 2017 |
PCT Filed: |
February 2, 2017 |
PCT NO: |
PCT/US2017/016175 |
371 Date: |
July 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62291673 |
Feb 5, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/241 20130101;
A61M 5/31591 20130101; A61K 2039/505 20130101; A61P 29/00 20180101;
C07K 2317/565 20130101; A61K 2039/545 20130101; A61M 5/003
20130101; A61P 3/10 20180101; A61M 5/31573 20130101; C07K 2317/21
20130101; A61M 5/31533 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61P 29/00 20060101 A61P029/00; A61P 3/10 20060101
A61P003/10; A61M 5/00 20060101 A61M005/00; A61M 5/315 20060101
A61M005/315 |
Claims
1-101. (canceled)
102. At least one isolated mammalian anti-TNF antibody having a
heavy chain (HC) comprising SEQ ID NO:36 and a light chain (LC)
comprising SEQ ID NO:37 for use in the treatment or prevention of
Type I Diabetes.
103. A method for treating or preventing a TNF related condition,
wherein the TNF related condition is Type 1 Diabetes, the method
comprising: (a) administering to a subject an isolated mammalian
anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO:36
and a light chain (LC) comprising SEQ ID NO:37.
104. The method according to claim 103, wherein said anti-TNF
antibody is administered subcutaneaously (SC) to the subject at an
induction dose of 60 mg/m.sup.2 at weeks 0 and 2 followed by a
maintenance dose of 30 mg/m.sup.2 at week 4 and q2w through week 52
when the subject weighs <45 kg and administered to the subject
at an induction dose of 100 mg/m.sup.2 at weeks 0 and 2 followed by
a maintenance dose of 50 mg/m.sup.2 at week 4 and q2w through week
52 when the subject weighs .gtoreq.45 kg.
105. The method according to claim 104, wherein said anti-TNF
antibody is administered with a medical device suitable for
self-administration and the device is selected from the group
consisting of: prefilled syringe, prefilled syringe with an
UltraSafe Passive Needle Guard, VarioJect, and combinations of
VarioJect and prefilled syringe with an UltraSafe Passive Needle
Guard.
106. The medical deviced according to claim 105, wherein the device
is a prefilled syringe with an UltraSafe Passive Needle Guard if
the subject weighs .gtoreq.45 kg and VarioJect if the subject
weighs <45 kg.
107. The medical deviced according to claim 105, wherein the device
is a VarioJect with a 4.5 mm needle length.
108. A composition comprising at least one isolated mammalian
anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO:36
and a light chain (LC) comprising SEQ ID NO:37, and at least one
pharmaceutically acceptable carrier or diluent for use in the
treatment or prevention of Type I Diabetes.
109. A method for treating or preventing a TNF related condition,
wherein the TNF related condition is Type 1 Diabetes, the method
comprising: (a) administering to a subject a composition comprising
an isolated mammalian anti-TNF antibody having a heavy chain (HC)
comprising SEQ ID NO:36 and a light chain (LC) comprising SEQ ID
NO:37.
110. The method according to claim 109, wherein said composition is
administered subcutaneaously (SC) to the subject such that said
anti-TNF antibody is administered at an induction dose of 60
mg/m.sup.2 at weeks 0 and 2 followed by a maintenance dose of 30
mg/m.sup.2 at week 4 and q2w through week 52 when the subject
weighs <45 kg and administered to the subject at an induction
dose of 100 mg/m.sup.2 at weeks 0 and 2 followed by a maintenance
dose of 50 mg/m.sup.2 at week 4 and q2w through week 52 when the
subject weighs .gtoreq.45 kg.
111. The method according to claim 110, wherein said composition is
administered with a medical device suitable for self-administration
and the device is selected from the group consisting of: prefilled
syringe, prefilled syringe with an UltraSafe Passive Needle Guard,
VarioJect, and combinations of VarioJect and prefilled syringe with
an UltraSafe Passive Needle Guard.
112. The medical deviced according to claim 111, wherein the device
is a prefilled syringe with an UltraSafe Passive Needle Guard if
the subject weighs .gtoreq.45 kg and VarioJect if the subject
weighs <45 kg.
113. The medical deviced according to claim 111, wherein the device
is a VarioJect with a 4.5 mm needle length.
114. A medical device for use in the treatment or prevention of
Type I Diabetes, comprising at least one isolated mammalian
anti-TNF antibody having a heavy chain (HC) comprising SEQ ID NO:36
and a light chain (LC) comprising SEQ ID NO:37, wherein said device
is suitable for subcutaneaously (SC) administering said at least
one anti-TNF antibody.
115. The medical deviced according to claim 114, wherein the device
is suitable for self-administration and the device is selected from
the group consisting of: prefilled syringe, prefilled syringe with
an UltraSafe Passive Needle Guard, and VarioJect.
116. The medical deviced according to claim 115, wherein the device
is a VarioJect with a 4.5 mm needle length.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
utilizing anti-TNF antibodies having a heavy chain (HC) comprising
SEQ ID NO:36 and a light chain (LC) comprising SEQ ID NO:37 for use
in the treatment or prevention of Type I Diabetes (T1D).
BACKGROUND OF THE INVENTION
[0002] TNF alpha is a soluble homotrimer of 17 kD protein subunits.
A membrane-bound 26 kD precursor form of TNF also exists.
[0003] Cells other than monocytes or macrophages also produce TNF
alpha. For example, human non-monocytic tumor cell lines produce
TNF alpha and CD4+ and CD8+ peripheral blood T lymphocytes and some
cultured T and B cell lines also produce TNF alpha.
[0004] TNF alpha causes pro-inflammatory actions which result in
tissue injury, such as degradation of cartilage and bone, induction
of adhesion molecules, inducing procoagulant activity on vascular
endothelial cells, increasing the adherence of neutrophils and
lymphocytes, and stimulating the release of platelet activating
factor from macrophages, neutrophils and vascular endothelial
cells.
[0005] TNF alpha has been associated with infections, immune
disorders, neoplastic pathologies, autoimmune pathologies and
graft-versus-host pathologies. The association of TNF alpha with
cancer and infectious pathologies is often related to the host's
catabolic state. Cancer patients suffer from weight loss, usually
associated with anorexia.
[0006] The extensive wasting which is associated with cancer, and
other diseases, is known as "cachexia". Cachexia includes
progressive weight loss, anorexia, and persistent erosion of lean
body mass in response to a malignant growth. The cachectic state
causes much cancer morbidity and mortality. There is evidence that
TNF alpha is involved in cachexia in cancer, infectious pathology,
and other catabolic states.
[0007] TNF alpha is believed to play a central role in
gram-negative sepsis and endotoxic shock, including fever, malaise,
anorexia, and cachexia. Endotoxin strongly activates
monocyte/macrophage production and secretion of TNF alpha and other
cytokines. TNF alpha and other monocyte-derived cytokines mediate
the metabolic and neurohormonal responses to endotoxin. Endotoxin
administration to human volunteers produces acute illness with
flu-like symptoms including fever, tachycardia, increased metabolic
rate and stress hormone release. Circulating TNF alpha increases in
patients suffering from Gram-negative sepsis.
[0008] Thus, TNF alpha has been implicated in inflammatory
diseases, autoimmune diseases, viral, bacterial and parasitic
infections, malignancies, and/or neurodegenerative diseases and is
a useful target for specific biological therapy in diseases, such
as rheumatoid arthritis and Crohn's disease. Beneficial effects in
open-label trials with a chimeric monoclonal antibody to TNF alpha
(cA2) have been reported with suppression of inflammation and with
successful retreatment after relapse in rheumatoid arthritis and in
Crohn's disease. Beneficial results in a randomized, double-blind,
placebo-controlled trial with cA2 have also been reported in
rheumatoid arthritis with suppression of inflammation.
[0009] Other investigators have described mAbs specific for
recombinant human TNF which had neutralizing activity in vitro.
Some of these mAbs were used to map epitopes of human TNF and
develop enzyme immunoassays and to assist in the purification of
recombinant TNF. However, these studies do not provide a basis for
producing TNF neutralizing antibodies that can be used for in vivo
diagnostic or therapeutic uses in humans, due to immunogenicity,
low specificity and/or pharmaceutical unsuitability.
[0010] Neutralizing antisera or mAbs to TNF have been shown in
mammals other than man to abrogate adverse phaysiological changes
and prevent death after lethal challenge in experimental
endotoxemia and bacteremia. This effect has been demonstrated,
e.g., in rodent lethality assays and in primate pathology model
systems.
[0011] Putative receptor binding loci of hTNF has been disclosed
and the receptor binding loci of TNF alpha as consisting of amino
acids 11-13, 37-42, 49-57 and 155-157 of TNF have been
disclosed.
[0012] Non-human mammalian, chimeric, polyclonal (e.g., anti-sera)
and/or monoclonal antibodies (Mabs) and fragments (e.g.,
proteolytic digestion or fusion protein products thereof) are
potential therapeutic agents that are being investigated in some
cases to attempt to treat certain diseases. However, such
antibodies or fragments can elicit an immune response when
administered to humans. Such an immune response can result in an
immune complex-mediated clearance of the antibodies or fragments
from the circulation, and make repeated administration unsuitable
for therapy, thereby reducing the therapeutic benefit to the
patient and limiting the readministration of the antibody or
fragment. For example, repeated administration of antibodies or
fragments comprising non-human portions can lead to serum sickness
and/or anaphylaxis. In order to avoid these and other problems, a
number of approaches have been taken to reduce the immunogenicity
of such antibodies and portions thereof, including chimerization
and humanization, as well known in the art. These and other
approaches, however, still can result in antibodies or fragments
having some immunogenicity, low affinity, low avidity, or with
problems in cell culture, scale up, production, and/or low yields.
Thus, such antibodies or fragments can be less than ideally suited
for manufacture or use as therapeutic proteins.
[0013] Accordingly, there is a need to provide anti-TNF antibodies
or fragments that overcome one more of these problems, as well as
improvements over known antibodies or fragments thereof.
SUMMARY OF THE INVENTION
[0014] The present invention provides isolated human, primate,
rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-TNF
antibodies comprising all of the heavy chain variable CDR regions
of SEQ ID NOS:1, 2 and 3 and/or all of the light chain variable CDR
regions of SEQ ID NOS:4, 5 and 6, immunoglobulins, cleavage
products and other specified portions and variants thereof, as well
as anti-TNF alpha antibody compositions, encoding or complementary
nucleic acids, vectors, host cells, compositions, formulations,
devices, transgenic animals, transgenic plants, and methods of
making and using thereof, as described and enabled herein, in
combination with what is known in the art.
[0015] The present invention also provides at least one isolated
anti-TNF antibody comprising all of the heavy chain variable CDR
regions of SEQ ID NOS:1, 2 and 3 and/or all of the light chain
variable CDR regions of SEQ ID NOS:4, 5 and 6 as described herein.
An 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
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, that can be incorporated
into an antibody of the present invention comprising all of the
heavy chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or
all of the light chain variable CDR regions of SEQ ID NOS:4, 5 and
6. An antibody of the invention can include or be derived from any
mammal, such as but not limited to a human, a mouse, a rabbit, a
rat, a rodent, a primate, or any combination thereof, and the
like.
[0016] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, or hybridizing
to, a polynucleotide encoding specific anti-TNF antibodies,
comprising at least one specified sequence, domain, portion or
variant thereof. The present invention further provides recombinant
vectors comprising said anti-TNF antibody nucleic acid molecules,
host cells containing such nucleic acids and/or recombinant
vectors, as well as methods of making and/or using such antibody
nucleic acids, vectors and/or host cells.
[0017] At least one antibody of the invention binds at least one
specified epitope specific to at least one TNF protein, subunit,
fragment, portion or any combination thereof. The at least one
epitope can comprise at least one antibody binding region that
comprises at least one portion of said protein, which epitope is
preferably comprised of at least 1-5 amino acids of at least one
portion thereof, such as but not limited to, at least one
functional, extracellular, soluble, hydrophillic, external or
cytoplasmic domain of said protein, or any portion thereof.
[0018] The at least one antibody can optionally comprise at least
one specified portion of at least one complementarity determining
region (CDR) (e.g., CDR1, CDR2 or CDR3 of the heavy or light chain
variable region) and/or at least one constant or variable framework
region or any portion thereof. The at least one antibody amino acid
sequence can further optionally comprise at least one specified
substitution, insertion or deletion as described herein or as known
in the art.
[0019] The present invention also provides at least one isolated
anti-TNF antibody as described herein, wherein the antibody has at
least one activity, such as, but not limited to inhibition of
TNF-induced cell adhesion molecules, inhibition of TNF binding to
receptor, Arthritic index improvement in mouse model, (see, e.g.,
Examples 3-7). A(n) anti-TNF antibody can thus be screened for a
corresponding activity according to known methods, such as but not
limited to, at least one biological activity towards a TNF
protein.
[0020] The present invention further provides at least one TNF
anti-idiotype antibody to at least one TNF antibody of the present
invention. The anti-idiotype antibody includes any protein or
peptide containing molecule that comprises at least a portion of an
immunoglobulin molecule, such as but not limited to at least one
complementarity 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, that can be incorporated
into an antibody of the present invention. An antibody of the
invention can include or be derived from any mammal, such as but
not limited to a human, a mouse, a rabbit, a rat, a rodent, a
primate, and the like.
[0021] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, or hybridizing
to, a polynucleotide encoding at least one TNF anti-idiotype
antibody, comprising at least one specified sequence, domain,
portion or variant thereof. The present invention further provides
recombinant vectors comprising said TNF anti-idiotype antibody
encoding nucleic acid molecules, host cells containing such nucleic
acids and/or recombinant vectors, as well as methods of making
and/or using such anti-idiotype antiobody nucleic acids, vectors
and/or host cells.
[0022] The present invention also provides at least one method for
expressing at least one anti-TNF antibody, or TNF anti-idiotype
antibody, in a host cell, comprising culturing a host cell as
described herein under conditions wherein at least one anti-TNF
antibody is expressed in detectable and/or recoverable amounts.
[0023] The present invention also provides at least one composition
comprising (a) an isolated anti-TNF antibody encoding nucleic acid
and/or antibody as described herein; and (b) a suitable carrier or
diluent. The carrier or diluent can optionally be pharmaceutically
acceptable, according to known carriers or diluents. The
composition can optionally further comprise at least one further
compound, protein or composition.
[0024] The present invention further provides at least one anti-TNF
antibody method or composition, for administering a therapeutically
effective amount to modulate or treat at least one TNF related
condition in a cell, tissue, organ, animal or patient and/or, prior
to, subsequent to, or during a related condition, as known in the
art and/or as described herein.
[0025] The present invention also provides at least one
composition, device and/or method of delivery of a therapeutically
or prophylactically effective amount of at least one anti-TNF
antibody, according to the present invention.
[0026] The present invention further provides at least one anti-TNF
antibody method or composition, for diagnosing at least one TNF
related condition in a cell, tissue, organ, animal or patient
and/or, prior to, subsequent to, or during a related condition, as
known in the art and/or as described herein.
[0027] The present invention also provides at least one
composition, device and/or method of delivery for diagnosing of at
least one anti-TNF antibody, according to the present
invention.
DESCRIPTION OF THE FIGURES
[0028] FIG. 1 shows a graphical representation showing an assay for
ability of TNV mAbs in hybridoma cell supernatants to inhibit
TNF.alpha. binding to recombinant TNF receptor. Varying amounts of
hybridoma cell supernatants containing known amounts of TNV mAb
were preincubated with a fixed concentration (5 ng/ml) of
.sup.125I-labeled TNF.alpha.. The mixture was transferred to
96-well Optiplates that had been previously coated with p55-sf2, a
recombinant TNF receptor/IgG fusion protein. The amount of
TNF.alpha. that bound to the p55 receptor in the presence of the
mAbs was determined after washing away the unbound material and
counting using a gamma counter. Although eight TNV mAb samples were
tested in these experiments, for simplicity three of the mAbs that
were shown by DNA sequence analyses to be identical to one of the
other TNV mAbs (see Section 5.2.2) are not shown here. Each sample
was tested in duplicate. The results shown are representative of
two independent experiments.
[0029] FIGS. 2A-B shows DNA sequences of the TNV mAb heavy chain
variable regions. The germline gene shown is the DP-46 gene. `TNVs`
indicates that the sequence shown is the sequence of TNV14, TNV15,
TNV148, and TNV196. The first three nucleotides in the TNV sequence
define the translation initiation Met codon. Dots in the TNV mAb
gene sequences indicate the nucleotide is the same as in the
germline sequence. The first 19 nucleotides (underlined) of the TNV
sequences correspond to the oligonucleotide used to PCR-amplify the
variable region. An amino acid translation (single letter
abbreviations) starting with the mature mAb is shown only for the
germline gene. The three CDR domains in the germline amino acid
translation are marked in bold and underlined. Lines labeled
TNV148(B) indicate that the sequence shown pertains to both TNV148
and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to
the sequence not being known or not existing in the germline gene.
The TNV mAb heavy chains use the J6 joining region.
[0030] FIG. 3 shows DNA sequences of the TNV mAb light chain
variable regions. The germline gene shown is a representative
member of the Vg/38K family of human kappa germline variable region
genes. Dots in the TNV mAb gene sequences indicate the nucleotide
is the same as in the germline sequence. The first 16 nucleotides
(underlined) of the TNV sequences correspond to the oligonucleotide
used to PCR-amplify the variable region. An amino acid translation
of the mature mAb (single letter abbreviations) is shown only for
the germline gene. The three CDR domains in the germline amino acid
translation are marked in bold and underlined. Lines labeled
TNV148(B) indicate that the sequence shown pertains to both TNV148
and TNV148B. Gaps in the germline DNA sequence (CDR3) are due to
the sequence not being known or not existing in the germline gene.
The TNV mAb light chains use the J3 joining sequence.
[0031] FIG. 4 shows deduced amino acid sequences of the TNV mAb
heavy chain variable regions. The amino acid sequences shown
(single letter abbreviations) were deduced from DNA sequence
determined from both uncloned PCR products and cloned PCR products.
The amino sequences are shown partitioned into the secretory signal
sequence (signal), framework (FW), and complementarity determining
region (CDR) domains. The amino acid sequence for the DP-46
germline gene is shown on the top line for each domain. Dots
indicate that the amino acid in the TNV mAb is identical to the
germline gene. TNV148(B) indicates that the sequence shown pertains
to both TNV148 and TNV148B. `TNVs` indicates that the sequence
shown pertains to all TNV mAbs unless a different sequence is
shown. Dashes in the germline sequence (CDR3) indicate that the
sequences are not known or do not exist in the germline gene.
[0032] FIG. 5 shows deduced amino acid sequences of the TNV mAb
light chain variable regions. The amino acid sequences shown
(single letter abbreviations) were deduced from DNA sequence
determined from both uncloned PCR products and cloned PCR products.
The amino sequences are shown partitioned into the secretory signal
sequence (signal), framework (FW), and complementarity determining
region (CDR) domains. The amino acid sequence for the Vg/38K-type
light chain germline gene is shown on the top line for each domain.
Dots indicate that the amino acid in the TNV mAb is identical to
the germline gene. TNV148(B) indicates that the sequence shown
pertains to both TNV148 and TNV148B. `All` indicates that the
sequence shown pertains to TNV14, TNV15, TNV148, TNV148B, and
TNV186.
[0033] FIG. 6 shows schematic illustrations of the heavy and light
chain expression plasmids used to make the rTNV148B-expressing C466
cells. p1783 is the heavy chain plasmid and p1776 is the light
chain plasmid. The rTNV148B variable and constant region coding
domains are shown as black boxes. The immunoglobulin enhancers in
the J-C introns are shown as gray boxes. Relevant restriction sites
are shown. The plasmids are shown oriented such that transcription
of the Ab genes proceeds in a clockwise direction. Plasmid p1783 is
19.53 kb in length and plasmid p1776 is 15.06 kb in length. The
complete nucleotide sequences of both plasmids are known. The
variable region coding sequence in p1783 can be easily replaced
with another heavy chain variable region sequence by replacing the
BsiWI/BstBI restriction fragment. The variable region coding
sequence in p1776 can be replaced with another variable region
sequence by replacing the SalI/AflII restriction fragment.
[0034] FIG. 7 shows graphical representation of growth curve
analyses of five rTNV148B-producing cell lines. Cultures were
initiated on day 0 by seeding cells into T75 flasks in 15Q+MHX
media to have a viable cell density of 1.0.times.10.sup.5 cells/ml
in a 30 ml volume. The cell cultures used for these studies had
been in continuous culture since transfections and subclonings were
performed. On subsequent days, cells in the T flasks were
thoroughly resuspended and a 0.3 ml aliquot of the culture was
removed. The growth curve studies were terminated when cell counts
dropped below 1.5.times.10.sup.5 cells/ml. The number of live cells
in the aliquot was determined by typan blue exclusion and the
remainder of the aliquot stored for later mAb concentration
determination. An ELISA for human IgG was performed on all sample
aliquots at the same time.
[0035] FIG. 8 shows a graphical representation of the comparison of
cell growth rates in the presence of varying concentrations of MHX
selection. Cell subclones C466A and C466B were thawed into MHX-free
media (IMDM, 5% FBS, 2 mM glutamine) and cultured for two
additional days. Both cell cultures were then divided into three
cultures that contained either no MHX, 0.2.times. MHX, or 1.times.
MHX. One day later, fresh T75 flasks were seeded with the cultures
at a starting density of 1.times.10.sup.5 cells/ml and cells
counted at 24 hour intervals for one week. Doubling times during
the first 5 days were calculated using the formula in SOP PD32.025
and are shown above the bars.
[0036] FIG. 9 shows graphical representations of the stability of
mAb production over time from two rTNV148B-producing cell lines.
Cell subclones that had been in continuous culture since performing
transfections and subclonings were used to start long-term serial
cultures in 24-well culture dishes. Cells were cultured in I5Q
media with and without MHX selection. Cells were continually
passaged by splitting the cultures every 4 to 6 days to maintain
new viable cultures while previous cultures were allowed to go
spent. Aliquots of spent cell supernatant were collected shortly
after cultures were spent and stored until the mAb concentrations
were determined. An ELISA for human IgG was performed on all sample
aliquots at the same time.
[0037] FIG. 10 shows arthritis mouse model mice Tg 197 weight
changes in response to anti-TNF antibodies of the present invention
as compared to controls in Example 4. At approximately 4 weeks of
age the Tg197 study mice were assigned, based on gender and body
weight, to one of 9 treatment groups and treated with a single
intraperitoneal bolus dose of Dulbecco's PBS (D-PBS) or an anti-TNF
anatibody of the present invention (TNV14, TNV148 or TNV196) at
either 1 mg/kg or 10 mg/kg. When the weights were analyzed as a
change from pre-dose, the animals treated with 10 mg/kg cA2 showed
consistently higher weight gain than the D-PBS-treated animals
throughout the study. This weight gain was significant at weeks
3-7. The animals treated with 10 mg/kg TNV148 also achieved
significant weight gain at week 7 of the study.
[0038] FIGS. 11A-C represent the progression of disease severity
based on the arthritic index as presented in Example 4. The 10
mg/kg cA2-treated group's arthritic index was lower then the D-PBS
control group starting at week 3 and continuing throughout the
remainder of the study (week 7). The animals treated with 1 mg/kg
TNV14 and the animals treated with 1 mg/kg cA2 failed to show
significant reduction in AI after week 3 when compared to the
D-PBS-treated Group. There were no significant differences between
the 10 mg/kg treatment groups when each was compared to the others
of similar dose (10 mg/kg cA2 compared to 10 mg/kg TNV14, 148 and
196). When the 1 mg/kg treatment groups were compared, the 1 mg/kg
TNV148 showed a significantly lower AI than 1 mg/kg cA2 at 3, 4 and
7 weeks. The 1 mg/kg TNV148 was also significantly lower than the 1
mg/kg TNV14-treated Group at 3 and 4 weeks. Although TNV196 showed
significant reduction in AI up to week 6 of the study (when
compared to the D-PBS-treated Group), TNV148 was the only 1 mg/kg
treatment that remained significant at the conclusion of the
study.
[0039] FIG. 12 shows arthritis mouse model mice Tg 197 weight
changes in response to anti-TNF antibodies of the present invention
as compared to controls in Example 5. At approximately 4 weeks of
age the Tg197 study mice were assigned, based on body weight, to
one of 8 treatment groups and treated with a intraperitoneal bolus
dose of control article (D-PBS) or antibody (TNV14, TNV148) at 3
mg/kg (week 0). Injections were repeated in all animals at weeks 1,
2, 3, and 4. Groups 1-6 were evaluated for test article efficacy.
Serum samples, obtained from animals in Groups 7 and 8 were
evaluated for immune response induction and pharmacokinetic
clearance of TNV14 or TNV148 at weeks 2, 3 and 4.
[0040] FIGS. 13A-C are graphs representing the progression of
disease severity in Example 5 based on the arthritic index. The 10
mg/kg cA2-treated group's arthritic index was significantly lower
then the D-PBS control group starting at week 2 and continuing
throughout the remainder of the study (week 5). The animals treated
with 1 mg/kg or 3 mg/kg of cA2 and the animals treated with 3 mg/kg
TNV14 failed to achieve any significant reduction in AI at any time
throughout the study when compared to the d-PBS control group. The
animals treated with 3 mg/kg TNV148 showed a significant reduction
when compared to the d-PBS-treated group starting at week 3 and
continuing through week 5. The 10 mg/kg cA2-treated animals showed
a significant reduction in AI when compared to both the lower doses
(1 mg/kg and 3 mg/kg) of cA2 at weeks 4 and 5 of the study and was
also significantly lower than the TNV14-treated animals at weeks
3-5. Although there appeared to be no significant differences
between any of the 3mg/kg treatment groups, the AI for the animals
treated with 3 mg/kg TNV14 were significantly higher at some time
points than the 10 mg/kg whereas the animals treated with TNV148
were not significantly different from the animals treated with 10
mg/kg of cA2.
[0041] FIG. 14 shows arthritis mouse model mice Tg 197 weight
changes in response to anti-TNF antibodies of the present invention
as compared to controls in Example 6. At approximately 4 weeks of
age the Tg197 study mice were assigned, based on gender and body
weight, to one of 6 treatment groups and treated with a single
intraperitoneal bolus dose of antibody (cA2, or TNV148) at either 3
mg/kg or 5 mg/kg. This study utilized the D-PBS and 10 mg/kg cA2
control Groups.
[0042] FIG. 15 represents the progression of disease severity based
on the arthritic index as presented in Example 6. All treatment
groups showed some protection at the earlier time points, with the
5 mg/kg cA2 and the 5 mg/kg TNV148 showing significant reductions
in AI at weeks 1-3 and all treatment groups showing a significant
reduction at week 2. Later in the study the animals treated with 5
mg/kg cA2 showed some protection, with significant reductions at
weeks 4, 6 and 7. The low dose (3 mg/kg) of both the cA2 and the
TNV148 showed significant reductions at 6 and all treatment groups
showed significant reductions at week 7. None of the treatment
groups were able to maintain a significant reduction at the
conclusion of the study (week 8). There were no significant
differences between any of the treatment groups (excluding the
saline control group) at any time point.
[0043] FIG. 16 shows arthritis mouse model mice Tg 197 weight
changes in response to anti-TNF antibodies of the present invention
as compared to controls in Example 7. To compare the efficacy of a
single intraperitoneal dose of TNV148 (derived from hybridoma
cells) and rTNV148B (derived from transfected cells). At
approximately 4 weeks of age the Tg197 study mice were assigned,
based on gender and body weight, to one of 9 treatment groups and
treated with a single intraperitoneal bolus dose of Dulbecco's PBS
(D-PBS) or antibody (TNV148, rTNV148B) at 1 mg/kg.
[0044] FIG. 17 represents the progression of disease severity based
on the arthritic index as presented in Example 7. The 10 mg/kg
cA2-treated group's arthritic index was lower then the D-PBS
control group starting at week 4 and continuing throughout the
remainder of the study (week 8). Both of the TNV148-treated Groups
and the 1 mg/kg cA2-treated Group showed a significant reduction in
AI at week 4. Although a previous study (P-099-017) showed that
TNV148 was slightly more effective at reducing the Arthritic Index
following a single 1 mg/kg intraperitoneal bolus, this study showed
that the AI from both versions of the TNV antibody-treated groups
was slightly higher. Although (with the exception of week 6) the 1
mg/kg cA2--treated Group was not significantly increased when
compared to the 10 mg/kg cA2 group and the TNV148-treated Groups
were significantly higher at weeks 7 and 8, there were no
significant differences in AI between the 1 mg/kg cA2, 1 mg/kg
TNV148 and 1 mg/kg TNV148B at any point in the study.
[0045] FIG. 18 shows study schema for study CNTO148DML2001.
[0046] FIG. 19 shows simulated golimumab concentrations (median and
95% Prediction Interval) through Week 24 for various dosing
regimens (without MTX) in virtual patients ages 6-21. Panel A: T1D
(red) vs JIA (blue) and Panel B: T1D (red) vs pedUC (green).
[0047] FIG. 20 shows simulated Time Course for Free TNF.alpha.
Suppression with Induction Period (1st month) in the left panel and
Maintenance Period (through 6 months) in the right panel.
[0048] FIG. 21 shows features of the Ultrasafe.
[0049] FIG. 22 shows an illustration of the SIMPONI.RTM.
VarioJect.TM. features.
[0050] FIG. 23 shows steps in the administration of the
SIMPONI.RTM. using the VarioJect.TM. device.
[0051] FIG. 24 shows proposed design modifications to the
VarioJect.TM. device.
DESCRIPTION OF THE INVENTION
[0052] The present invention provides isolated, recombinant and/or
synthetic anti-TNF human, primate, rodent, mammalian, chimeric,
humanized or CDR-grafted, antibodies comprising all of the heavy
chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all of
the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 and
TNF anti-idiotype antibodies thereto, as well as compositions and
encoding nucleic acid molecules comprising at least one
polynucleotide encoding at least one anti-TNF antibody or
anti-idiotype antibody. The present invention further includes, but
is not limited to, methods of making and using such nucleic acids
and antibodies and anti-idiotype antibodies, including diagnostic
and therapeutic compositions, methods and devices.
[0053] As used herein, an "anti-tumor necrosis factor alpha
antibody," "anti-TNF antibody," "anti-TNF antibody portion," or
"anti-TNF antibody fragment" and/or "anti-TNF 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 TNF 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, angonizes, mitigates,
aleviates, blocks, inhibits, abrogates and/or interferes with at
least one TNF activity or binding, or with TNF receptor activity or
binding, in vitro, in situ and/or in vivo. As a non-limiting
example, a suitable anti-TNF antibody, specified portion or variant
of the present invention can bind at least one TNF, or specified
portions, variants or domains thereof. A suitable anti-TNF
antibody, specified portion, or variant can also optionally affect
at least one of TNF activity or function, such as but not limited
to, RNA, DNA or protein synthesis, TNF release, TNF receptor
signaling, membrane TNF cleavage, TNF activity, TNF production
and/or synthesis. 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 TNF. For
example, antibody fragments capable of binding to TNF 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).
[0054] Such fragments can be produced by enzymatic cleavage,
synthetic or recombinant techniques, as known in the art and/or as
described herein antibodies can also be produced in a variety of
truncated forms using antibody genes in which one or more stop
codons have been introduced upstream of the natural stop site. For
example, a combination gene encoding a F(ab').sub.2 heavy chain
portion can be designed to include DNA sequences encoding the
CH.sub.1 domain and/or hinge region of the heavy chain. The various
portions of antibodies can be joined together chemically by
conventional techniques, or can be prepared as a contiguous protein
using genetic engineering techniques.
[0055] As used herein, the term "human antibody" refers to an
antibody in which substantially every part of the protein (e.g.,
CDR, framework, C.sub.L, C.sub.H domains (e.g., C.sub.H1, C.sub.H2,
C.sub.H3), hinge, (V.sub.L, V.sub.H)) is substantially
non-immunogenic in humans, with only minor sequence changes or
variations. Similarly, antibodies designated primate (monkey,
babboon, chimpanzee, etc.), rodent (mouse, rat, rabbit, guinea pid,
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.
[0056] Bispecific, heterospecific, heteroconjugate or similar
antibodies can also be used that are monoclonal, preferably human
or humanized, antibodies that have binding specificities for at
least two different antigens. In the present case, one of the
binding specificities is for at least one TNF protein, the other
one is for any other antigen. Methods for making bispecific
antibodies are known in the art. Traditionally, the recombinant
production of bispecific antibodies is based on the co-expression
of two immunoglobulin heavy chain-light chain pairs, where the two
heavy chains have different specificities (Milstein and Cuello,
Nature 305:537 (1983)). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of
which only one has the correct bispecific structure. The
purification of the correct molecule, which is usually done by
affinity chromatography steps, is rather cumbersome, and the
product yields are low. Similar procedures are disclosed, e.g., in
WO 93/08829, U.S. Pat. Nos. 6,210,668, 6,193,967, 6,132,992,
6,106,833, 6,060,285, 6,037,453, 6,010,902, 5,989,530, 5,959,084,
5,959,083, 5,932,448, 5,833,985, 5,821,333, 5,807,706, 5,643,759,
5,601,819, 5,582,996, 5,496,549, 4,676,980, WO 91/00,360, WO
92/00,373, EP 03,089, Traunecker et al., EMBO J. 10:3655 (1991),
Suresh et al., Methods in Enzymology 121:210 (1986), each entirely
incorporated herein by reference.
[0057] Anti-TNF antibodies (also termed TNF antibodies) useful in
the methods and compositions of the present invention can
optionally be characterized by high affinity binding to TNF 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).
[0058] Utility: The isolated nucleic acids of the present invention
can be used for production of at least one anti-TNF 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 TNF
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.
[0059] Such a method can comprise administering an effective amount
of a composition or a pharmaceutical composition comprising at
least one anti-TNF antibody to a cell, tissue, organ, animal or
patient in need of such modulation, treatment, alleviation,
prevention, or reduction in symptoms, effects or mechanisms. The
effective amount can comprise an amount of about 0.001 to 500 mg/kg
per single (e.g., bolus), multiple or continuous administration, or
to achieve a serum concentration of 0.01-5000 .mu.g/ml serum
concentration per single, multiple, or continuous adminstration, or
any effective range or value therein, as done and determined using
known methods, as described herein or known in the relevant arts.
Citations. All publications or patents cited herein are entirely
incorporated herein by reference as they show the state of the art
at the time of the present invention and/or to provide description
and enablement of the present invention. Publications refer to any
scientific or patent publications, or any other information
available in any media format, including all recorded, electronic
or printed formats. The following references are entirely
incorporated herein by reference: Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,
N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow
and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.
(1989); Colligan, et al., eds., Current Protocols in Immunology,
John Wiley & Sons, Inc., N.Y. (1994-2001); Colligan et al.,
Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001).
[0060] Antibodies of the Present Invention: At least one anti-TNF
antibody of the present invention comprising all of the heavy chain
variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all of the
light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 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., N.Y. (1994-2001); Colligan et al.,
Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2001), each entirely incorporated herein by
reference.
[0061] Human antibodies that are specific for human TNF proteins or
fragments thereof can be raised against an appropriate immunogenic
antigen, such as isolated and/or TNF protein or a portion thereof
(including synthetic molecules, such as synthetic peptides). Other
specific or general mammalian antibodies can be similarly raised.
Preparation of immunogenic antigens, and monoclonal antibody
production can be performed using any suitable technique.
[0062] In one approach, a hybridoma is produced by fusing a
suitable immortal cell line (e.g., a myeloma cell line such as, but
not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5,
>243, P3X63Ag8.653, Sp2 SA3, Sp2 MAl, Sp2 SS1, Sp2 SAS, U937,
MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH
3T3, HL-60, MLA 144, NAMAIWA, 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.
[0063] 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).
[0064] 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, Del.;
Biovation, Aberdeen, Scotland, UK; Bioinvent, Lund, Sweden; Dyax
Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, Calif.; Ixsys. See,
e.g., EP 368,684, PCT/GB91/01134; PCT/GB92/01755; PCT/GB92/002240;
PCT/GB92/00883; PCT/GB93/00605; US 08/350260(May 12, 1994);
PCT/GB94/01422; PCT/GB94/02662; PCT/GB97/01835; (CAT/MRC);
WO90/14443; WO90/14424; WO90/14430; PCT/U594/1234; WO92/18619;
WO96/07754; (Scripps); EP 614 989 (MorphoSys); WO95/16027
(Bioinvent); WO88/06630; WO90/3809 (Dyax); U.S. Pat. No. 4,704,692
(Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371 998; EP 550
400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); or stochastically
generated peptides or proteins--U.S. Pat. Nos. 5,723,323,
5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803,
EP 590 689 (Ixsys, now Applied Molecular Evolution (AME), each
entirely incorporated herein by reference) or that rely upon
immunization of transgenic animals (e.g., SCID mice, Nguyen et al.,
Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.
Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161
(1998), each entirely incorporated by reference as well as related
patents and applications) that are capable of producing a
repertoire of human antibodies, as known in the art and/or as
described herein. Such techniques, include, but are not limited to,
ribosome display (Hanes et al., Proc. Natl. Acad. Sci. USA,
94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,
95:14130-14135 (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)).
[0065] Methods for engineering or humanizing non-human or human
antibodies can also be used and are well known in the art.
Generally, a humanized or engineered antibody has one or more amino
acid residues from a source which is non-human, e.g., but not
limited to mouse, rat, rabbit, non-human primate or other mammal.
These human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable,
constant or other domain of a known human sequence. Known human Ig
sequences are disclosed, e.g., [0066]
www.ncbi.nlm.nih.gov/entrez/query.fcgi;
www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/;
www.antibodyresource.com/onlinecomp.html;
www.public.iastate.edu/.about.pedro/research_tools.html;
www.mgen.uni-heidelberg.de/SD/IT/IT.html;
www.whfreeman.com/immunology/CH05/kuby05.htm;
www.library.thinkquest.org/12429/Immune/Antibody.html;
www.hhmi.org/grants/lectures/1996/vlab/;
www.path.cam.ac.uk/.about.mrc7/mikeimages.html;
www.antibodyresource.com/;
mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/;
pathbox.wustl.edu/.about.hcenter/index.html;
www.biotech.ufl.edu/.about.hcl/;
www.pebio.com/pa/340913/340913.html;
www.nal.usda.gov/awic/pubs/antibody/;
www.m.ehime-u.ac.jp/.about.yasuhito/Elisa.html;
www.biodesign.com/table.asp;
www.icnet.uk/axp/facs/davies/links.html;
www.biotech.ufl.edu/.about.fccl/protocol.html;
www.isac-net.org/sites_geo.html;
aximtl.imt.uni-marburg.de/.about.rek/AEPStart.html;
baserv.uci.kun.nl/.about.jraats/links1.html;
www.recab.uni-hd.de/immuno.bme.nwu.edu/;
www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;
www.ibt.unam.mx/vir/_mice.html; imgt.cnusc.fr:8104/;
www.biochem.ucl.ac.uk/.about.martin/abs/index.html;
antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;
www.unizh.ch/.about.honegger/AHOseminar/Slide01.html;
www.cryst.bbk.ac.uk/.about.ubcg07s/;
www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;
www.path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;
www.ibt.unam.mx/vir/structure/stat_aim.html;
www.biosci.missouri.edu/smithgp/index.html;
www.cryst.bioc.cam.ac.uk/.about.fmolina/Web-pages/Pept/spottech.html;
www.jerini.de/fr_products.htm; www.patents.ibm.com/ibm.html.Kabat
et al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983), each entirely incorporated herein by reference.
[0067] Such imported sequences can be used to reduce immunogenicity
or reduce, enhance or modify binding, affinity, on-rate, off-rate,
avidity, specificity, half-life, or any other suitable
characteristic, as known in the art. Generally part or all of the
non-human or human CDR sequences are maintained while the non-human
sequences of the variable and constant regions are replaced with
human or other amino acids. antibodies can also optionally be
humanized with retention of high affinity for the antigen and other
favorable biological properties. To achieve this goal, humanized
antibodies can be optionally prepared by a process of analysis of
the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the consensus and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen(s), is achieved. In general, the CDR residues are
directly and most substantially involved in influencing antigen
binding. Humanization or engineering of antibodies of the present
invention can be performed using any known method, such as but not
limited to those described in, Winter (Jones et al., Nature 321:522
(1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al.,
Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296
(1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et
al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al.,
J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862,
5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886,
5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089,
5,225,539; 4,816,567, PCT/: US98/16280, US96/18978, US91/09630,
US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755;
WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirely
incorporated herein by reference, included references cited
therein.
[0068] The anti-TNF 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-TNF antibody can be isolated
from such animals and immortalized using suitable methods, such as
the methods described herein.
[0069] 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. US. 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.
[0070] 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.
Other systems for generating libraries of peptides have aspects of
both in vitro chemical synthesis and recombinant methods. See, PCT
Patent Publication Nos. 92/05258, 92/14843, and 96/19256. See also,
U.S. Pat. Nos. 5,658,754; and 5,643,768. Peptide display libraries,
vector, and screening kits are commercially available from such
suppliers as Invitrogen (Carlsbad, Calif.), and Cambridge antibody
Technologies (Cambridgeshire, UK). See, e.g., U.S. Pat. Nos.
4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889,
5,534,621, 5,656,730, 5,763,733, 5,767,260, 5,856,456, assigned to
Enzon; U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 5,837,500,
assigned to Dyax, U.S. Pat. Nos. 5,427,908, 5,580,717, assigned to
Affymax; U.S. Pat. No. 5,885,793, assigned to Cambridge antibody
Technologies; U.S. Pat. No. 5,750,373, assigned to Genentech, U.S.
Pat. Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5693493,
5698417, assigned to Xoma, Colligan, supra; Ausubel, supra; or
Sambrook, supra, each of the above patents and publications
entirely incorporated herein by reference.
[0071] Antibodies of the present invention can also be prepared
using at least one anti-TNF antibody encoding nucleic acid to
provide transgenic animals or mammals, such as goats, cows, horses,
sheep, and the like, that produce such antibodies in their milk.
Such animals can be provided using known methods. See, e.g., but
not limited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316;
5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of
which is entirely incorporated herein by reference.
[0072] Antibodies of the present invention can additionally be
prepared using at least one anti-TNF 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 reference
cited therein. Thus, antibodies of the present invention can also
be produced using transgenic plants, according to know methods. See
also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108
(October, 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma
et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem.
Soc. Trans. 22:940-944 (1994); and references cited therein. See,
also generally for plant expression of antibodies, but not limited
to, Each of the above references is entirely incorporated herein by
reference.
[0073] The antibodies of the invention can bind human TNF with a
wide range of affinities (K.sub.D). In a preferred embodiment, at
least one human mAb of the present invention can optionally bind
human TNF with high affinity. For example, a human mAb can bind
human TNF with a K.sub.D equal to or less than about 10.sup.-7 M,
such as but not limited to, 0.1-9.9 (or any range or value therein)
X 10.sup.-7, 10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11,
10.sup.-12, 10.sup.-13 or any range or value therein.
[0074] 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.
[0075] Nucleic Acid Molecules. Using the information provided
herein, such as the nucleotide sequences encoding at least 70-100%
of the contiguous amino acids of at least one of SEQ ID NOS:1, 2,
3, 4, 5, 6, 7, 8, specified fragments, variants or consensus
sequences thereof, or a deposited vector comprising at least one of
these sequences, a nucleic acid molecule of the present invention
encoding at least one anti-TNF antibody comprising all of the heavy
chain variable CDR regions of SEQ ID NOS:1, 2 and 3 and/or all of
the light chain variable CDR regions of SEQ ID NOS:4, 5 and 6 can
be obtained using methods described herein or as known in the
art.
[0076] 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.
[0077] Isolated nucleic acid molecules of the present invention can
include nucleic acid molecules comprising an open reading frame
(ORF), optionally with one or more introns, e.g., but not limited
to, at least one specified portion of at least one CDR, as CDR1,
CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS:1-3)
or light chain (e.g., SEQ ID NOS: 4-6); nucleic acid molecules
comprising the coding sequence for an anti-TNF antibody or variable
region (e.g., SEQ ID NOS:7,8); and nucleic acid 1molecules 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-TNF 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-TNF antibodies of the present invention. See,
e.g., Ausubel, et al., supra, and such nucleic acid variants are
included in the present invention. Non-limiting examples of
isolated nucleic acid molecules of the present inveniton include
SEQ ID NOS:10, 11, 12, 13, 14, 15, corresponding to non-limiting
examples of a nucleic acid encoding, respectively, HC CDR1, HC
CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3, HC variable region and LC
variable region.
[0078] As indicated herein, nucleic acid molecules of the present
invention which comprise a nucleic acid encoding an anti-TNF
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.
[0079] Polynucleotides Which Selectively Hybridize to a
Polynucleotide as Described Herein. The present invention provides
isolated nucleic acids that hybridize under selective hybridization
conditions to a polynucleotide disclosed herein. Thus, the
polynucleotides of this embodiment can be used for isolating,
detecting, and/or quantifying nucleic acids comprising such
polynucleotides. For example, polynucleotides of the present
invention can be used to identify, isolate, or amplify partial or
full-length clones in a deposited library. In some embodiments, the
polynucleotides are genomic or cDNA sequences isolated, or
otherwise complementary to, a cDNA from a human or mammalian
nucleic acid library.
[0080] 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.
[0081] Optionally, polynucleotides of this invention will encode at
least a portion of an antibody encoded by the polynucleotides
described herein. The polynucleotides of this invention embrace
nucleic acid sequences that can be employed for selective
hybridization to a polynucleotide encoding an antibody of the
present invention. See, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by reference.
[0082] Construction of Nucleic Acids. The isolated nucleic acids of
the present invention can be made using (a) recombinant methods,
(b) synthetic techniques, (c) purification techniques, or
combinations thereof, as well-known in the art.
[0083] 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.
[0084] 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).
[0085] Recombinant Methods for Constructing Nucleic Acids. The
isolated nucleic acid compositions of this invention, such as RNA,
cDNA, genomic DNA, or any combination thereof, can be obtained from
biological sources using any number of cloning methodologies known
to those of skill in the art. In some embodiments, oligonucleotide
probes that selectively hybridize, under stringent conditions, to
the polynucleotides of the present invention are used to identify
the desired sequence in a cDNA or genomic DNA library. The
isolation of RNA, and construction of cDNA and genomic libraries,
is well known to those of ordinary skill in the art. (See, e.g.,
Ausubel, supra; or Sambrook, supra).
[0086] Nucleic Acid Screening and Isolation Methods. A cDNA or
genomic library can be screened using a probe based upon the
sequence of a polynucleotide of the present invention, such as
those disclosed herein. Probes can be used to hybridize with
genomic DNA or cDNA sequences to isolate homologous genes in the
same or different organisms. Those of skill in the art will
appreciate that various degrees of stringency of hybridization can
be employed in the assay; and either the hybridization or the wash
medium can be stringent. As the conditions for hybridization become
more stringent, there must be a greater degree of complementarity
between the probe and the target for duplex formation to occur. The
degree of stringency can be controlled by one or more of
temperature, ionic strength, pH and the presence of a partially
denaturing solvent such as formamide. For example, the stringency
of hybridization is conveniently varied by changing the polarity of
the reactant solution through, for example, manipulation of the
concentration of formamide within the range of 0% to 50%. The
degree of complementarity (sequence identity) required for
detectable binding will vary in accordance with the stringency of
the hybridization medium and/or wash medium. The degree of
complementarity will optimally be 100%, or 70-100%, or any range or
value therein. However, it should be understood that minor sequence
variations in the probes and primers can be compensated for by
reducing the stringency of the hybridization and/or wash
medium.
[0087] 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.
[0088] 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. No.
4,795,699 and U.S. Pat. No. 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.)
[0089] For instance, polymerase chain reaction (PCR) technology can
be used to amplify the sequences of polynucleotides of the present
invention and related genes directly from genomic DNA or cDNA
libraries. PCR and other in vitro amplification methods can also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of the desired mRNA in samples, for
nucleic acid sequencing, or for other purposes. Examples of
techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, supra, Sambrook, supra,
and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No.
4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to
Methods and Applications, Eds., Academic Press Inc., San Diego,
Calif. (1990). Commercially available kits for genomic PCR
amplification are known in the art. See, e.g., Advantage-GC Genomic
PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein
(Boehringer
[0090] Mannheim) can be used to improve yield of long PCR
products.
[0091] Synthetic Methods for Constructing Nucleic Acids. The
isolated nucleic acids of the present invention can also be
prepared by direct chemical synthesis by known methods (see, e.g.,
Ausubel, et al., supra). Chemical synthesis generally produces a
single-stranded oligonucleotide, which can be converted into
double-stranded DNA by hybridization with a complementary sequence,
or by polymerization with a DNA polymerase using the single strand
as a template. One of skill in the art will recognize that while
chemical synthesis of DNA can be limited to sequences of about 100
or more bases, longer sequences can be obtained by the ligation of
shorter sequences.
[0092] Recombinant Expression Cassettes. The present invention
further provides recombinant expression cassettes comprising a
nucleic acid of the present invention. A nucleic acid sequence of
the present invention, for example a cDNA or a genomic sequence
encoding an antibody of the present invention, can be used to
construct a recombinant expression cassette that can be introduced
into at least one desired host cell. A recombinant expression
cassette will typically comprise a polynucleotide of the present
invention operably linked to transcriptional initiation regulatory
sequences that will direct the transcription of the polynucleotide
in the intended host cell. Both heterologous and non-heterologous
(i.e., endogenous) promoters can be employed to direct expression
of the nucleic acids of the present invention.
[0093] In some embodiments, isolated nucleic acids that serve as
promoter, enhancer, or other elements can be introduced in the
appropriate position (upstream, downstream or in intron) of a
non-heterologous form of a polynucleotide of the present invention
so as to up or down regulate expression of a polynucleotide of the
present invention. For example, endogenous promoters can be altered
in vivo or in vitro by mutation, deletion and/or substitution.
[0094] Vectors And Host Cells. The present invention also relates
to vectors that include isolated nucleic acid molecules of the
present invention, host cells that are genetically engineered with
the recombinant vectors, and the production of at least one
anti-TNF 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.
[0095] 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.
[0096] 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 initating site 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.
[0097] Expression vectors will preferably but optionally include at
least one selectable marker. Such markers include, e.g., but not
limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S.
Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636;
5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or
glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359;
5,827,739) resistance for eukaryotic cell culture, and tetracycline
or ampicillin resistance genes for culturing in E. coli and other
bacteria or prokaryotics (the above patents are entirely
incorporated hereby by reference). Appropriate culture mediums and
conditions for the above-described host cells are known in the art.
Suitable vectors will be readily apparent to the skilled artisan.
Introduction of a vector construct into a host cell can be effected
by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other known methods.
Such methods are described in the art, such as Sambrook, supra,
Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15,
16.
[0098] At least one antibody of the present invention can be
expressed in a modified form, such as a fusion protein, and can
include not only secretion signals, but also additional
heterologous functional regions. For instance, a region of
additional amino acids, particularly charged amino acids, can be
added to the N-terminus of an antibody to improve stability and
persistence in the host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties can be
added to an antibody of the present invention to facilitate
purification. Such regions can be removed prior to final
preparation of an antibody or at least one fragment thereof. Such
methods are described in many standard laboratory manuals, such as
Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel,
supra, Chapters 16, 17 and 18.
[0099] Those of ordinary skill in the art are knowledgeable in the
numerous expression systems available for expression of a nucleic
acid encoding a protein of the present invention.
[0100] Alternatively, nucleic acids of the present invention can be
expressed in a host cell by turning on (by manipulation) in a host
cell that contains endogenous DNA encoding an antibody of the
present invention. Such methods are well known in the art, e.g., as
described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and
5,733,761, entirely incorporated herein by reference.
[0101] Illustrative of cell cultures useful for the production of
the antibodies, specified portions or variants thereof, are
mammalian cells. Mammalian cell systems often will be in the form
of monolayers of cells although mammalian cell suspensions or
bioreactors can also be used. A number of suitable host cell lines
capable of expressing intact glycosylated proteins have been
developed in the art, and include the COS-1 (e.g., ATCC CRL 1650),
COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO
(e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines,
Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293
cells, HeLa cells and the like, which are readily available from,
for example, American Type Culture Collection, Manassas, Va.
(www.atcc.org). Preferred host cells include cells of lymphoid
origin such as myeloma and lymphoma cells. Particularly preferred
host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580)
and SP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a
particularly preferred embodiment, the recombinant cell is a
P3X63Ab8.653 or a SP2/0-Ag14 cell.
[0102] 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.
[0103] 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.
[0104] Purification of an Antibody. An anti-TNF 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.
[0105] Antibodies of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a eukaryotic host,
including, for example, yeast, higher plant, insect and mammalian
cells. Depending upon the host employed in a recombinant production
procedure, the antibody of the present invention can be
glycosylated or can be non-glycosylated, with glycosylated
preferred. Such methods are described in many standard laboratory
manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel,
supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein
Science, supra, Chapters 12-14, all entirely incorporated herein by
reference.
[0106] Anti-TNF Antibodies
[0107] The isolated antibodies of the present invention, comprising
all of the heavy chain variable CDR regions of SEQ ID NOS:1, 2 and
3 and/or all of the light chain variable CDR regions of SEQ ID
NOS:4, 5 and 6, comprise 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 TNF 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 TNF protein or fragment can
bind the protein or fragment and thereby inhibit activitys mediated
through the binding of TNF to the TNF receptor or through other
TNF-dependent or mediated mechanisms. As used herein, the term
"neutralizing antibody" refers to an antibody that can inhibit an
TNF-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-TNF antibody to inhibit an TNF-dependent
activity is preferably assessed by at least one suitable TNF
protein or receptor assay, as described herein and/or as known in
the art. A human antibody of the invention can be of any class
(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa
or lambda light chain. In one embodiment, the human antibody
comprises an IgG heavy chain or defined fragment, for example, at
least one of isotypes, IgG1, IgG2, IgG3 or IgG4. Antibodies of this
type can be prepared by employing a transgenic mouse or other
trangenic non-human mammal comprising at least one human light
chain (e.g., IgG, IgA.quadrature. and IgM (e.g., .gamma.1,
.gamma.2, .gamma.3, .gamma.4) transgenes as described herein and/or
as known in the art. In another embodiment, the anti-human TNF
human antibody comprises an IgG1 heavy chain and a IgG1 light
chain.
[0108] At least one antibody of the invention binds at least one
specified epitope specific to at least one TNF protein, subunit,
fragment, portion or any combination thereof. The at least one
epitope can comprise at least one antibody binding region that
comprises at least one portion of said protein, which epitope is
preferably comprised of at least one extracellular, soluble,
hydrophillic, external or cytoplasmic portion of said protein. The
at least one specified epitope can comprise any combination of at
least one amino acid sequence of at least 1-3 amino acids to the
entire specified portion of contiguous amino acids of the SEQ ID
NO:9.
[0109] Generally, the human antibody or antigen-binding fragment of
the present invention 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. As a non-limiting example, the antibody or
antigen-binding portion or variant can comprise at least one of the
heavy chain CDR3 having the amino acid sequence of SEQ ID NO:3,
and/or a light chain CDR3 having the amino acid sequence of SEQ ID
NO:6. In a particular embodiment, the antibody or antigen-binding
fragment can have an antigen-binding region that comprises at least
a portion of at least one heavy chain CDR (i.e., CDR1, CDR2 and/or
CDR3) having the amino acid sequence of the corresponding CDRs 1, 2
and/or 3 (e.g., SEQ ID NOS:1, 2, and/or 3). In another particular
embodiment, the antibody or antigen-binding portion or variant can
have an antigen-binding region that comprises at least a portion of
at least one light chain CDR (i.e., CDR1, CDR2 and/or CDR3) having
the amino acid sequence of the corresponding CDRs 1, 2 and/or 3
(e.g., SEQ ID NOS: 4, 5, and/or 6). In a preferred embodiment the
three heavy chain CDRs and the three light chain CDRs of the
antibody or antigen-binding fragment have the amino acid sequence
of the corresponding CDR of at least one of mAb TNV148, TNV14,
TNV15, TNV196, TNV118, TNV32, TNV86, as described herein. Such
antibodies can be prepared by chemically joining together the
various portions (e.g., CDRs, framework) of the antibody using
conventional techniques, by preparing and expressing a (i.e., one
or more) nucleic acid molecule that encodes the antibody using
conventional techniques of recombinant DNA technology or by using
any other suitable method.
[0110] The anti-TNF 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-TNF
antibody comprises at least one of heavy chain variable region,
optionally having the amino acid sequence of SEQ ID NO:7 and/or at
least one light chain variable region, optionally having the amino
acid sequence of SEQ ID NO:8. antibodies that bind to human TNF 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 TNF 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.
[0111] 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 TNF with high affinity (e.g., K.sub.D less than or equal to
about 10.sup.-9 M). Amino acid sequences that are substantially the
same as the sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions. A conservative amino acid substitution
refers to the replacement of a first amino acid by a second amino
acid that has chemical and/or physical properties (e.g., charge,
structure, polarity, hydrophobicity/ hydrophilicity) that are
similar to those of the first amino acid. Conservative
substitutions include replacement of one amino acid by another
within the following groups: lysine (K), arginine (R) and histidine
(H); aspartate (D) and glutamate (E); asparagine (N), glutamine
(Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E;
alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),
phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and
glycine (G); F, W and Y; C, S and T.
[0112] Amino Acid Codes. The amino acids that make up anti-TNF
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 LETTER THREE THREE NUCLEOTIDE CODE LETTER
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
[0113] An anti-TNF antibody of the present invention can include
one or more amino acid substitutions, deletions or additions,
either from natural mutations or human manipulation, as specified
herein.
[0114] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions, insertions or deletions for any given anti-TNF
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.
[0115] Amino acids in an anti-TNF antibody of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and
Wells, Science 244:1081-1085 (1989)). The latter procedure
introduces single alanine mutations at every residue in the
molecule. The resulting mutant molecules are then tested for
biological activity, such as, but not limited to at least one TNF
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)).
[0116] Anti-TNF antibodies of the present invention can include,
but are not limited to, at least one portion, sequence or
combination selected from 1 to all of the contiguous amino acids of
at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6.
[0117] A(n) anti-TNF antibody can further optionally comprise a
polypeptide of at least one of 70-100% of the contiguous amino
acids of at least one of SEQ ID NOS:7, 8.
[0118] In one embodiment, the amino acid sequence of an
immunoglobulin chain, or portion thereof (e.g., variable region,
CDR) has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the
amino acid sequence of the corresponding chain of at least one of
SEQ ID NOS:7, 8. For example, the amino acid sequence of a light
chain variable region can be compared with the sequence of SEQ ID
NO:8, or the amino acid sequence of a heavy chain CDR3 can be
compared with SEQ ID NO:7. Preferably, 70-100% amino acid identity
(i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or
value therein) is determined using a suitable computer algorithm,
as known in the art.
[0119] Exemplary heavy chain and light chain variable regions
sequences are provided in SEQ ID NOS: 7, 8. The antibodies of the
present invention, or specified variants thereof, can comprise any
number of contiguous amino acid residues from an antibody of the
present invention, wherein that number is selected from the group
of integers consisting of from 10-100% of the number of contiguous
residues in an anti-TNF 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.
[0120] As those of skill will appreciate, the present invention
includes at least one biologically active antibody of the present
invention. Biologically active antibodies have a specific activity
at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or
70%, and most preferably at least 80%, 90%, or 95%-1000% 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.
[0121] 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.
[0122] The modified antibodies and antigen-binding fragments of the
invention can comprise one or more organic moieties that are
covalently bonded, directly or indirectly, to the antibody. Each
organic moiety that is bonded to an antibody or antigen-binding
fragment of the invention can independently be a hydrophilic
polymeric group, a fatty acid group or a fatty acid ester group. As
used herein, the term "fatty acid" encompasses mono-carboxylic
acids and di-carboxylic acids. A "hydrophilic polymeric group," as
the term is used herein, refers to an organic polymer that is more
soluble in water than in octane. For example, polylysine is more
soluble in water than in octane. Thus, an antibody modified by the
covalent attachment of polylysine is encompassed by the invention.
Hydrophilic polymers suitable for modifying antibodies of the
invention can be linear or branched and include, for example,
polyalkane glycols (e.g., PEG, monomethoxy--polyethylene glycol
(mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose,
oligosaccharides, polysaccharides and the like), polymers of
hydrophilic amino acids (e.g., polylysine, polyarginine,
polyaspartate and the like), polyalkane oxides (e.g., polyethylene
oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
Preferably, the hydrophilic polymer that modifies the antibody of
the invention has a molecular weight of about 800 to about 150,000
Daltons as a separate molecular entity. For example PEG.sub.5000
and PEG.sub.20,000, wherein the subscript is the average molecular
weight of the polymer in Daltons, can be used. The hydrophilic
polymeric group can be substituted with one to about six alkyl,
fatty acid or fatty acid ester groups. Hydrophilic polymers that
are substituted with a fatty acid or fatty acid ester group can be
prepared by employing suitable methods. For example, a polymer
comprising an amine group can be coupled to a carboxylate of the
fatty acid or fatty acid ester, and an activated carboxylate (e.g.,
activated with N,N-carbonyl diimidazole) on a fatty acid or fatty
acid ester can be coupled to a hydroxyl group on a polymer.
[0123] Fatty acids and fatty acid esters suitable for modifying
antibodies of the invention can be saturated or can contain one or
more units of unsaturation. Fatty acids that are suitable for
modifying antibodies of the invention include, for example,
n-dodecanoate (C.sub.12, laurate), n-tetradecanoate (C.sub.14,
myristate), n-octadecanoate (C.sub.18, stearate), n-eicosanoate
(C.sub.20, arachidate), n-docosanoate (C.sub.22, behenate),
n-triacontanoate (C.sub.30), n-tetracontanoate (C.sub.40),
cis-.DELTA.9-octadecanoate (C.sub.18, oleate), all
cis-.DELTA.5,8,11,14-eicosatetraenoate (C.sub.20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid,
docosanedioic acid, and the like. Suitable fatty acid esters
include mono-esters of dicarboxylic acids that comprise a linear or
branched lower alkyl group. The lower alkyl group can comprise from
one to about twelve, preferably one to about six, carbon atoms.
[0124] 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.)
[0125] The modified antibodies of the invention can be produced by
reacting a human antibody or antigen-binding fragment with a
modifying agent. For example, the organic moieties can be bonded to
the antibody in a non-site specific manner by employing an
amine-reactive modifying agent, for example, an NHS ester of PEG.
Modified human antibodies or antigen-binding fragments can also be
prepared by reducing disulfide bonds (e.g., intra-chain disulfide
bonds) of an antibody or antigen-binding fragment. The reduced
antibody or antigen-binding fragment can then be reacted with a
thiol-reactive modifying agent to produce the modified antibody of
the invention. Modified human antibodies and antigen-binding
fragments comprising an organic moiety that is bonded to specific
sites of an antibody of the present invention can be prepared using
suitable methods, such as reverse proteolysis (Fisch et al.,
Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate
Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.
6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68
(1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463
(1997)), and the methods described in Hermanson, G. T.,
Bioconjugate Techniques, Academic Press: San Diego, Calif.
(1996).
[0126] Anti-Idiotype Antibodies To Anti-Tnf Antibody Compositions.
In addition to monoclonal or chimeric anti-TNF antibodies, the
present invention is also directed to an anti-idiotypic (anti-Id)
antibody specific for such antibodies of the invention. An anti-Id
antibody is an antibody which recognizes unique determinants
generally associated with the antigen-binding region of another
antibody. The anti-Id can be prepared by immunizing an animal of
the same species and genetic type (e.g. mouse strain) as the source
of the Id antibody with the antibody or a CDR containing region
thereof. The immunized animal will recognize and respond to the
idiotypic determinants of the immunizing antibody and produce an
anti-Id antibody. The anti-Id antibody may also be used as an
"immunogen" to induce an immune response in yet another animal,
producing a so-called anti-anti-Id antibody.
[0127] Anti-Tnf Antibody Compositions. The present invention also
provides at least one anti-TNF antibody composition comprising at
least one, at least two, at least three, at least four, at least
five, at least six or more anti-TNF 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-TNF antibody amino acid sequence selected from the group
consisting of 70-100% of the contiguous amino acids of SEQ ID
NOS:1, 2, 3, 4, 5, 6, 7, 8, or specified fragments, domains or
variants thereof. Preferred anti-TNF antibody compositions include
at least one or two full length, fragments, domains or variants as
at least one CDR or LBR containing portions of the anti-TNF
antibody sequence of 70-100% of SEQ ID NOS:1, 2, 3, 4, 5, 6, or
specified fragments, domains or variants thereof. Further preferred
compositions comprise 40-99% of at least one of 70-100% of SEQ ID
NOS:1, 2, 3, 4, 5, 6, or specified fragments, domains or variants
thereof. Such composition percentages are by weight, volume,
concentration, molarity, or molality as liquid or dry solutions,
mixtures, suspension, emulsions or colloids, as known in the art or
as described herein.
[0128] Anti-TNF antibody compositions of the present invention can
further comprise at least one of any suitable and effective amount
of a composition or pharmaceutical composition comprising at least
one anti-TNF antibody to a cell, tissue, organ, animal or patient
in need of such modulation, treatment or therapy, optionally
further comprising at least one selected from at least one TNF
antagonist (e.g., but not limited to a TNF antibody or fragment, a
soluble TNF receptor or fragment, fusion proteins thereof, or a
small molecule TNF antagonist), an antirheumatic (e.g.,
methotrexate, auranofin, aurothioglucose, azathioprine, etanercept,
gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide,
sulfasalzine), a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anethetic, a neuromuscular blocker, an
antimicrobial (e.g., aminoglycoside, an antifungal, an
antiparasitic, an antiviral, a carbapenem, cephalosporin, a
flurorquinolone, a macrolide, a penicillin, a sulfonamide, a
tetracycline, another antimicrobial), an antipsoriatic, a
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 erythropieitin
(e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a
sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin,
an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab),
a growth hormone, a hormone replacement drug, an estrogen receptor
modulator, a mydriatic, a cycloplegic, an alkylating agent, an
antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an
antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a
hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an
asthma medication, a beta agonist, an inhaled steroid, a
leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine
or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine
antagonist. Non-limiting examples of such cytokines include, but
are not limted to, any of IL-1 to IL-23. 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.
[0129] Such anti-cancer or anti-infectives can also include toxin
molecules that are associated, bound, co-formulated or
co-administered with at least one antibody of the present
invention. The toxin can optionally act to selectively kill the
pathologic cell or tissue. The pathologic cell can be a cancer or
other cell. Such toxins can be, but are not limited to, purified or
recombinant toxin or toxin fragment comprising at least one
functional cytotoxic domain of toxin, e.g., selected from at least
one of ricin, diphtheria toxin, a venom toxin, or a bacterial
toxin. The term toxin also includes both endotoxins and exotoxins
produced by any naturally occurring, mutant or recombinant bacteria
or viruses which may cause any pathological condition in humans and
other mammals, including toxin shock, which can result in death.
Such toxins may include, but are not limited to, enterotoxigenic E.
coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST),
Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome
toxin-1 (TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C
(SEC), Streptococcal enterotoxins and the like. Such bacteria
include, but are not limited to, strains of a species of
enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g.,
strains of serotype 0157:H7), Staphylococcus species (e.g.,
Staphylococcus aureus, Staphylococcus pyogenes), Shigella species
(e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii,
and Shigella sonnei), Salmonella species (e.g., Salmonella typhi,
Salmonella cholera-suis, Salmonella enteritidis), Clostridium
species (e.g., Clostridium perfringens, Clostridium dificile,
Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter
jejuni, Camphlobacter fetus), Heliocbacter species, (e.g.,
Heliocbacter pylori), Aeromonas species (e.g., Aeromonas sobria,
Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,
Yersinia enterocolitica, Vibrio species (e.g., Vibrio cholerae,
Vibrio parahemolyticus), Klebsiella species, Pseudomonas
aeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL
MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990);
Evans et al., eds., Bacterial Infections of Humans: Epidemiology
and Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., N.Y.
(1991); Mandell et al, Principles and Practice of Infectious
Diseases, 3d. Ed., Churchill Livingstone, N.Y. (1990); Berkow et
al, eds., The Merck Manual, 16th edition, Merck and Co., Rahway,
N.J., 1992; Wood et al, FEMS Microbiology Immunology, 76:121-134
(1991); Marrack et al, Science, 248:705-711 (1990), the contents of
which references are incorporated entirely herein by reference.
[0130] Anti-TNF antibody compounds, compositions or combinations of
the present invention can further comprise at least one of any
suitable auxiliary, such as, but not limited to, diluent, binder,
stabilizer, buffers, salts, lipophilic solvents, preservative,
adjuvant or the like. Pharmaceutically acceptable auxiliaries are
preferred. Non-limiting examples of, and methods of preparing such
sterile solutions are well known in the art, such as, but limited
to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18.sup.th
Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically
acceptable carriers can be routinely selected that are suitable for
the mode of administration, solubility and/or stability of the
anti-TNF antibody, fragment or variant composition as well known in
the art or as described herein.
[0131] 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.
[0132] 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.
[0133] Anti-TNF 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.
[0134] Additionally, anti-TNF antibody compositions of the
invention can include polymeric excipients/additives such as
polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates
(e.g., cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin),
polyethylene glycols, flavoring agents, antimicrobial agents,
sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and
chelating agents (e.g., EDTA).
[0135] These and additional known pharmaceutical excipients and/or
additives suitable for use in the anti-TNF antibody, portion or
variant compositions according to the invention are known in the
art, e.g., as listed in "Remington: The Science & Practice of
Pharmacy", 19.sup.th ed., Williams & Williams, (1995), and in
the "Physician's Desk Reference", 52.sup.nd ed., Medical Economics,
Montvale, N.J. (1998), the disclosures of which are entirely
incorporated herein by reference. Preferrred carrier or excipient
materials are carbohydrates (e.g., saccharides and alditols) and
buffers (e.g., citrate) or polymeric agents.
[0136] Formulations. As noted above, the invention provides for
stable formulations, which is preferably a phosphate buffer with
saline or a chosen salt, as well as preserved solutions and
formulations containing a preservative as well as multi-use
preserved formulations suitable for pharmaceutical or veterinary
use, comprising at least one anti-TNF 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.
[0137] As noted above, the invention provides an article of
manufacture, comprising packaging material and at least one vial
comprising a solution of at least one anti-TNF antibody with the
prescribed buffers and/or preservatives, optionally in an aqueous
diluent, wherein said packaging material comprises a label that
indicates that such solution can be held over a period of 1, 2, 3,
4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or
greater. The invention further comprises an article of manufacture,
comprising packaging material, a first vial comprising lyophilized
at least one anti-TNF antibody, and a second vial comprising an
aqueous diluent of prescribed buffer or preservative, wherein said
packaging material comprises a label that instructs a patient to
reconstitute the at least one anti-TNF antibody in the aqueous
diluent to form a solution that can be held over a period of
twenty-four hours or greater.
[0138] The at least one anti-TNFantibody 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.
[0139] The range of at least one anti-TNF antibody in the product
of the present invention includes amounts yielding upon
reconstitution, if in a wet/dry system, concentrations from about
1.0 .mu.g/ml to about 1000 mg/ml, although lower and higher
concentrations are operable and are dependent on the intended
delivery vehicle, e.g., solution formulations will differ from
transdermal patch, pulmonary, transmucosal, or osmotic or micro
pump methods.
[0140] 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.
[0141] Other excipients, e.g. isotonicity agents, buffers,
antioxidants, preservative enhancers, can be optionally and
preferably added to the diluent. An isotonicity agent, such as
glycerin, is commonly used at known concentrations. A
physiologically tolerated buffer is preferably added to provide
improved pH control. The formulations can cover a wide range of
pHs, such as from about pH 4 to about pH 10, and preferred ranges
from about pH 5 to about pH 9, and a most preferred range of about
6.0 to about 8.0. Preferably the formulations of the present
invention have pH between about 6.8 and about 7.8. Preferred
buffers include phosphate buffers, most preferably sodium
phosphate, particularly phosphate buffered saline (PBS).
[0142] 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.
polyols, 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.
[0143] The formulations of the present invention can be prepared by
a process which comprises mixing at least one anti-TNF 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-TNF 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-TNF 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.
[0144] The claimed formulations can be provided to patients as
clear solutions or as dual vials comprising a vial of lyophilized
at least one anti-TNF 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.
[0145] The present claimed articles of manufacture are useful for
administration over a period of immediately to twenty-four hours or
greater. Accordingly, the presently claimed articles of manufacture
offer significant advantages to the patient. Formulations of the
invention can optionally be safely stored at temperatures of from
about 2 to about 40.degree. C. and retain the biologically activity
of the protein for extended periods of time, thus, allowing a
package label indicating that the solution can be held and/or used
over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.
If preserved diluent is used, such label can include use up to 1-12
months, one-half, one and a half, and/or two years.
[0146] The solutions of at least one anti-TNF antibody in the
invention can be prepared by a process that comprises mixing at
least one antibody in an aqueous diluent. Mixing is carried out
using conventional dissolution and mixing procedures. To prepare a
suitable diluent, for example, a measured amount of at least one
antibody in water or buffer is combined in quantities sufficient to
provide the protein and optionally a preservative or buffer at the
desired concentrations. Variations of this process would be
recognized by one of ordinary skill in the art. For example, the
order the components are added, whether additional additives are
used, the temperature and pH at which the formulation is prepared,
are all factors that can be optimized for the concentration and
means of administration used.
[0147] The claimed products can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized at
least one anti-TNF 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.
[0148] 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-TNF 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.
[0149] Recognized devices comprising these single vial systems
include those pen-injector devices for delivery of a solution such
as BD Pens, BD Autojector.RTM., Humaject.RTM., NovoPen.RTM.,
B-D.RTM.Pen, AutoPen.RTM., and OptiPen.RTM., GenotropinPen.RTM.,
Genotronorm Pen.RTM., Humatro Pen.RTM., Reco-Pen.RTM., Roferon
Pen.RTM., Biojector.RTM., iject.RTM., J-tip Needle-Free
Injector.RTM., Intraject.RTM., Medi-Ject.RTM., e.g., as made or
developed by Becton Dickensen (Franklin Lakes, N.J.,
www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,
www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com);
National Medical Products , Weston Medical (Peterborough, UK,
www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,
www.mediject.com). Recognized devices comprising a dual vial system
include those pen-injector systems for reconstituting a lyophilized
drug in a cartridge for delivery of the reconstituted solution such
as the HumatroPen.RTM..
[0150] The products presently claimed include packaging material.
The packaging material provides, in addition to the information
required by the regulatory agencies, the conditions under which the
product can be used. The packaging material of the present
invention provides instructions to the patient to reconstitute the
at least one anti-TNF antibody in the aqueous diluent to form a
solution and to use the solution over a period of 2-24 hours or
greater for the two vial, wet/dry, product. For the single vial,
solution product, the label indicates that such solution can be
used over a period of 2-24 hours or greater. The presently claimed
products are useful for human pharmaceutical product use.
[0151] The formulations of the present invention can be prepared by
a process that comprises mixing at least one anti-TNF antibody and
a selected buffer, preferably a phosphate buffer containing saline
or a chosen salt. Mixing the at least one antibody and buffer in an
aqueous diluent is carried out using conventional dissolution and
mixing procedures. To prepare a suitable formulation, for example,
a measured amount of at least one antibody in water or buffer is
combined with the desired buffering agent in water in quantities
sufficient to provide the protein and buffer at the desired
concentrations. Variations of this process would be recognized by
one of ordinary skill in the art. For example, the order the
components are added, whether additional additives are used, the
temperature and pH at which the formulation is prepared, are all
factors that can be optimized for the concentration and means of
administration used.
[0152] The claimed stable or preserved formulations can be provided
to patients as clear solutions or as dual vials comprising a vial
of lyophilized at least one anti-TNF 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.
[0153] At least one anti-TNF 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.
[0154] Therapeutic Applications. The present invention also
provides a method for modulating or treating at least one TNF
related disease, in a cell, tissue, organ, animal, or patient, as
known in the art or as described herein, using at least one dual
integrin antibody of the present invention.
[0155] The present invention also provides a method for modulating
or treating at least one TNF related disease, in a cell, tissue,
organ, animal, or patient including, but not limited to, at least
one of obesity, an immune related disease, a cardiovascular
disease, an infectious disease, a malignant disease or a neurologic
disease.
[0156] The present invention also provides a method for modulating
or treating at least one immune related disease, in a cell, tissue,
organ, animal, or patient including, but not limited to, at least
one of rheumatoid arthritis, juvenile, systemic onset juvenile
rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,
gastric ulcer, seronegative arthropathies, osteoarthritis,
inflammatory bowel disease, ulcerative colitis, systemic lupus
erythematosis, antiphospholipid syndrome,
iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary
fibrosis, systemic vasculitis/wegener's granulomatosis,
sarcoidosis, orchitis/vasectomy reversal procedures,
allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic contact dermatitis, allergic conjunctivitis,
hypersensitivity pneumonitis, transplants, organ transplant
rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis,
neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage,
burns, ionizing radiation exposure, acute pancreatitis, adult
respiratory distress syndrome, alcohol-induced hepatitis, chronic
inflammatory pathologies, sarcoidosis, Crohn's pathology, sickle
cell anemia, diabetes, nephrosis, atopic diseases, hypersensitity
reactions, allergic rhinitis, hay fever, perennial rhinitis,
conjunctivitis, endometriosis, asthma, urticaria, systemic
anaphylaxis, dermatitis, pernicious anemia, hemolytic disesease,
thrombocytopenia, graft rejection of any organ or tissue, kidney
translplant rejection, heart transplant rejection, liver transplant
rejection, pancreas transplant rejection, lung transplant
rejection, bone marrow transplant (BMT) rejection, skin allograft
rejection, cartilage transplant rejection, bone graft rejection,
small bowel transplant rejection, fetal thymus implant rejection,
parathyroid transplant rejection, xenograft rejection of any organ
or tissue, allograft rejection, anti-receptor hypersensitivity
reactions, Graves disease, Raynoud's disease, type B
insulin-resistant diabetes, asthma, myasthenia gravis,
antibody-meditated cytotoxicity, type III hypersensitivity
reactions, systemic lupus erythematosus, POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, skin changes
syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed
connective tissue disease, idiopathic Addison's disease, diabetes
mellitus, chronic active hepatitis, primary billiary cirrhosis,
vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV
hypersensitivity, contact dermatitis, hypersensitivity pneumonitis,
allograft rejection, granulomas due to intracellular organisms,
drug sensitivity, metabolic/idiopathic, Wilson's disease,
hemachromatosis, alpha-1-antitrypsin deficiency, diabetic
retinopathy, hashimoto's thyroiditis, osteoporosis, primary biliary
cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic
fibrosis, neonatal chronic lung disease, chronic obstructive
pulmonary disease (COPD), familial hematophagocytic
lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,
nephrotic syndrome, nephritis, glomerular nephritis, acute renal
failure, hemodialysis, uremia, toxicity, preeclampsia, okt3
therapy, anti-cd3 therapy, cytokine therapy, chemotherapy,
radiation therapy (e.g., including but not limited toasthenia,
anemia, cachexia, and the like), chronic salicylate intoxication,
and the like. See, e.g., the Merck Manual, 12th-17th Editions,
Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992,
1999), Pharmacotherapy Handbook, Wells et al., eds., Second
Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each
entirely incorporated by reference.
[0157] The present invention also provides a method for modulating
or treating at least one cardiovascular disease in a cell, tissue,
organ, animal, or patient, including, but not limited to, at least
one of cardiac stun syndrome, myocardial infarction, congestive
heart failure, stroke, ischemic stroke, hemorrhage,
arteriosclerosis, atherosclerosis, restenosis, diabetic
ateriosclerotic disease, hypertension, arterial hypertension,
renovascular hypertension, syncope, shock, syphilis of the
cardiovascular system, heart failure, cor pulmonale, primary
pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats,
atrial flutter, atrial fibrillation (sustained or paroxysmal), post
perfusion syndrome, cardiopulmonary bypass inflammation response,
chaotic or multifocal atrial tachycardia, regular narrow QRS
tachycardia, specific arrythmias, ventricular fibrillation, His
bundle arrythmias, atrioventricular block, bundle branch block,
myocardial ischemic disorders, coronary artery disease, angina
pectoris, myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart
diseases, endocarditis, pericardial disease, cardiac tumors, aortic
and peripheral aneuryisms, aortic dissection, inflammation of the
aorta, occulsion of the abdominal aorta and its branches,
peripheral vascular disorders, occulsive arterial disorders,
peripheral atherlosclerotic disease, thromboangitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous
thrombosis, varicose veins, arteriovenous fistula, lymphedema,
lipedema, unstable angina, reperfusion injury, post pump syndrome,
ischemia-reperfusion injury, and the like. Such a method can
optionally comprise administering an effective amount of a
composition or pharmaceutical composition comprising at least one
anti-TNF antibody to a cell, tissue, organ, animal or patient in
need of such modulation, treatment or therapy.
[0158] The present invention also provides a method for modulating
or treating at least one infectious disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: acute or chronic bacterial infection, acute and chronic
parasitic or infectious processes, including bacterial, viral and
fungal infections, HIV infection/HIV neuropathy, meningitis,
hepatitis (A, B or C, or the like), septic arthritis, peritonitis,
pneumonia, epiglottitis, e. coli 0157:h7, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue
hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome,
streptococcal myositis, gas gangrene, mycobacterium tuberculosis,
mycobacterium avium intracellulare, pneumocystis carinii pneumonia,
pelvic inflammatory disease, orchitis/epidydimitis, legionella,
lyme disease, influenza a, epstein-barr virus, viral-associated
hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and
the like.
[0159] The present invention also provides a method for modulating
or treating at least one malignant disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: leukemia, acute leukemia, acute lymphoblastic leukemia
(ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),
chromic myelocytic leukemia (CML), chronic lymphocytic leukemia
(CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a
lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's
lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma,
colorectal carcinoma, pancreatic carcinoma, nasopharyngeal
carcinoma, malignant histiocytosis, paraneoplastic
syndrome/hypercalcemia of malignancy, solid tumors,
adenocarcinomas, sarcomas, malignant melanoma, hemangioma,
metastatic disease, cancer related bone resorption, cancer related
bone pain, and the like.
[0160] The present invention also provides a method for modulating
or treating at least one neurologic disease in a cell, tissue,
organ, animal or patient, including, but not limited to, at least
one of: neurodegenerative diseases, multiple sclerosis, migraine
headache, AIDS dementia complex, demyelinating diseases, such as
multiple sclerosis and acute transverse myelitis; extrapyramidal
and cerebellar disorders' such as lesions of the corticospinal
system; disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs which block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; Progressive
supranucleo Palsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia, telangiectasiaa, and mitochondrial
multi. system disorder); demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit' such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease; and
Dementia pugilistica, and the like. Such a method can optionally
comprise administering an effective amount of a composition or
pharmaceutical composition comprising at least one TNF antibody or
specified portion or variant to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy. See,
e.g., the Merck Manual, 16.sup.th Edition, Merck & Company,
Rahway, N.J. (1992)
[0161] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising at least one anti-TNF
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 immune diseases, wherein the administering of said at
least one anti-TNF 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 antibody or fragment, a soluble TNF receptor
or fragment, fusion proteins thereof, or a small molecule TNF
antagonist), an antirheumatic (e.g., methotrexate, auranofin,
aurothioglucose, azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, 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 erythropieitin (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), each of which references are entirely
incorporated herein by reference.
[0162] TNF antagonists suitable for compositions, combination
therapy, co-administration, devices and/or methods of the present
invention (further comprising at least one anti body, specified
portion and variant thereof, of the present invention), include,
but are not limited to, anti-TNF antibodies, antigen-binding
fragments thereof, and receptor molecules which bind specifically
to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF
release or its action on target cells, such as thalidomide,
tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and
rolipram), A2b adenosine receptor agonists and A2b adenosine
receptor enhancers; compounds which prevent and/or inhibit TNF
receptor signalling, such as mitogen activated protein (MAP) kinase
inhibitors; compounds which block and/or inhibit membrane TNF
cleavage, such as metalloproteinase inhibitors; compounds which
block and/or inhibit TNF activity, such as angiotensin converting
enzyme (ACE) inhibitors (e.g., captopril); and compounds which
block and/or inhibit TNF production and/or synthesis, such as MAP
kinase inhibitors.
[0163] As used herein, a "tumor necrosis factor antibody," "TNF
antibody," "TNF.alpha. antibody," or fragment and the like
decreases, blocks, inhibits, abrogates or interferes with
TNF.alpha. activity in vitro, in situ and/or preferably in vivo.
For example, a suitable TNF human antibody of the present invention
can bind TNF.alpha. and includes anti-TNF antibodies,
antigen-binding fragments thereof, and specified mutants or domains
thereof that bind specifically to TNF.alpha.. A suitable TNF
antibody or fragment can also decrease block, abrogate, interfere,
prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF
release, TNF receptor signaling, membrane TNF cleavage, TNF
activity, TNF production and/or synthesis.
[0164] Chimeric antibody cA2 consists of the antigen binding
variable region of the high-affinity neutralizing mouse anti-human
TNF.alpha. IgG1 antibody, designated A2, and the constant regions
of a human IgG1, kappa immunoglobulin. The human IgG1 Fc region
improves allogeneic antibody effector function, increases the
circulating serum half-life and decreases the immunogenicity of the
antibody. The avidity and epitope specificity of the chimeric
antibody cA2 is derived from the variable region of the murine
antibody A2. In a particular embodiment, a preferred source for
nucleic acids encoding the variable region of the murine antibody
A2 is the A2 hybridoma cell line.
[0165] Chimeric A2 (cA2) neutralizes the cytotoxic effect of both
natural and recombinant human TNF.alpha. in a dose dependent
manner. From binding assays of chimeric antibody cA2 and
recombinant human TNF.alpha., the affinity constant of chimeric
antibody cA2 was calculated to be 1.04.times.10.sup.10M.sup.-1.
Preferred methods for determining monoclonal antibody specificity
and affinity by competitive inhibition can be found in Harlow, et
al., antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988; Colligan et al., eds.,
Current Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1992-2000); Kozbor et al., Immunol. Today,
4:72-79 (1983); Ausubel et al., eds. Current Protocols in Molecular
Biology, Wiley Interscience, N.Y. (1987-2000); and Muller, Meth.
Enzymol., 92:589-601 (1983), which references are entirely
incorporated herein by reference.
[0166] In a particular embodiment, murine monoclonal antibody A2 is
produced by a cell line designated c134A. Chimeric antibody cA2 is
produced by a cell line designated c168A.
[0167] Additional examples of monoclonal anti-TNF antibodies that
can be used in the present invention are described in the art (see,
e.g., U.S. Pat. No. 5,231,024; Moller, A. et al., Cytokine
2(3):162-169 (1990); U.S. application Ser. No. 7/943,852 (filed
Sep. 11, 1992); Rathjen et al., International Publication No. WO
91/02078 (published Feb. 21, 1991); Rubin et al., EPO Patent
Publication No. 0 218 868 (published Apr. 22, 1987); Yone et al.,
EPO Patent Publication No. 0 288 088 (Oct. 26, 1988); Liang, et
al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et
al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369
(1987); Bringman, et al., Hybridoma 6:489-507 (1987); and Hirai, et
al., J. Immunol. Meth. 96:57-62 (1987), which references are
entirely incorporated herein by reference).
[0168] TNF Receptor Molecules. Preferred TNF receptor molecules
useful in the present invention are those that bind TNF.alpha. with
high affinity (see, e.g., Feldmann et al., International
Publication No. WO 92/07076 (published Apr. 30, 1992); Schall et
al., Cell 61:361-370 (1990); and Loetscher et al., Cell 61:351-359
(1990), which references are entirely incorporated herein by
reference) and optionally possess low immunogenicity. In
particular, the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF
cell surface receptors are useful in the present invention.
Truncated forms of these receptors, comprising the extracellular
domains (ECD) of the receptors or functional portions thereof (see,
e.g., Corcoran et al., Eur. J. Biochem. 223:831-840 (1994)), are
also useful in the present invention. Truncated forms of the TNF
receptors, comprising the ECD, have been detected in urine and
serum as 30 kDa and 40 kDa TNF.alpha. inhibitory binding proteins
(Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)). TNF
receptor multimeric molecules and TNF immunoreceptor fusion
molecules, and derivatives and fragments or portions thereof, are
additional examples of TNF receptor molecules which are useful in
the methods and compositions of the present invention. The TNF
receptor molecules which can be used in the invention are
characterized by their ability to treat patients for extended
periods with good to excellent alleviation of symptoms and low
toxicity. Low immunogenicity and/or high affinity, as well as other
undefined properties, can contribute to the therapeutic results
achieved.
[0169] TNF receptor multimeric molecules useful in the present
invention comprise all or a functional portion of the ECD of two or
more TNF receptors linked via one or more polypeptide linkers or
other nonpeptide linkers, such as polyethylene glycol (PEG). The
multimeric molecules can further comprise a signal peptide of a
secreted protein to direct expression of the multimeric molecule.
These multimeric molecules and methods for their production have
been described in U.S. application Ser. No. 8/437,533 (filed May 9,
1995), the content of which is entirely incorporated herein by
reference.
[0170] TNF immunoreceptor fusion molecules useful in the methods
and compositions of the present invention comprise at least one
portion of one or more immunoglobulin molecules and all or a
functional portion of one or more TNF receptors. These
immunoreceptor fusion molecules can be assembled as monomers, or
hetero- or homo-multimers. The immunoreceptor fusion molecules can
also be monovalent or multivalent. An example of such a TNF
immunoreceptor fusion molecule is TNF receptor/IgG fusion protein.
TNF immunoreceptor fusion molecules and methods for their
production have been described in the art (Lesslauer et al., Eur.
J. Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl.
Acad. Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med.
174:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219 (1994); Butler et al., Cytokine 6(6):616-623 (1994);
Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al.,
U.S. Pat. No. 5,447,851; and U.S. application Ser. No. 8/442,133
(filed May 16, 1995), each of which references are entirely
incorporated herein by reference). Methods for producing
immunoreceptor fusion molecules can also be found in Capon et al.,
U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538; and
Capon et al., Nature 337:525-531 (1989), which references are
entirely incorporated herein by reference.
[0171] A functional equivalent, derivative, fragment or region of
TNF receptor molecule refers to the portion of the TNF receptor
molecule, or the portion of the TNF receptor molecule sequence
which encodes TNF receptor molecule, that is of sufficient size and
sequences to functionally resemble TNF receptor molecules that can
be used in the present invention (e.g., bind TNF.quadrature. with
high affinity and possess low immunogenicity). A functional
equivalent of TNF receptor molecule also includes modified TNF
receptor molecules that functionally resemble TNF receptor
molecules that can be used in the present invention (e.g., bind
TNF.alpha. with high affinity and possess low immunogenicity). For
example, a functional equivalent of TNF receptor molecule can
contain a "SILENT" codon or one or more amino acid substitutions,
deletions or additions (e.g., substitution of one acidic amino acid
for another acidic amino acid; or substitution of one codon
encoding the same or different hydrophobic amino acid for another
codon encoding a hydrophobic amino acid). See Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing Assoc.
and Wiley-Interscience, N.Y. (1987-2000).
[0172] Cytokines include any known cytokine. See, e.g.,
CopewithCytokines.com. Cytokine antagonists include, but are not
limited to, any antibody, fragment or mimetic, any soluble
receptor, fragment or mimetic, any small molecule antagonist, or
any combination thereof.
[0173] Therapeutic Treatments. Any method of the present invention
can comprise a method for treating a TNF mediated disorder,
comprising administering an effective amount of a composition or
pharmaceutical composition comprising at least one anti-TNF
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 immune diseases, wherein the administering of said at
least one anti-TNF 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 antibody or fragment, a soluble TNF receptor
or fragment, fusion proteins thereof, or a small molecule TNF
antagonist), an antirheumatic (e.g., methotrexate, auranofin,
aurothioglucose, azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anethetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a flurorquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, 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 erythropieitin (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.
[0174] Typically, treatment of pathologic conditions is effected by
administering an effective amount or dosage of at least one
anti-TNF antibody composition that total, on average, a range from
at least about 0.01 to 500 milligrams of at least one
anti-TNFantibody per kilogram of patient per dose, and preferably
from at least about 0.1 to 100 milligrams antibody /kilogram of
patient per single or multiple administration, depending upon the
specific activity of contained in the composition. Alternatively,
the effective serum concentration can comprise 0.1-5000 .mu.g/ml
serum concentration per single or multiple adminstration. Suitable
dosages are known to medical practitioners and will, of course,
depend upon the particular disease state, specific activity of the
composition being administered, and the particular patient
undergoing treatment. In some instances, to achieve the desired
therapeutic amount, it can be necessary to provide for repeated
administration, i.e., repeated individual administrations of a
particular monitored or metered dose, where the individual
administrations are repeated until the desired daily dose or effect
is achieved.
[0175] 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, 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.
[0176] 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.
[0177] 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.
[0178] Dosage forms (composition) suitable for internal
administration generally contain from about 0.1 milligram to about
500 milligrams of active ingredient per unit or container. In these
pharmaceutical compositions the active ingredient will ordinarily
be present in an amount of about 0.5-99.999% by weight based on the
total weight of the composition.
[0179] For parenteral administration, the antibody can be
formulated as a solution, suspension, emulsion or lyophilized
powder in association, or separately provided, with a
pharmaceutically acceptable parenteral vehicle. Examples of such
vehicles are water, saline, Ringer's solution, dextrose solution,
and 1-10% human serum albumin. Liposomes and nonaqueous vehicles
such as fixed oils can also be used. The vehicle or lyophilized
powder can contain additives that maintain isotonicity (e.g.,
sodium chloride, mannitol) and chemical stability (e.g., buffers
and preservatives). The formulation is sterilized by known or
suitable techniques.
[0180] Suitable pharmaceutical carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
[0181] Alternative Administration. Many known and developed modes
of administration can be used according to the present invention
for administering pharmaceutically effective amounts of at least
one anti-TNF antibody according to the present invention. While
pulmonary administration is used in the following description,
other modes of administration can be used according to the present
invention with suitable results.
[0182] TNF 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.
[0183] Parenteral Formulations and Administration. Formulations for
parenteral administration can contain as common excipients sterile
water or saline, polyalkylene glycols such as polyethylene glycol,
oils of vegetable origin, hydrogenated naphthalenes and the like.
Aqueous or oily suspensions for injection can be prepared by using
an appropriate emulsifier or humidifier and a suspending agent,
according to known methods. Agents for injection can be a
non-toxic, non-orally administrable diluting agent such as aquous
solution or a sterile injectable solution or suspension in a
solvent. As the usable vehicle or solvent, water, Ringer's
solution, isotonic saline, etc. are allowed; as an ordinary
solvent, or suspending solvent, sterile involatile oil can be used.
For these purposes, any kind of involatile oil and fatty acid can
be used, including natural or synthetic or semisynthetic fatty oils
or fatty acids; natural or synthetic or semisynthtetic mono- or di-
or tri-glycerides. Parental administration is known in the art and
includes, but is not limited to, conventional means of injections,
a gas pressured needle-less injection device as described in U.S.
Pat. No. 5,851,198, and a laser perforator device as described in
U.S. Pat. No. 5,839,446 entirely incorporated herein by
reference.
[0184] Alternative Delivery. The invention further relates to the
administration of at least one anti-TNF antibody by parenteral,
subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal
means. At least one anti-TNF 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).
[0185] Pulmonary/Nasal Administration. For pulmonary
administration, preferably at least one anti-TNF antibody
composition is delivered in a particle size effective for reaching
the lower airways of the lung or sinuses. According to the
invention, at least one anti-TNF antibody can be delivered by any
of a variety of inhalation or nasal devices known in the art for
administration of a therapeutic agent by inhalation. These devices
capable of depositing aerosolized formulations in the sinus cavity
or alveoli of a patient include metered dose inhalers, nebulizers,
dry powder generators, sprayers, and the like. Other devices
suitable for directing the pulmonary or nasal administration of
antibodies are also known in the art. All such devices can use of
formulations suitable for the administration for the dispensing of
antibody in an aerosol. Such aerosols can be comprised of either
solutions (both aqueous and non aqueous) or solid particles.
Metered dose inhalers like the Ventolin.RTM. metered dose inhaler,
typically use a propellent gas and require actuation during
inspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder
inhalers like Turbuhaler.TM. (Astra), Rotahaler.RTM. (Glaxo),
Diskus.RTM. (Glaxo), Spiros.TM. inhaler (Dura), devices marketed by
Inhale Therapeutics, and the Spinhaler.RTM. powder inhaler
(Fisons), use breath-actuation of a mixed powder (U.S. Pat. No.
4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552
Dura, U.S. Pat. No. 5,458,135 Inhale, WO 94/06498 Fisons, entirely
incorporated herein by reference). Nebulizers like AERx.TM.
Aradigm, the Ultravent.RTM. nebulizer (Mallinckrodt), and the Acorn
II.RTM. nebulizer (Marquest Medical Products) (U.S. Pat. No.
5,404,871 Aradigm, WO 97/22376), the above references entirely
incorporated herein by reference, produce aerosols from solutions,
while metered dose inhalers, dry powder inhalers, etc. generate
small particle aerosols. These specific examples of commercially
available inhalation devices are intended to be a representative of
specific devices suitable for the practice of this invention, and
are not intended as limiting the scope of the invention.
[0186] Preferably, a composition comprising at least one anti-TNF
antibody is delivered by a dry powder inhaler or a sprayer. There
are a several desirable features of an inhalation device for
administering at least one antibody of the present invention. For
example, delivery by the inhalation device is advantageously
reliable, reproducible, and accurate. The inhalation device can
optionally deliver small dry particles, e.g. less than about 10
.mu.m, preferably about 1-5 .mu.m, for good respirability.
[0187] Administration of TNF antibody Compositions as a Spray. A
spray including TNF antibody composition protein can be produced by
forcing a suspension or solution of at least one anti-TNF antibody
through a nozzle under pressure. The nozzle size and configuration,
the applied pressure, and the liquid feed rate can be chosen to
achieve the desired output and particle size. An electrospray can
be produced, for example, by an electric field in connection with a
capillary or nozzle feed. Advantageously, particles of at least one
anti-TNF antibody composition protein delivered by a sprayer have a
particle size less than about 10 .mu.m, preferably in the range of
about 1 .mu.m to about 5 .mu.m, and most preferably about 2 .mu.m
to about 3 .mu.m.
[0188] Formulations of at least one anti-TNF antibody composition
protein suitable for use with a sprayer typically include antibody
composition protein in an aqueous solution at a concentration of
about 0.1 mg to about 100 mg of at least one anti-TNF antibody
composition protein per ml of solution or mg/gm, or any range or
value therein, e.g., but not lmited to, 0.1, 0.2., 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40,
45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. The formulation can
include agents such as an excipient, a buffer, an isotonicity
agent, a preservative, a surfactant, and, preferably, zinc. The
formulation can also include an excipient or agent for
stabilization of the antibody composition protein, such as a
buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk
proteins useful in formulating antibody composition proteins
include albumin, protamine, or the like. Typical carbohydrates
useful in formulating antibody composition proteins include
sucrose, mannitol, lactose, trehalose, glucose, or the like. The
antibody composition protein formulation can also include a
surfactant, which can reduce or prevent surface-induced aggregation
of the antibody composition protein caused by atomization of the
solution in forming an aerosol. Various conventional surfactants
can be employed, such as polyoxyethylene fatty acid esters and
alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts
will generally range between 0.001 and 14% by weight of the
formulation. Especially preferred surfactants for purposes of this
invention are polyoxyethylene sorbitan monooleate, polysorbate 80,
polysorbate 20, or the like. Additional agents known in the art for
formulation of a protein such as TNF antibodies, or specified
portions or variants, can also be included in the formulation.
[0189] Administration of TNF antibody compositions by a Nebulizer.
Antibody composition protein can be administered by a nebulizer,
such as jet nebulizer or an ultrasonic nebulizer. Typically, in a
jet nebulizer, a compressed air source is used to create a
high-velocity air jet through an orifice. As the gas expands beyond
the nozzle, a low-pressure region is created, which draws a
solution of antibody composition protein through a capillary tube
connected to a liquid reservoir. The liquid stream from the
capillary tube is sheared into unstable filaments and droplets as
it exits the tube, creating the aerosol. A range of configurations,
flow rates, and baffle types can be employed to achieve the desired
performance characteristics from a given jet nebulizer. In an
ultrasonic nebulizer, high-frequency electrical energy is used to
create vibrational, mechanical energy, typically employing a
piezoelectric transducer. This energy is transmitted to the
formulation of antibody composition protein either directly or
through a coupling fluid, creating an aerosol including the
antibody composition protein. Advantageously, particles of antibody
composition protein delivered by a nebulizer have a particle size
less than about 10 .mu.am, preferably in the range of about 1 .mu.m
to about 5 .mu.m, and most preferably about 2 .mu.m to about 3
.mu.m.
[0190] Formulations of at least one anti-TNF antibody suitable for
use with a nebulizer, either jet or ultrasonic, typically include a
concentration of about 0.1 mg to about 100 mg of at least one
anti-TNF antibody protein per ml of solution. The formulation can
include agents such as an excipient, a buffer, an isotonicity
agent, a preservative, a surfactant, and, preferably, zinc. The
formulation can also include an excipient or agent for
stabilization of the at least one anti-TNF antibody composition
protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk proteins useful in formulating at least one
anti-TNF antibody composition proteins include albumin, protamine,
or the like. Typical carbohydrates useful in formulating at least
one anti-TNF antibody include sucrose, mannitol, lactose,
trehalose, glucose, or the like. The at least one anti-TNF antibody
formulation can also include a surfactant, which can reduce or
prevent surface-induced aggregation of the at least one anti-TNF
antibody caused by atomization of the solution in forming an
aerosol. Various conventional surfactants can be employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene
sorbital fatty acid esters. Amounts will generally range between
0.001 and 4% by weight of the formulation. Especially preferred
surfactants for purposes of this invention are polyoxyethylene
sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.
Additional agents known in the art for formulation of a protein
such as antibody protein can also be included in the
formulation.
[0191] Administration of TNF antibody compositions By A Metered
Dose Inhaler.
[0192] In a metered dose inhaler (MDI), a propellant, at least one
anti-TNF antibody, and any excipients or other additives are
contained in a canister as a mixture including a liquefied
compressed gas. Actuation of the metering valve releases the
mixture as an aerosol, preferably containing particles in the size
range of less than about 10 .mu.m, preferably about 1 .mu.m to
about 5 .mu.m, and most preferably about 2 .mu.m to about 3
.mu.m.
[0193] The desired aerosol particle size can be obtained by
employing a formulation of antibody composition protein produced by
various methods known to those of skill in the art, including
jet-milling, spray drying, critical point condensation, or the
like. Preferred metered dose inhalers include those manufactured by
3M or Glaxo and employing a hydrofluorocarbon propellant.
[0194] Formulations of at least one anti-TNF antibody for use with
a metered-dose inhaler device will generally include a finely
divided powder containing at least one anti-TNF antibody as a
suspension in a non-aqueous medium, for example, suspended in a
propellant with the aid of a surfactant. The propellant can be any
conventional material employed for this purpose, such as
chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon,
or a hydrocarbon, including trichlorofluoromethane,
dichlorodifluoromethane, dichlorotetrafluoroethanol and
1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a),
HFA-227 (hydrofluroalkane-227), or the like. Preferably the
propellant is a hydrofluorocarbon. The surfactant can be chosen to
stabilize the at least one anti-TNF antibody as a suspension in the
propellant, to protect the active agent against chemical
degradation, and the like. Suitable surfactants include sorbitan
trioleate, soya lecithin, oleic acid, or the like. In some cases
solution aerosols are preferred using solvents such as ethanol.
Additional agents known in the art for formulation of a protein can
also be included in the formulation.
[0195] One of ordinary skill in the art will recognize that the
methods of the current invention can be achieved by pulmonary
administration of at least one anti-TNF antibody compositions via
devices not described herein.
[0196] Oral Formulations and Administration. Formulations for oral
rely on the co-administration of adjuvants (e.g., resorcinols and
nonionic surfactants such as polyoxyethylene oleyl ether and
n-hexadecylpolyethylene ether) to increase artificially the
permeability of the intestinal walls, as well as the
co-administration of enzymatic inhibitors (e.g., pancreatic trypsin
inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to
inhibit enzymatic degradation. The active constituent compound of
the solid-type dosage form for oral administration can be mixed
with at least one additive, including sucrose, lactose, cellulose,
mannitol, trehalose, raffinose, maltitol, dextran, starches, agar,
arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic,
gelatin, collagen, casein, albumin, synthetic or semisynthetic
polymer, and glyceride. These dosage forms can also contain other
type(s) of additives, e.g., inactive diluting agent, lubricant such
as magnesium stearate, paraben, preserving agent such as sorbic
acid, ascorbic acid, .alpha.-tocopherol, antioxidant such as
cysteine, disintegrator, binder, thickener, buffering agent,
sweetening agent, flavoring agent, perfuming agent, etc.
[0197] Tablets and pills can be further processed into
enteric-coated preparations. The liquid preparations for oral
administration include emulsion, syrup, elixir, suspension and
solution preparations allowable for medical use. These preparations
can contain inactive diluting agents ordinarily used in said field,
e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More
recently, microspheres of artificial polymers of mixed amino acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat.
No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to
deliver biologically active agents orally are known in the art.
[0198] Mucosal Formulations and Administration. For absorption
through mucosal surfaces, compositions and methods of administering
at least one anti-TNF antibody include an emulsion comprising a
plurality of submicron particles, a mucoadhesive macromolecule, a
bioactive peptide, and an aqueous continuous phase, which promotes
absorption through mucosal surfaces by achieving mucoadhesion of
the emulsion particles (U.S. Pat. Nos. 5,514,670). Mucous surfaces
suitable for application of the emulsions of the present invention
can include corneal, conjunctival, buccal, sublingual, nasal,
vaginal, pulmonary, stomachic, intestinal, and rectal routes of
administration. Formulations for vaginal or rectal administration,
e.g. suppositories, can contain as excipients, for example,
polyalkyleneglycols, vaseline, cocoa butter, and the like.
Formulations for intranasal administration can be solid and contain
as excipients, for example, lactose or can be aqueous or oily
solutions of nasal drops. For buccal administration excipients
include sugars, calcium stearate, magnesium stearate,
pregelinatined starch, and the like (U.S. Pat. No. 5,849,695).
[0199] Transdermal Formulations and Administration. For transdermal
administration, the at least one anti-TNF antibody is encapsulated
in a delivery device such as a liposome or polymeric nanoparticles,
microparticle, microcapsule, or microspheres (referred to
collectively as microparticles unless otherwise stated). A number
of suitable devices are known, including microparticles made of
synthetic polymers such as polyhydroxy acids such as polylactic
acid, polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and polyphosphazenes, and natural polymers such as
collagen, polyamino acids, albumin and other proteins, alginate and
other polysaccharides, and combinations thereof (U.S. Pat. No.
5,814,599).
[0200] Prolonged Administration and Formulations. It can be
sometimes desirable to deliver the compounds of the present
invention to the subject over prolonged periods of time, for
example, for periods of one week to one year from a single
administration. Various slow release, depot or implant dosage forms
can be utilized. For example, a dosage form can contain a
pharmaceutically acceptable non-toxic salt of the compounds that
has a low degree of solubility in body fluids, for example, (a) an
acid addition salt with a polybasic acid such as phosphoric acid,
sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic
acid, alginic acid, polyglutamic acid, naphthalene mono- or
di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt
with a polyvalent metal cation such as zinc, calcium, bismuth,
barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and
the like, or with an organic cation formed from e.g.,
N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c)
combinations of (a) and (b) e.g. a zinc tannate salt. Additionally,
the compounds of the present invention or, preferably, a relatively
insoluble salt such as those just described, can be formulated in a
gel, for example, an aluminum monostearate gel with, e.g. sesame
oil, suitable for injection. Particularly preferred salts are zinc
salts, zinc tannate salts, pamoate salts, and the like. Another
type of slow release depot formulation for injection would contain
the compound or salt dispersed for encapsulated in a slow
degrading, non-toxic, non-antigenic polymer such as a polylactic
acid/polyglycolic acid polymer for example as described in U.S.
Pat. No. 3,773,919. The compounds or, preferably, relatively
insoluble salts such as those described above can also be
formulated in cholesterol matrix silastic pellets, particularly for
use in animals. Additional slow release, depot or implant
formulations, e.g. gas or liquid liposomes are known in the
literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled
Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker,
Inc., N.Y., 1978).
[0201] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
Cloning and Expression of TNF antibody in Mammalian Cells
[0202] A typical mammalian expression vector contains at least one
promoter element, which mediates the initiation of transcription of
mRNA, the antibody coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pIRES1neo, pRetro-Off,
pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.,
pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-)
(Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat
(ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
Mammalian host cells that could be used include human Hela 293, H9
and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV
1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO)
cells.
[0203] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[0204] The transfected gene can also be amplified to express large
amounts of the encoded antibody. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et
al., Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
antibodies.
[0205] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec.
Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer
(Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, Xbal and
Asp718, facilitate the cloning of the gene of interest. The vectors
contain in addition the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene.
[0206] Cloning and Expression in CHO Cells. The vector pC4 is used
for the expression of TNF antibody. Plasmid pC4 is a derivative of
the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid
contains the mouse DHFR gene under control of the SV40 early
promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids can
be selected by growing the cells in a selective medium (e.g., alpha
minus MEM, Life Technologies, Gaithersburg, Md.) supplemented with
the chemotherapeutic agent methotrexate. The amplification of the
DHFR genes in cells resistant to methotrexate (MTX) has been well
documented (see, e.g., F. W. Alt, et al., J. Biol. Chem.
253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys.
Acta 1097:107-143 (1990); and M. J. Page and M. A. Sydenham,
Biotechnology 9:64-68 (1991)). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach can be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained that contain the
amplified gene integrated into one or more chromosome(s) of the
host cell.
[0207] 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, Xbal, 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 be used to express
the TNF 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.
[0208] The plasmid pC4 is digested with restriction enzymes and
then dephosphorylated using calf intestinal phosphatase by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0209] The isolated variable and constant region encoding DNA and
the dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
[0210] Chinese hamster ovary (CHO) cells lacking an active DHFR
gene are used for transfection. 5 .mu.g of the expression plasmid
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using
lipofectin. The plasmid pSV2neo contains a dominant selectable
marker, the neo gene from Tn5 encoding an enzyme that confers
resistance to a group of antibiotics including G418. The cells are
seeded in alpha minus MEM supplemented with 1 .mu.g/ml G418. After
2 days, the cells are trypsinized and seeded in hybridoma cloning
plates (Greiner, Germany) in alpha minus MEM supplemented with 10,
25, or 50 ng/ml of methotrexate plus 1 .mu.g/ml G418. After about
10-14 days single clones are trypsinized and then seeded in 6-well
petri dishes or 10 ml flasks using different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained that grow
at a concentration of 100-200 mM. Expression of the desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or
by reverse phase HPLC analysis.
EXAMPLE 2
Generation of High Affinity Human IgG Monoclonal Antibodies
Reactive with Human TNF using Transgenic Mice
SUMMARY
[0211] Transgenic mice have been used that contain human heavy and
light chain immunoglobulin genes to generate high affinity,
completely human, monoclonal antibodies that can be used
therapeutically to inhibit the action of TNF for the treatment of
one or more TNF-mediated disease. (CBA/J.times.C57/BL6/J) F.sub.2
hybrid mice containing human variable and constant region antibody
transgenes for both heavy and light chains are immunized with human
recombinant TNF (Taylor et al., Intl. Immunol. 6:579-591 (1993);
Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M., Nature
Biotech. 14:826 (1996); Fishwild, et al., Nature Biotechnology
14:845-851 (1996)). Several fusions yielded one or more panels of
completely human TNF reactive IgG monoclonal antibodies. The
completely human anti-TNF antibodies are further characterized. All
are IgG1.kappa.. Such antibodies are found to have affinity
constants somewhere between 1.times.10.sup.9 and 9.times.10.sup.12.
The unexpectedly high affinities of these fully human monoclonal
antibodies make them suitable candidates for therapeutic
applications in TNF related diseases, pathologies or disorders.
[0212] Abbreviations. BSA--bovine serum albumin; CO.sub.2--carbon
dioxide; DMSO--dimethyl sulfoxide; EIA--enzyme immunoassay;
FBS--fetal bovine serum; H.sub.2O.sub.2 --hydrogen peroxide;
HRP--horseradish peroxidase; ID--interadermal; Ig--immunoglobulin;
TNF--tissue necrosis factor alpha; IP--intraperitoneal;
IV--intravenous; Mab--monoclonal antibody; OD--optical density;
OPD--o-Phenylenediamine dihydrochloride; PEG--polyethylene glycol;
PSA--penicillin, streptomycin, amphotericin; RT--room temperature;
SQ--subcutaneous; v/v--volume per volume; w/v--weight per
volume.
Materials and Methods
[0213] Animals. Transgenic mice that can express human antibodies
are known in the art (and are commecially available (e.g., from
GenPharm International, San Jose, Calif.; Abgenix, Freemont,
Calif., and others) that express human immunoglobulins but not
mouse IgM or Ig.kappa.. For example, such transgenic mice contain
human sequence transgenes that undergo V(D)Jjoining, heavy-chain
class switching, and somatic mutation to generate a repertoire of
human sequence immunoglobulins (Lonberg, et al., Nature 368:856-859
(1994)). The light chain transgene can be derived, e.g., in part
from a yeast artificial chromosome clone that includes nearly half
of the germline human V.kappa. region. In addition, the heavy-chain
transgene can encode both human .mu. and human .gamma.1(Fishwild,
et al., Nature Biotechnology 14:845-851 (1996)) and/or .gamma.3
constant regions. Mice derived from appropriate genotopic lineages
can be used in the immunization and fusion processes to generate
fully human monoclonal antibodies to TNF.
[0214] Immunization. One or more immunization schedules can be used
to generate the anti-TNF human hybridomas. The first several
fusions can be performed after the following exemplary immunization
protocol, but other similar known protocols can be used. Several
14-20 week old female and/or surgically castrated transgenic male
mice are immunized IP and/or ID with 1-1000 .mu.g of recombinant
human TNF emulsified with an equal volume of TITERMAX or complete
Freund's adjuvant in a final volume of 100-400 .mu.L (e.g., 200).
Each mouse can also optionally receive 1-10 .mu.g in 100 .mu.L
physiological saline at each of 2 SQ sites. The mice can then be
immunized 1-7, 5-12, 10-18, 17-25 and/or 21-34 days later IP (1-400
.mu.g) and SQ (1-400 .mu.g.times.2) with TNF emulsified with an
equal volume of TITERMAX or incomplete Freund's adjuvant. Mice can
be bled 12-25 and 25-40 days later by retro-orbital puncture
without anti-coagulant. The blood is then allowed to clot at RT for
one hour and the serum is collected and titered using an TNF EIA
assay according to known methods. Fusions are performed when
repeated injections do not cause titers to increase. At that time,
the mice can be given a final IV booster injection of 1-400 .mu.g
TNF diluted in 100 .mu.L physiological saline. Three days later,
the mice can be euthanized by cervical dislocation and the spleens
removed aseptically and immersed in 10 mL of cold phosphate
buffered saline (PBS) containing 100 U/mL penicillin, 100 .mu.g/mL
streptomycin, and 0.25 .mu.g/mL amphotericin B (PSA). The
splenocytes are harvested by sterilely perfusing the spleen with
PSA-PBS. The cells are washed once in cold PSA-PBS, counted using
Trypan blue dye exclusion and resuspended in RPMI 1640 media
containing 25 mM Hepes.
[0215] Cell Fusion. Fusion can be carried out at a 1:1 to 1:10
ratio of murine myeloma cells to viable spleen cells according to
known methods, e.g., as known in the art. As a non-limiting
example, spleen cells and myeloma cells can be pelleted together.
The pellet can then be slowly resuspended, over 30 seconds, in 1 mL
of 50% (w/v) PEG/PBS solution (PEG molecular weight 1,450, Sigma)
at 37.quadrature.C. The fusion can then be stopped by slowly adding
10.5 mL of RPMI 1640 medium containing 25 mM Hepes
(37.quadrature.C) over 1 minute. The fused cells are centrifuged
for 5 minutes at 500-1500 rpm. The cells are then resuspended in
HAT medium (RPMI 1640 medium containing 25 mM Hepes, 10% Fetal
Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10
.mu.g/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 10%
653-conditioned RPMI 1640/Hepes media, 50 .mu.M 2-mercaptoethanol,
100 .mu.M hypoxanthine, 0.4 .mu.M aminopterin, and 16 .mu.M
thymidine) and then plated at 200 .mu.L/well in fifteen 96-well
flat bottom tissue culture plates. The plates are then placed in a
humidified 37.quadrature.C incubator containing 5% CO.sub.2 and 95%
air for 7-10 days.
[0216] Detection of Human IgG Anti-TNF Antibodies in Mouse Serum.
Solid phase EIA's can be used to screen mouse sera for human IgG
antibodies specific for human TNF. Briefly, plates can be coated
with TNF at 2.mu.g/mL in PBS overnight. After washing in 0.15 M
saline containing 0.02% (v/v) Tween 20, the wells can be blocked
with 1% (w/v) BSA in PBS, 200 .mu.L/well for 1 hour at RT. Plates
are used immediately or frozen at -20.quadrature.C for future use.
Mouse serum dilutions are incubated on the TNF coated plates at 50
.mu.L/well at RT for 1 hour. The plates are washed and then probed
with 50 .mu.L/well HRP-labeled goat anti-human IgG, Fc specific
diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT. The plates can
again be washed and 100 .mu.L/well of the citrate-phosphate
substrate solution (0.1M citric acid and 0.2M sodium phosphate,
0.01% H.sub.2O.sub.2 and 1 mg/mL OPD) is added for 15 minutes at
RT. Stop solution (4N sulfuric acid) is then added at 25 .mu.L/well
and the OD's are read at 490 nm via an automated plate
spectrophotometer.
[0217] Detection of Completely Human Immunoglobulins in Hybridoma
Supernates. Growth positive hybridomas secreting fully human
immunoglobulins can be detected using a suitable EIA. Briefly, 96
well pop-out plates (VWR, 610744) can be coated with 10 .mu.g/mL
goat anti-human IgG Fc in sodium carbonate buffer overnight at
4.quadrature.C. The plates are washed and blocked with 1% BSA-PBS
for one hour at 37.degree. C. and used immediately or frozen at
-20.quadrature.C. Undiluted hybridoma supernatants are incubated on
the plates for one hour at 37.degree. C. The plates are washed and
probed with HRP labeled goat anti-human kappa diluted 1:10,000 in
1% BSA-PBS for one hour at 37.degree. C. The plates are then
incubated with substrate solution as described above.
[0218] Determination of Fully Human Anti-TNF Reactivity.
Hybridomas, as above, can be simultaneously assayed for reactivity
to TNF using a suitable RIA or other assay. For example,
supernatants are incubated on goat anti-human IgG Fc plates as
above, washed and then probed with radiolabled TNF with appropriate
counts per well for 1 hour at RT. The wells are washed twice with
PBS and bound radiolabled TNF is quantitated using a suitable
counter.
[0219] Human IgG1.kappa. anti-TNF secreting hybridomas can be
expanded in cell culture and serially subcloned by limiting
dilution. The resulting clonal populations can be expanded and
cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in
liquid nitrogen.
[0220] Isotyping. Isotype determination of the antibodies can be
accomplished using an EIA in a format similar to that used to
screen the mouse immune sera for specific titers. TNF can be coated
on 96-well plates as described above and purified antibody at 2
.mu.g/mL can be incubated on the plate for one hour at RT. The
plate is washed and probed with HRP labeled goat anti-human
IgG.sub.1 or HRP labeled goat anti-human IgG.sub.3 diluted at
1:4000 in 1% BSA-PBS for one hour at RT. The plate is again washed
and incubated with substrate solution as described above.
[0221] Binding Kinetics of Human Anti-Human TNF Antibodies With
Human TNF. Binding characteristics for antibodies can be suitably
assessed using an TNF capture EIA and BlAcore technology, for
example. Graded concentrations of purified human TNF antibodies can
be assessed for binding to EIA plates coated with 2 .mu.g/mL of TNF
in assays as described above. The OD's can be then presented as
semi-log plots showing relative binding efficiencies.
[0222] Quantitative binding constants can be obtained, e.g., as
follows, or by any other known suitable method. A BIAcore CM-5
(carboxymethyl) chip is placed in a BIAcore 2000 unit. HBS buffer
(0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant,
pH 7.4) is flowed over a flow cell of the chip at 5 .mu.L/minute
until a stable baseline is obtained. A solution (100 .mu.L) of 15
mg of EDC (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide
hydrochloride) in 200 .mu.L water is added to 100 .mu.L of a
solution of 2.3 mg of NHS (N-hydroxysuccinimide) in 200 .mu.L
water. Forty (40) .mu.L of the resulting solution is injected onto
the chip. Six .mu.L of a solution of human TNF (15 .mu.g/mL in 10
mM sodium acetate, pH 4.8) is injected onto the chip, resulting in
an increase of ca. 500 RU. The buffer is changed to TBS/Ca/Mg/BSA
running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mM calcium
chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 .mu.g/mL
BSA, pH 7.4) and flowed over the chip overnight to equilibrate it
and to hydrolyze or cap any unreacted succinimide esters.
[0223] Antibodies are dissolved in the running buffer at 33.33,
16.67, 8.33, and 4.17 nM. The flow rate is adjusted to 30 .mu.L/min
and the instrument temperature to 25.quadrature.C. Two flow cells
are used for the kinetic runs, one on which TNF had been
immobilized (sample) and a second, underivatized flow cell (blank).
120 .mu.L of each antibody concentration is injected over the flow
cells at 30 .mu.L/min (association phase) followed by an
uninterrupted 360 seconds of buffer flow (dissociation phase). The
surface of the chip is regenerated (tissue necrosis factor
alpha/antibody complex dissociated) by two sequential injections of
30 .mu.L each of 2 M guanidine thiocyanate.
[0224] Analysis of the data is done using BIA evaluation 3.0 or
CLAMP 2.0, as known in the art. For each antibody concentration the
blank sensogram is subtracted from the sample sensogram. A global
fit is done for both dissociation (k.sub.d, sec.sup.-1) and
association (k.sub.a, mol.sup.-1 sec.sup.-1) and the dissociation
constant (K.sub.D, mol) calculated (k.sub.d/k.sub.a). Where the
antibody affinity is high enough that the RUs of antibody captured
are >100, additional dilutions of the antibody are run.
Results and Discussion
[0225] Generation of Anti-Human TNF Monoclonal Antibodies. Several
fusions are performed and each fusion is seeded in 15 plates (1440
wells/fusion) that yield several dozen antibodies specific for
human TNF. Of these, some are found to consist of a combination of
human and mouse Ig chains. The remaining hybridomas secret anti-TNF
antibodies consisting solely of human heavy and light chains. Of
the human hybridomas all are expected to be IgG1.kappa..
[0226] Binding Kinetics of Human Anti-Human TNF Antibodies. ELISA
analysis confirms that purified antibody from most or all of these
hybridomas bind TNF in a concentration-dependent manner. FIGS. 1-2
show the results of the relative binding efficiency of these
antibodies. In this case, the avidity of the antibody for its
cognate antigen (epitope) is measured. It should be noted that
binding TNF directly to the EIA plate can cause denaturation of the
protein and the apparent binding affinities cannot be reflective of
binding to undenatured protein. Fifty percent binding is found over
a range of concentrations.
[0227] Quantitative binding constants are obtained using BlAcore
analysis of the human antibodies and reveals that several of the
human monoclonal antibodies are very high affinity with K.sub.D in
the range of 1.times.10.sup.-9 to 7.times.10.sup.-12.
Conclusions
[0228] Several fusions are performed utilizing splenocytes from
hybrid mice containing human variable and constant region antibody
transgenes that are immunized with human TNF. A set of several
completely human TNF reactive IgG monoclonal antibodies of the
IgG1.kappa. isotype are generated. The completely human anti-TNF
antibodies are further characterized. Several of generated
antibodies have affinity constants between 1.times.10.sup.9 and
9.times.10.sup.12. The unexpectedly high affinities of these fully
human monoclonal antibodies make them suitable for therapeutic
applications in TNF-dependent diseases, pathologies or related
conditions.
EXAMPLE 3
Generation of Human IgG Monoclonal Antibodies Reactive to Human
TNF.alpha.
[0229] Summary. (CBA/J.times.C57BL/6J) F.sub.2 hybrid mice (1-4)
containing human variable and constant region antibody transgenes
for both heavy and light chains were immunized with recombinant
human TNF.alpha.. One fusion, named GenTNV, yielded eight totally
human IgG1.kappa. monoclonal antibodies that bind to immobilized
recombinant human TNF.alpha.. Shortly after identification, the
eight cell lines were transferred to Molecular Biology for further
characterization. As these Mabs are totally human in sequence, they
are expected to be less immunogenic than cA2 (Remicade) in
humans.
[0230] Abbreviations. BSA--bovine serum albumin; CO.sub.2--carbon
dioxide; DMSO--dimethyl sulfoxide; EIA--enzyme immunoassay;
FBS--fetal bovine serum; H.sub.2O.sub.2--hydrogen peroxide;
HC--heavy chain; HRP--horseradish peroxidase; ID--interadermal;
Ig--immunoglobulin; TNF--tissue necrosis factor alpha;
IP--intraperitoneal; IV--intravenous; Mab--monoclonal antibody;
OD--optical density; OPD--o-Phenylenediamine dihydrochloride;
PEG--polyethylene glycol; PSA--penicillin, streptomycin,
amphotericin; RT--room temperature; SQ--subcutaneous;
TNF.alpha.tumor necrosis factor alpha ; v/v--volume per volume;
w/v--weight per volume.
[0231] Introduction. Transgenic mice that contain human heavy and
light chain immunoglobulin genes were utilized to generate totally
human monoclonal antibodies that are specific to recombinant human
TNF.alpha.. It is hoped that these unique antibodies can be used,
as cA2 (Remicade) is used to therapeutically inhibit the
inflammatory processes involved in TNF.alpha.-mediated disease with
the benefit of increased serum half-life and decreased side effects
relating to immunogenicity.
Materials and Methods
[0232] Animals. Transgenic mice that express human immunoglobulins,
but not mouse IgM or Ig.kappa., have been developed by GenPharm
International. These mice contain functional human antibody
transgenes that undergo V(D)J joining, heavy-chain class switching
and somatic mutation to generate a repertoire of antigen-specific
human immunoglobulins (1). The light chain transgenes are derived
in part from a yeast artificial chromosome clone that includes
nearly half of the germline human V.kappa. locus. In addition to
several VH genes, the heavy-chain (HC) transgene encodes both human
.mu. and human .gamma.1 (2) and/or .gamma.3 constant regions. A
mouse derived from the HCo12/KCo5 genotypic lineage was used in the
immunization and fusion process to generate the monoclonal
antibodies described here.
[0233] Purification of Human TNF.alpha.. Human TNF.alpha. was
purified from tissue culture supernatant from C237A cells by
affinity chromatography using a column packed with the TNF.alpha.
receptor-Fc fusion protein (p55-sf2) (5) coupled to Sepharose 4B
(Pharmacia). The cell supernatant was mixed with one-ninth its
volume of 10.times. Dulbecco's PBS (D-PBS) and passed through the
column at 4.degree. C. at 4 mL/min. The column was then washed with
PBS and the TNF.alpha. was eluted with 0.1 M sodium citrate, pH 3.5
and neutralized with 2 M Tris-HCl pH 8.5. The purified TNF.alpha.
was buffer exchanged into 10 mM Tris, 0.12 M sodium chloride pH 7.5
and filtered through a 0.2 um syringe filter.
[0234] Immunizations. A female GenPharm mouse, approximately 16
weeks old, was immunized IP (200 .mu.L) and ID (100 .mu.L at the
base of the tail) with a total of 100 .mu.g of TNF.beta. (lot
JG102298 or JG102098) emulsified with an equal volume of Titermax
adjuvant on days 0, 12 and 28. The mouse was bled on days 21 and 35
by retro-orbital puncture without anti-coagulant. The blood was
allowed to clot at RT for one hour and the serum was collected and
titered using TNF.alpha. solid phase EIA assay. The fusion, named
GenTNV, was performed after the mouse was allowed to rest for seven
weeks following injection on day 28. The mouse, with a specific
human IgG titer of 1:160 against TNF.alpha., was then given a final
IV booster injection of 50 .mu.g TNF.alpha. diluted in 100 .mu.L
physiological saline. Three days later, the mouse was euthanized by
cervical dislocation and the spleen was removed aseptically and
immersed in 10 mL of cold phosphate-buffered saline (PBS)
containing 100 U/mL penicillin, 100 .mu.g/mL streptomycin, and 0.25
.mu.g/mL amphotericin B (PSA). The splenocytes were harvested by
sterilely perfusing the spleen with PSA-PBS. The cells were washed
once in cold PSA-PBS, counted using a Coulter counter and
resuspended in RPMI 1640 media containing 25 mM Hepes.
[0235] Cell Lines. The non-secreting mouse myeloma fusion partner,
653 was received into Cell Biology Services (CBS) group on 5-14-97
from Centocor's Product Development group. The cell line was
expanded in RPMI medium (JRH Biosciences) supplemented with 10%
(v/v) FBS (Cell Culture Labs), 1 mM sodium pyruvate, 0.1 mM NEAA, 2
mM L-glutamine (all from JRH Biosciences) and cryopreserved in 95%
FBS and 5% DMSO (Sigma), then stored in a vapor phase liquid
nitrogen freezer in CBS. The cell bank was sterile (Quality Control
Centocor, Malvern) and free of mycoplasma (Bionique Laboratories).
Cells were maintained in log phase culture until fusion. They were
washed in PBS, counted, and viability determined (>95%) via
trypan blue dye exclusion prior to fusion.
[0236] Human TNF.alpha. was produced by a recombinant cell line,
named C237A, generated in Molecular Biology at Centocor. The cell
line was expanded in IMDM medium (JRH Biosciences) supplemented
with 5% (v/v) FBS (Cell Culture Labs), 2 mM L-glutamine (all from
JRH Biosciences), and 0.5:g/mL mycophenolic acid, and cryopreserved
in 95% FBS and 5% DMSO (Sigma), then stored in a vapor phase liquid
nitrogen freezer in CBS (13). The cell bank was sterile (Quality
Control Centocor, Malvern) and free of mycoplasma (Bionique
Laboratories).
[0237] Cell Fusion. The cell fusion was carried out using a 1:1
ratio of 653 murine myeloma cells and viable murine spleen cells.
Briefly, spleen cells and myeloma cells were pelleted together. The
pellet was slowly resuspended over a 30 second period in 1 mL of
50% (w/v) PEG/PBS solution (PEG molecular weight of 1,450 g/mole,
Sigma) at 37.degree. C. The fusion was stopped by slowly adding
10.5 mL of RPMI media (no additives) (JRH) (37.degree. C.) over 1
minute. The fused cells were centrifuged for 5 minutes at 750 rpm.
The cells were then resuspended in HAT medium (RPMI/HEPES medium
containing 10% Fetal Bovine Serum (JRH), 1 mM sodium pyruvate, 2 mM
L-glutamine, 10 .mu.g/mL gentamicin, 2.5% Origen culturing
supplement (Fisher), 50 .mu.M 2-mercaptoethanol, 1% 653-conditioned
RPMI media, 100 .mu.M hypoxanthine, 0.4 .mu.M aminopterin, and 16
.mu.M thymidine) and then plated at 200 .mu.L/well in five 96-well
flat bottom tissue culture plates. The plates were then placed in a
humidified 37.degree. C. incubator containing 5% CO.sub.2 and 95%
air for 7-10 days.
[0238] Detection of Human IgG Anti-TNF.alpha. Antibodies in Mouse
Serum. Solid phase EIAs were used to screen mouse sera for human
IgG antibodies specific for human TNF.alpha.. Briefly, plates were
coated with TNF.alpha. at 1 .mu.g/mL in PBS overnight. After
washing in 0.15 M saline containing 0.02% (v/v) Tween 20, the wells
were blocked with 1% (w/v) BSA in PBS, 200 .mu.L/well for 1 hour at
RT. Plates were either used immediately or frozen at -20 .degree.
C. for future use. Mouse sera were incubated in two-fold serial
dilutions on the human TNF.alpha.-coated plates at 50 .mu.L/well at
RT for 1 hour. The plates were washed and then probed with 50
.mu.L/well HRP-labeled goat anti-human IgG, Fc specific (Accurate)
diluted 1:30,000 in 1% BSA-PBS for 1 hour at RT. The plates were
again washed and 100 .mu.L/well of the citrate-phosphate substrate
solution (0.1 M citric acid and 0.2 M sodium phosphate, 0.01%
H.sub.2O.sub.2 and 1 mg/mL OPD) was added for 15 minutes at RT.
Stop solution (4N sulfuric acid) was then added at 25 .mu.L/well
and the OD's were read at 490 nm using an automated plate
spectrophotometer.
[0239] Detection of Totally Human Immunoglobulins in Hybridoma
Supernatants. Because the GenPharm mouse is capable of generating
both mouse and human immunoglobulin chains, two separate EIA assays
were used to test growth-positive hybridoma clones for the presence
of both human light chains and human heavy chains. Plates were
coated as described above and undiluted hybridoma supernatants were
incubated on the plates for one hour at 37.degree. C. The plates
were washed and probed with either HRP-conjugated goat anti-human
kappa (Southern Biotech) antibody diluted 1:10,000 in 1% BSA-HBSS
or HRP-conjugated goat anti-human IgG Fc specific antibody diluted
to 1:30,000 in 1% BSA-HBSS for one hour at 37.degree. C. The plates
were then incubated with substrate solution as described above.
Hybridoma clones that did not give a positive signal in both the
anti-human kappa and anti-human IgG Fc EIA formats were
discarded.
[0240] Isotyping. Isotype determination of the antibodies was
accomplished using an EIA in a format similar to that used to
screen the mouse immune sera for specific titers. EIA plates were
coated with goat anti-human IgG (H+L) at 10 :g/mL in sodium
carbonate buffer overnight at 4EC and blocked as described above.
Neat supernatants from 24 well cultures were incubated on the plate
for one hour at RT. The plate was washed and probed with
HRP-labeled goat anti-human IgG.sub.1, IgG.sub.2, IgG.sub.3 or
IgG.sub.4 (Binding Site) diluted at 1:4000 in 1% BSA-PBS for one
hour at RT. The plate was again washed and incubated with substrate
solution as described above.
[0241] Results and Discussion. Generation of Totally Human
Anti-Human TNF.alpha. Monoclonal Antibodies. One fusion, named
GenTNV, was performed from a GenPharm mouse immunized with
recombinant human TNF.alpha. protein. From this fusion, 196
growth-positive hybrids were screened. Eight hybridoma cell lines
were identified that secreted totally human IgG antibodies reactive
with human TNF.alpha.. These eight cell lines each secreted
immunoglobulins of the human IgG1.kappa. isotype and all were
subcloned twice by limiting dilution to obtain stable cell lines
(>90% homogeneous). Cell line names and respective C code
designations are listed in Table 1. Each of the cell lines was
frozen in 12-vial research cell banks stored in liquid
nitrogen.
[0242] Parental cells collected from wells of a 24-well culture
dish for each of the eight cell lines were handed over to Molecular
Biology group on 2-18-99 for transfection and further
characterization.
TABLE-US-00002 TABLE 1 GenTNV Cell Line Designations C Code Name
Designation GenTNV14.17.12 C414A GenTNV15.28.11 C415A GenTNV32.2.16
C416A GenTNV86.14.34 C417A GenTNV118.3.36 C418A GenTNV122.23.2
C419A GenTNV148.26.12 C420A GenTNV196.9.1 C421A
Conclusion
[0243] The GenTNV fusion was performed utilizing splenocytes from a
hybrid mouse containing human variable and constant region antibody
transgenes that was immunized with recombinant human TNF.alpha.
prepared at Centocor. Eight totally human, TNF.alpha.-reactive IgG
monoclonal antibodies of the IgG1.kappa. isotype were generated.
Parental cell lines were transferred to Molecular Biology group for
further characterization and development. One of these new human
antibodies may prove useful in anti-inflammatory with the potential
benefit of decreased immunogenicity and allergic-type complications
as compared with Remicade.
[0244] References
[0245] Taylor, et al.,. International Immunology 6:579-591
(1993).
[0246] Lonberg, et al., Nature 368:856-859 (1994).
[0247] Neuberger, M. Nature Biotechnology 14:826 (1996).
[0248] Fishwild, et al., Nature Biotechnology 14:845-851
(1996).
[0249] Scallon, et al., Cytokine 7:759-770 (1995).
Example 4
Cloning and Preparation of Cell lines Expressing Human
Anti-TNF.alpha. Antibody
[0250] Summary. A panel of eight human monoclonal antibodies (mAbs)
with a TNV designation were found to bind immobilized human
TNF.alpha. with apparently high avidity. Seven of the eight mAbs
were shown to efficiently block huTNF.alpha. binding to a
recombinant TNF receptor. Sequence analysis of the DNA encoding the
seven mAbs confirmed that all the mAbs had human V regions. The DNA
sequences also revealed that three pairs of the mAbs were identical
to each other, such that the original panel of eight mAbs contained
only four distinct mAbs, represented by TNV14, TNV15, TNV148, and
TNV196. Based on analyses of the deduced amino acid sequences of
the mAbs and results of in vitro TNF.alpha. neutralization data,
mAb TNV148 and TNV14 were selected for further study.
[0251] Because the proline residue at position 75 (framework 3) in
the TNV148 heavy chain was not found at that position in other
human antibodies of the same subgroup during a database search,
site-directed DNA mutagenesis was performed to encode a serine
residue at that position in order to have it conform to known
germline framework e sequences. The serine modified mAb was
designated TNV148B. PCR-amplified DNA encoding the heavy and light
chain variable regions of TNV148B and TNV14 was cloned into newly
prepared expression vectors that were based on the recently cloned
heavy and light chain genes of another human mAb (12B75), disclosed
in US patent application No. 60/236,827, filed Oct. 7, 2000,
entitled IL-12 Antibodies, Compositions, Methods and Uses,
published as WO 02/12500which is entirely incorporated herein by
reference.
[0252] P3X63Ag8.653 (653) cells or Sp2/0-Ag14 (Sp2/0) mouse myeloma
cells were transfected with the respective heavy and light chain
expression plasmids and screened through two rounds of subcloning
for cell lines producing high levels of recombinant TNV148B and
TNV14 (rTNV148B and rTNV14) mAbs. Evaluations of growth curves and
stability of mAb production over time indicated that
653-transfectant clones C466D and C466C stably produced
approximately 125 :g/ml of rTNV148B mAb in spent cultures whereas
Sp2/0 transfectant 1.73-12-122 (C467A) stably produced
approximately 25 :g/ml of rTNV148B mAb in spent cultures. Similar
analyses indicated that Sp2/0-transfectant clone C476A produced 18
:g/ml of rTNV14 in spent cultures.
[0253] Introduction. A panel of eight mAbs derived from human
TNF.alpha.-immunized GenPharm/Medarex mice (HCo12/KCoS genotype)
were previously shown to bind human TNF.alpha. and to have a
totally human IgG1, kappa isotype. A simple binding assay was used
to determine whether the exemplary mAbs of the invention were
likely to have TNF.alpha.-neutralizing activity by evaluating their
ability to block TNF.alpha. from binding to recombinant TNF
receptor. Based on those results, DNA sequence results, and in
vitro characterizations of several of the mAbs, TNV148 was selected
as the mAb to be further characterized.
[0254] DNA sequences encoding the TNV148 mAb were cloned, modified
to fit into gene expression vectors that encode suitable constant
regions, introduced into the well-characterized 653 and Sp2/0 mouse
myeloma cells, and resulting transfected cell lines screened until
subclones were identified that produced 40-fold more mAb than the
original hybridoma cell line.
Materials and Methods.
[0255] Reagents and Cells. TRIZOL reagent was purchased from Gibco
BRL. Proteinase K was obtained from Sigma Chemical Company. Reverse
Transcriptase was obtained from Life Sciences, Inc. Taq DNA
Polymerase was obtained from either Perkin Elmer Cetus or Gibco
BRL. Restriction enzymes were purchased from New England Biolabs.
QlAquick PCR Purification Kit was from Qiagen. A QuikChange
Site-Directed Mutagenesis Kit was purchased from Stratagene. Wizard
plasmid miniprep kits and RNasin were from Promega. Optiplates were
obtained from Packard. .sup.125Iodine was purchased from Amersham.
Custom oligonucleotides were purchased from Keystone/Biosource
International. The names, identification numbers, and sequences of
the oligonucleotides used in this work are shown in Table 2.
TABLE-US-00003 TABLE 2 Oligonucleotides used to clone, engineer, or
sequence the TNV mAb genes. The amino acids encoded by
oligonucleotide 5'14s and HuH-J6 are shown above the sequence. The
`M` amino acid residue represents the translation start codon. The
underlined sequences in oligo- nucleotides 5'14s and HuH-J6 mark
the BsiWI and BstBI restriction sites, respectively. The slash in
HuH-J6 corresponds to the exon/intron boundary. Note that
oligonucleotides whose sequence cor- responds to the minus strand
are written in a 3'-5' orientation. Name I.D. Sequence HG1-4b 119
3'-TTGGTCCAGTCGGACTGG-5' (SEQ ID NO: 10) HG1-5b 354
3'-CACCTGCACTCGGTGCTT-5' (SEQ ID NO: 11) HG1hg 360
3'-CACTGTTTTGAGTGTGTACGGGCTTAAGTT-5' (SEQ ID NO: 12) HG1-6 35
3'-GCCGCACGTGTGGAAGGG-5' (SEQ ID NO: 13) HCK1-3E 117
3'-AGTCAAGGTCGGACTGGCTTAAGTT-5' (SEQ ID NO: 14) HuK-3'Hd 208
3'-GTTGTCCCCTCTCACAATCTTCGAATTT-5' (SEQ ID NO: 15) HVKRNAseq 34
3'-GGCGGTAGACTACTCGTC-5' (SEQ ID NO: 16) BsiWI M D W T W S I (SEQ
ID NO: 17) 5'14s 366 5-TTTCGTACGCCACCATGGACTGGACCT GGAGCATC-3' (SEQ
ID NO: 18) 5'46s 367 5'-TTTCGTACGCCACCATGGGGTTTGGG CTGAGCTG-3' (SEQ
ID NO: 19) 5'47s 368 5'-TTTCGTACGCCACCATGGAGTTTGGG CTGAGCATG-3'
(SEQ ID NO: 20) 5'63s 369 5'-TTTCGTACGCCACCATGAAACACCTG
TGGTTCTTC-3' (SEQ ID NO: 21) 5'73s 370
5'-TTTCGTACGCCACCATGGGGTCAACC GCCATCCTC-3' (SEQ ID NO: 22) T V T V
S S BstBI (SEQ ID NO: 23) HuH-J6 388 3'GTGCCAGTGGCAGAGGAGTCCATTC
AAGCTTAAGTT-5' (SEQ ID NO: 24) SalI M D M R V (SEQ ID NO: 25) LK7s
362 5'-TTTGTCGACACCATGGACATGAGGGT CC(TC)C-3' (SEQ ID NO: 26) LVgs
363 5'-TTTGTCGACACCATGGAAGCCCCAGCTC-3' (SEQ ID NO: 27) T K V D I K
(SEQ ID NO: 28) Afl2 HuL-J3 380 3'CTGGTTTCACCTATAGTTTG/CATTCA
GAATTCGGCGCCTTT (SEQ ID NO: 29) V148-QC1 399
5'-CATCTCCAGAGACAATtCCAAGAACA CGCTGTATC-3' (SEQ ID NO: 30) V148-QC2
400 3'-GTAGAGGTCTCTGTTAaGGTTCTTGT GCGACATAG-5' (SEQ ID NO: 31)
[0256] A single frozen vial of 653 mouse myeloma cells was
obtained. The vial was thawed that day and expanded in T flasks in
IMDM, 5% FBS, 2 mM glutamine (media). These cells were maintained
in continuous culture until they were transfected 2 to 3 weeks
later with the anti-TNF DNA described here. Some of the cultures
were harvested 5 days after the thaw date, pelleted by
centrifugation, and resuspended in 95% FBS, 5% DMSO, aliquoted into
30 vials, frozen, and stored for future use. Similarly, a single
frozen vial of Sp2/0 mouse myeloma cells was obtained. The vial was
thawed, a new freeze-down prepared as described above, and the
frozen vials stored in CBC freezer boxes AA and AB. These cells
were thawed and used for all Sp2/0 transfections described
here.
[0257] Assay for Inhibition of TNF Binding to Receptor. Hybridoma
cell supernatants containing the TNV mAbs were used to assay for
the ability of the mAbs to block binding of .sup.125I-labeled
TNF.alpha. to the recombinant TNF receptor fusion protein, p55-sf2
(Scallon et al. (1995) Cytokine 7:759-770). 50 :1 of p55-sf2 at 0.5
:g/ml in PBS was added to Optiplates to coat the wells during a
one-hour incubation at 37.degree. C. Serial dilutions of the eight
TNV cell supernatants were prepared in 96-well round-bottom plates
using PBS/0.1% BSA as diluent. Cell supernatant containing
anti-IL-18 mAb was included as a negative control and the same
anti-IL-18 supernatant spiked with cA2 (anti-TNF chimeric antibody,
Remicade, U.S. Pat. No. 5,770,198, entirely incorporated herein by
reference) was included as a positive control. .sup.125I-labeled
TNF.alpha. (58 :Ci/:g, D. Shealy) was added to 100:1 of cell
supernatants to have a final TNF.alpha. concentration of 5 ng/ml.
The mixture was preincubated for one hour at RT. The coated
Optiplates were washed to remove unbound p55-sf2 and 50:1 of the
.sup.125I-TNF.alpha./cell supernatant mixture was transferred to
the Optiplates. After 2 hrs at RT, Optiplates were washed three
times with PBS-Tween. 100:1 of Microscint-20 was added and the cpm
bound determined using the TopCount gamma counter.
[0258] Amplification of V Genes and DNA Sequence Analysis.
Hybridoma cells were washed once in PBS before addition of TRIZOL
reagent for RNA preparation. Between 7.times.10.sup.6 and
1.7.times.10.sup.7 cells were resuspended in 1 ml TRIZOL. Tubes
were shaken vigorously after addition of 200 .mu.l of chloroform.
Samples were centrifuged at 4.degree. C. for 10 minutes. The
aqueous phase was transferred to a fresh microfuge tube and an
equal volume of isopropanol was added. Tubes were shaken vigorously
and allowed to incubate at room temperature for 10 minutes. Samples
were then centrifuged at 4.degree. C. for 10 minutes. The pellets
were washed once with 1 ml of 70% ethanol and dried briefly in a
vacuum dryer. The RNA pellets were resuspended with 40 .mu.l of
DEPC-treated water. The quality of the RNA preparations was
determined by fractionating 0.5 .mu.l in a 1% agarose gel. The RNA
was stored in a -80.degree. C. freezer until used.
[0259] To prepare heavy and light chain cDNAs, mixtures were
prepared that included 3 .mu.l of RNA and 1 .mu.g of either
oligonucleotide 119 (heavy chain) or oligonucleotide 117 (light
chain) (see Table 1) in a volume of 11.5 .mu.l . The mixture was
incubated at 70.degree. C. for 10 minutes in a water bath and then
chilled on ice for 10 minutes. A separate mixture was prepared that
was made up of 2.5 .mu.l of 10.times. reverse transcriptase buffer,
10 .mu.l of 2.5 mM dNTPs, 1 .mu.l of reverse transcriptase (20
units), and 0.4 .mu.l of ribonuclease inhibitor RNasin (1 unit).
13.5 .mu.l of this mixture was added to the 11.5 .mu.l of the
chilled RNA/oligonucleotide mixture and the reaction incubated for
40 minutes at 42.degree. C. The cDNA synthesis reaction was then
stored in a -20.degree. C. freezer until used.
[0260] The unpurified heavy and light chain cDNAs were used as
templates to PCR-amplify the variable region coding sequences. Five
oligonucleotide pairs (366/354, 367/354, 368/354, 369/354, and
370/354, Table 1) were simultaneously tested for their ability to
prime amplification of the heavy chain DNA. Two oligonucleotide
pairs (362/208 and 363/208) were simultaneously tested for their
ability to prime amplification of the light chain DNA. PCR
reactions were carried out using 2 units of PLATINUM.TM. high
fidelity (HIFI) Taq DNA polymerase in a total volume of 50 .mu.l.
Each reaction included 2 .mu.l of a cDNA reaction, 10 pmoles of
each oligonucleotide, 0.2 mM dNTPs, 5 .mu.l of 10.times. HIFI
Buffer, and 2 mM magnesium sulfate. The thermal cycler program was
95.degree. C. for 5 minutes followed by 30 cycles of (94.degree. C.
for 30 seconds, 62.degree. C. for 30 seconds, 68.degree. C. for 1.5
minutes). There was then a final incubation at 68.degree. C. for 10
minutes.
[0261] To prepare the PCR products for direct DNA sequencing, they
were purified using the QlAquick.TM. PCR Purification Kit according
to the manufacturer's protocol. The DNA was eluted from the spin
column using 50 .mu.l of sterile water and then dried down to a
volume of 10 .mu.l using a vacuum dryer. DNA sequencing reactions
were then set up with 1 .mu.l of purified PCR product, 10 .mu.M
oligonucleotide primer, 4 .mu.l BigDye Terminator.TM. ready
reaction mix, and 14 .mu.l sterile water for a total volume of 20
.mu.l. Heavy chain PCR products made with oligonucleotide pair
367/354 were sequenced with oligonucleotide primers 159 and 360.
Light chain PCR products made with oligonucleotide pair 363/208
were sequenced with oligonucleotides 34 and 163. The thermal cycler
program for sequencing was 25 cycles of (96.degree. C. for 30
seconds, 50.degree. C. for 15 seconds, 60.degree. C. for 4 minutes)
followed by overnight at 4.degree. C. The reaction products were
fractionated through a polyacrylamide gel and detected using an ABI
377 DNA Sequencer.
[0262] Site-directed Mutagenesis to Change an Amino Acid. A single
nucleotide in the TNV148 heavy chain variable region DNA sequence
was changed in order to replace Pro.sup.75 with a Serine residue in
the TNV148 mAb. Complimentary oligonucleotides, 399 and 400 (Table
1), were designed and ordered to make this change using the
QuikChange.TM. site-directed mutagenesis method as described by the
manufacturer. The two oligonucleotides were first fractionated
through a 15% polyacrylamide gel and the major bands purified.
Mutagenesis reactions were prepared using either 10 ng or 50 ng of
TNV148 heavy chain plasmid template (p1753), 5 .mu.l of 10.times.
reaction buffer, 1 .mu.l of dNTP mix, 125 ng of primer 399, 125 ng
of primer 400, and 1 .mu.l of Pfu DNA Polymerase. Sterile water was
added to bring the total volume to 50 .mu.l. The reaction mix was
then incubated in a thermal cycler programmed to incubate at
95.degree. C. for 30 seconds, and then cycle 14 times with
sequential incubations of 95.degree. C. for 30 seconds, 55.degree.
C. for 1 minute, 64.degree. C. for 1 minute, and 68.degree. C. for
7 minutes, followed by 30.degree. C. for 2 minutes (1 cycle). These
reactions were designed to incorporate the mutagenic
oligonucleotides into otherwise identical, newly synthesized
plasmids. To rid of the original TNV148 plasmids, samples were
incubated at 37.degree. C. for 1 hour after addition of 1 .mu.l of
Dpnl endonuclease, which cleaves only the original methylated
plasmid. One .mu.l of the reaction was then used to transform
Epicurian Coli XL1-Blue supercompetent E. coli by standard
heat-shock methods and transformed bacteria identified after
plating on LB-ampicillin agar plates. Plasmid minipreps were
prepared using the Wizard.TM. kits as described by the
manufacturer. After elution of sample from the Wizard.TM. column,
plasmid DNA was precipitated with ethanol to further purify the
plasmid DNA and then resuspended in 20 .mu.l of sterile water. DNA
sequence analysis was then performed to identify plasmid clones
that had the desired base change and to confirm that no other base
changes were inadvertently introduced into the TNV148 coding
sequence. One .mu.l of plasmid was subjected to a cycle sequencing
reaction prepared with 3 .mu.l of BigDye mix, 1 .mu.l of pUC19
Forward primer, and 10 .mu.l of sterile water using the same
parameters described in Section 4.3.
[0263] Construction of Expression Vectors from 12B75 Genes. Several
recombinant DNA steps were performed to prepare a new human IgG1
expression vector and a new human kappa expression vector from the
previously-cloned genomic copies of the 12B75-encoding heavy and
light chain genes, respectively, disclosed in US patent application
No. 60/236,827, filed Oct. 7, 2000, entitled IL-12 Antibodies,
Compositions, Methods and Uses, published as WO 02/12500 which is
entirely incorporated herein by reference. The final vectors were
designed to permit simple, one-step replacement of the existing
variable region sequences with any appropriately-designed,
PCR-amplified, variable region.
[0264] To modify the 12B75 heavy chain gene in plasmid p1560, a
6.85 kb BamHI/HindIII fragment containing the promoter and variable
region was transferred from p1560 to pUC19 to make p1743. The
smaller size of this plasmid compared to p1560 enabled use of
QuikChange.TM. mutagenesis (using oligonucleotides BsiWI-1 and
BsiWI-2) to introduce a unique BsiWI cloning site just upstream of
the translation initiation site, following the manufacturer's
protocol. The resulting plasmid was termed p1747. To introduce a
BstBI site at the 3' end of the variable region, a 5'
oligonucleotide primer was designed with SalI and BstBI sites. This
primer was used with the pUC reverse primer to amplify a 2.75 kb
fragment from p1747. This fragment was then cloned back into the
naturally-occurring SalI site in the 12B75 variable region and a
HindIII site, thereby introducing the unique BstB1 site. The
resulting intermediate vector, designated p1750, could accept
variable region fragments with BsiWII and BstBI ends. To prepare a
version of heavy chain vector in which the constant region also
derived from the 12B75 gene, the BamHI-HindIII insert in p1750 was
transferred to pBR322 in order to have an EcoRl site downstream of
the HindIII site. The resulting plasmid, p1768, was then digested
with HindIII and EcoRI and ligated to a 5.7 kb HindIII-EcoRI
fragment from p1744, a subclone derived by cloning the large
BamHI-BamHI fragment from p1560 into pBC. The resulting plasmid,
p1784, was then used as vector for the TNV Ab cDNA fragments with
BsiWI and BstBI ends. Additional work was done to prepare
expression vectors, p1788 and p1798, which include the IgG1
constant region from the 12B75 gene and differ from each other by
how much of the 12B75 heavy chain J-C intron they contain.
[0265] To modify the 12B75 light chain gene in plasmid p1558, a 5.7
kb SalI/AflII fragment containing the 12B75 promoter and variable
region was transferred from p1558 into the XhoI/AfLII sites of
plasmid L28. This new plasmid, p1745, provided a smaller template
for the mutagenesis step. Oligonucleotides (C340salI and C340sal2)
were used to introduce a unique SalI restriction site at the 5' end
of the variable region by QuikChange.TM. mutagenesis. The resulting
intermediate vector, p1746, had unique SalI and AflII restriction
sites into which variable region fragments could be cloned. Any
variable region fragment cloned into p1746 would preferably be
joined with the 3' half of the light chain gene. To prepare a
restriction fragment from the 3' half of the 12B75 light chain gene
that could be used for this purpose, oligonucleotides BAHN-1 and
BAHN-2 were annealed to each other to form a double-stranded linker
containing the restriction sites BsiW1, AflII, HindII, and Notl and
which contained ends that could be ligated into KpnI and SacI
sites. This linker was cloned between the KpnI and SacI sites of
pBC to give plasmid p1757. A 7.1 kb fragment containing the 12B75
light chain constant region, generated by digesting p1558 with
AflII, then partially digesting with HindIII, was cloned between
the AflII and HindII sites of p1757 to yield p1762. This new
plasmid contained unique sites for BsiWI and AflII into which the
BsiWI/AflII fragment containing the promoter and variable regions
could be transferred uniting the two halves of the gene.
[0266] cDNA Cloning and Assembly of Expression Plasmids. All RT-PCR
reactions (see above) were treated with Klenow enzyme to further
fill in the DNA ends. Heavy chain PCR fragments were digested with
restriction enzymes BsiWI and BstBI and then cloned between the
BsiWI and BstBI sites of plasmid L28 (L28 used because the
12B75-based intermediate vector p1750 had not been prepared yet).
DNA sequence analysis of the cloned inserts showed that the
resulting constructs were correct and that there were no errors
introduced during PCR amplifications. The assigned identification
numbers for these L28 plasmid constructs (for TNV14, TNV15, TNV148,
TNV148B, and TNV196) are shown in Table 3.
[0267] The BsiWI/BstBI inserts for TNV14, TNV148, and TNV148B heavy
chains were transferred from the L28 vector to the newly prepared
intermediate vector, p1750. The assigned identification numbers for
these intermediate plasmids are shown in Table 2. This cloning step
and subsequent steps were not done for TNV15 and TNV196. The
variable regions were then transferred into two different human
IgG1 expression vectors. Restriction enzymes EcoRI and HindIII were
used to transfer the variable regions into Centocor's
previously-used IgG1 vector, p104. The resulting expression
plasmids, which encode an IgG1 of the Gm(f+) allotype, were
designated p1781 (TNV14), p1782 (TNV148), and p1783 (TNV148B) (see
Table 2). The variable regions were also cloned upstream of the
IgG1 constant region derived from the 12B75 (GenPharm) gene. Those
expression plasmids, which encode an IgG1 of the Glm(z) allotype,
are also listed in Table 3.
TABLE-US-00004 TABLE 3 Plasmid identification numbers for various
heavy and light chain plasmids. The L28 vector or pBC vector
represents the initial Ab cDNA clone. The inserts in those plasmids
were transferred to an incomplete 12B75-based vector to make the
intermediate plasmids. One additional transfer step resulted in the
final expression plasmids that were either introduced into cells
after being linearized or used to purify the mAb gene inserts prior
to cell transfection. Gm(f+) G1m(z) 128 vector Intermediate
Expression Expression Mab Plasmid ID Plasmid ID Plasmid ID Plasmid
ID Heavy Chains TNV14 p1751 p1777 p1781 p1786 TNV15 p1752 (ND) (ND)
(ND) TNV148 p1753 p1778 p1782 p1787 TNV148B p1760 p1779 p1783 p1788
TNV196 p1754 (ND) (ND) (ND) Intermediate Expression pBC vector
Plasmid ID Plasmid ID Plasmid ID Light Chains TNV14 p1748 p1755
p1775 TNV15 p1748 p1755 p1775 TNV148 p1749 p1756 p1776 TNV196 p1749
p1756 p1776 (ND) = not done.
[0268] Light chain PCR products were digested with restriction
enzymes SalI and SacII and then cloned between the SalI and SaclI
sites of plasmid pBC. The two different light chain versions, which
differed by one amino acid, were designated p1748 and p1749 (Table
2). DNA sequence analysis confirmed that these constructs had the
correct sequences. The SalI/AflII fragments in p1748 and p1749 were
then cloned between the SalI and AflII sites of intermediate vector
p1746 to make p1755 and p1756, respectively. These 5' halves of the
light chain genes were then joined to the 3' halves of the gene by
transferring the BsiWI/AflII fragments from p1755 and p1756 to the
newly prepared construct p1762 to make the final expression
plasmids p1775 and p1776, respectively (Table 2).
[0269] Cell Transfections, Screening, and Subcloning. A total of 15
transfections of mouse myeloma cells were performed with the
various TNV expression plasmids (see Table 3 in the Results and
Discussion section). These transfections were distinguished by
whether (1) the host cells were Sp2/0 or 653; (2) the heavy chain
constant region was encoded by Centocor's previous IgG1 vector or
the 12B75 heavy chain constant region; (3) the mAb was TNV148B,
TNV148, TNV14, or a new HC/LC combination; (4) whether the DNA was
linearized plasmid or purified Ab gene insert; and (5) the presence
or absence of the complete J-C intron sequence in the heavy chain
gene. In addition, several of the transfections were repeated to
increase the likelihood that a large number of clones could be
screened.
[0270] Sp2/0 cells and 653 cells were each transfected with a
mixture of heavy and light chain DNA (8-12 :g each) by
electroporation under standard conditions as previously described
(Knight D M et al. (1993) Molecular Immunology 30:1443-1453). For
transfection numbers 1, 2, 3, and 16, the appropriate expression
plasmids were linearized by digestion with a restriction enzyme
prior to transfection. For example, SalI and Notl restriction
enzymes were used to linearize TNV148B heavy chain plasmid p1783
and light chain plasmid p1776, respectively. For the remaining
transfections, DNA inserts that contained only the mAb gene were
separated from the plasmid vector by digesting heavy chain plasmids
with BamHI and light chain plasmids with BsiWI and NotI. The mAb
gene inserts were then purified by agarose gel electrophoresis and
Qiex purification resins. Cells transfected with purified gene
inserts were simultaneously transfected with 3-5 :g of
Pstl-linearized pSV2gpt plasmid (p13) as a source of selectable
marker. Following electroporation, cells were seeded in 96-well
tissue culture dishes in IMDM, 15% FBS, 2 mM glutamine and
incubated at 37.degree. C. in a 5% CO.sub.2 incubator . Two days
later, an equal volume of IMDM, 5% FBS, 2mM glutamine, 2 X MHX
selection (1 X MHX =0.5 :g/ml mycophenolic acid, 2.5:g/ml
hypoxanthine, 50 :g/ml xanthine) was added and the plates incubated
for an additional 2 to 3 weeks while colonies formed.
[0271] Cell supernatants collected from wells with colonies were
assayed for human IgG by ELISA as described. In brief, varying
dilutions of the cell supernatants were incubated in 96-well EIA
plates coated with polyclonal goat anti-human IgG Fc fragment and
then bound human IgG was detected using Alkaline
Phosphatase-conjugated goat anti-human IgG(H+L) and the appropriate
color substrates. Standard curves, which used as standard the same
purified mAb that was being measured in the cell supernatants, were
included on each EIA plate to enable quantitation of the human IgG
in the supernatants. Cells in those colonies that appeared to be
producing the most human IgG were passaged into 24-well plates for
additional production determinations in spent cultures and the
highest-producing parental clones were subsequently identified.
[0272] The highest-producing parental clones were subcloned to
identify higher-producing subclones and to prepare a more
homogenous cell line. 96-well tissue culture plates were seeded
with one cell per well or four cells per well in of IMDM, 5% FBS, 2
mM glutamine, 1.times.MHX and incubated at 37.degree. C. in a 5%
CO.sub.2 incubator for 12 to 20 days until colonies were apparent.
Cell supernatants were collected from wells that contained one
colony per well and analyzed by ELISA as described above. Selected
colonies were passaged to 24-well plates and the cultures allowed
to go spent before identifying the highest-producing subclones by
quantitating the human IgG levels in their supernatants. This
process was repeated when selected first-round subclones were
subjected to a second round of subcloning. The best second-round
subclones were selected as the cell lines for development.
[0273] Characterization of Cell Subclones. The best second-round
subclones were chosen and growth curves performed to evaluate mAb
production levels and cell growth characteristics. T75 flasks were
seeded with 1.times.10.sup.5 cells/ml in 30 ml IMDM, 5% FBS, 2 mM
glutamine, and 1.times.MHX (or serum-free media). Aliquots of 300
.mu.l were taken at 24 hr intervals and live cell density
determined. The analyses continued until the number of live cells
was less than 1.times.10.sup.5 cells/ml. The collected aliquots of
cell supernatants were assayed for the concentration of antibody
present. ELISA assays were performed using as standard rTNV148B or
rTNV14 JG92399. Samples were incubated for 1 hour on ELISA plates
coated with polyclonal goat anti-human IgG Fc and bound mAb
detected with Alkaline Phosphatase-conjugated goat anti-human
IgG(H+L) at a 1:1000 dilution.
[0274] A different growth curve analysis was also done for two cell
lines for the purpose of comparing growth rates in the presence of
varying amounts of MHX selection. Cell lines C466A and C466B were
thawed into MHX-free media (IMDM, 5% FBS, 2 mM glutamine) and
cultured for two additional days. Both cell cultures were then
divided into three cultures that contained either no MHX,
0.2.times.MHX, or 1.times.MHX (1.times.MHX =0.5 :g/ml mycophenolic
acid, 2.5 :g/ml hypoxanthine, 50 :g/ml xanthine). One day later,
fresh T75 flasks were seeded with the cultures at a starting
density of 1.times.10.sup.5 cells/ml and cells counted at 24 hour
intervals for one week. Aliquots for mAb production were not
collected. Doubling times were calculated for these samples using
the formula provided in SOP PD32.025.
[0275] Additional studies were performed to evaluate stability of
mAb production over time. Cultures were grown in 24-well plates in
IMDM, 5% FBS, 2 mM glutamine, either with or without MHX selection.
Cultures were split into fresh cultures whenever they became
confluent and the older culture was then allowed to go spent. At
this time, an aliquot of supernatant was taken and stored at
4.degree. C. Aliquots were taken over a 55-78 day period. At the
end of this period, supernatants were tested for amount of antibody
present by the anti-human IgG Fc ELISA as outlined above.
Results and Discussion.
[0276] Inhibition of TNF binding to Recombinant Receptor.
[0277] A simple binding assay was done to determine whether the
eight TNV mAbs contained in hybridoma cell supernatant were capable
of blocking TNF.alpha. binding to receptor. The concentrations of
the TNV mAbs in their respective cell supernatants were first
determined by standard ELISA analysis for human IgG. A recombinant
p55 TNF receptor/IgG fusion protein, p55-sf2, was then coated on
EIA plates and .sup.125I-labeled TNF.alpha. allowed to bind to the
p55 receptor in the presence of varying amounts of TNV mAbs. As
shown in FIG. 1, all but one (TNV122) of the eight TNV mAbs
efficiently blocked TNF.alpha. binding to p55 receptor. In fact,
the TNV mAbs appeared to be more effective at inhibiting TNF.alpha.
binding than cA2 positive control mAb that had been spiked into
negative control hybridoma supernatant. These results were
interpreted as indicating that it was highly likely that the TNV
mAbs would block TNF.alpha. bioactivity in cell-based assays and in
vivo and therefore additional analyses were warranted.
DNA Sequence Analysis.
[0278] Confirmation that the RNAs Encode Human mAbs.
[0279] As a first step in characterizing the seven TNV mAbs (TNV14,
TNV15, TNV32, TNV86, TNV118, TNV148, and TNV196) that showed
TNF.alpha.-blocking activity in the receptor binding assay, total
RNA was isolated from the seven hybridoma cell lines that produce
these mAbs. Each RNA sample was then used to prepare human antibody
heavy or light chain cDNA that included the complete signal
sequence, the complete variable region sequence, and part of the
constant region sequence for each mAb. These cDNA products were
then amplified in PCR reactions and the PCR-amplified DNA was
directly sequenced without first cloning the fragments. The heavy
chain cDNAs sequenced were >90% identical to one of the five
human germline genes present in the mice, DP-46 (FIG. 2).
Similarly, the light chain cDNAs sequenced were either 100% or 98%
identical to one of the human germline genes present in the mice
(FIG. 3). These sequence results confirmed that the RNA molecules
that were transcribed into cDNA and sequenced encoded human
antibody heavy chains and human antibody light chains. It should be
noted that, because the variable regions were PCR-amplified using
oligonucleotides that map to the 5' end of the signal sequence
coding sequence, the first few amino acids of the signal sequence
may not be the actual sequence of the original TNV translation
products but they do represent the actual sequences of the
recombinant TNV mAbs.
Unique Neutralizing mAbs.
[0280] Analyses of the cDNA sequences for the entire variable
regions of both heavy and light chains for each mAb revealed that
TNV32 is identical to TNV15, TNV118 is identical to TNV14, and
TNV86 is identical to TNV148. The results of the receptor binding
assay were consistent with the DNA sequence analyses, i.e. both
TNV86 and TNV148 were approximately 4-fold better than both TNV118
and TNV14 at blocking TNF binding. Subsequent work was therefore
focused on only the four unique TNV mAbs, TNV14, TNV15, TNV148, and
TNV196.
Relatedness of the Four mAbs
[0281] The DNA sequence results revealed that the genes encoding
the heavy chains of the four TNV mAbs were all highly homologous to
each other and appear to have all derived from the same germline
gene, DP-46 (FIG. 2). In addition, because each of the heavy chain
CDR3 sequences are so similar and of the same length, and because
they all use the J6 exon, they apparently arose from a single VDJ
gene rearrangement event that was then followed by somatic changes
that made each mAb unique. DNA sequence analyses revealed that
there were only two distinct light chain genes among the four mAbs
(FIG. 3). The light chain variable region coding sequences in TNV14
and TNV15 are identical to each other and to a representative
germline sequence of the Vg/38K family of human kappa chains. The
TNV148 and TNV196 light chain coding sequences are identical to
each other but differ from the germline sequence at two nucleotide
positions (FIG. 3).
[0282] The deduced amino acid sequences of the four mAbs revealed
the relatedness of the actual mAbs. The four mAbs contain four
distinct heavy chains (FIG. 4) but only two distinct light chains
(FIG. 5). Differences between the TNV mAb sequences and the
germline sequences were mostly confined to CDR domains but three of
the mAb heavy chains also differed from the germline sequence in
the framework regions (FIG. 4). Compared to the DP-46
germline-encoded Ab framework regions, TNV14 was identical, TNV15
differed by one amino acid, TNV148 differed by two amino acids, and
TNV196 differed by three amino acids.
[0283] Cloning of cDNAs, Site-specific Mutagenesis, and Assembly of
Final Expression Plasmids. Cloning of cDNAs. Based on the DNA
sequence of the PCR-amplified variable regions, new
oligonucleotides were ordered to perform another round of PCR
amplification for the purpose of adapting the coding sequence to be
cloned into expression vectors. In the case of the heavy chains,
the products of this second round of PCR were digested with
restriction enzymes BsiWI and BstBI and cloned into plasmid vector
L28 (plasmid identification numbers shown in Table 2). In the case
of the light chains, the second-round PCR products were digested
with SalI and AflII and cloned into plasmid vector pBC. Individual
clones were then sequenced to confirm that their sequences were
identical to the previous sequence obtained from direct sequencing
of PCR products, which reveals the most abundant nucleotide at each
position in a potentially heterogeneous population of
molecules.
[0284] Site-specific Mutagenesis to Change TNV148. mAbs TNV148 and
TNV196 were being consistently observed to be four-fold more potent
than the next best mAb (TNV14) at neutralizing TNF.alpha.
bioactivity. However, as described above, the TNV148 and TNV196
heavy chain framework sequences differed from the germline
framework sequences. A comparison of the TNV148 heavy chain
sequence to other human antibodies indicated that numerous other
human mAbs contained an Ile residue at position 28 in framework 1
(counting mature sequence only) whereas the Pro residue at position
75 in framework 3 was an unusual amino acid at that position.
[0285] A similar comparison of the TNV196 heavy chain suggested
that the three amino acids by which it differs from the germline
sequence in framework 3 may be rare in human mAbs. There was a
possibility that these differences may render TNV148 and TNV196
immunogenic if administered to humans. Because TNV148 had only one
amino acid residue of concern and this residue was believed to be
unimportant for TNF.alpha. binding, a site-specific mutagenesis
technique was used to change a single nucleotide in the TNV148
heavy chain coding sequence (in plasmid p1753) so that a germline
Ser residue would be encoded in place of the Pro residue at
position 75. The resulting plasmid was termed p1760 (see Table 2).
The resulting gene and mAb were termed TNV148B to distinguish it
from the original TNV148 gene and mAb (see FIG. 5).
[0286] Assembly of Final Expression Plasmids. New antibody
expression vectors were prepared that were based on the 12B75 heavy
chain and light chain genes previously cloned as genomic fragments.
Although different TNV expression plasmids were prepared (see Table
2), in each case the 5' flanking sequences, promoter, and intron
enhancer derived from the respective 12B75 genes. For the light
chain expression plasmids, the complete J-C intron, constant region
coding sequence and 3' flanking sequence were also derived from the
12B75 light chain gene. For the heavy chain expression plasmids
that resulted in the final production cell lines (p1781 and p1783,
see below), the human IgG1 constant region coding sequences derived
from Centocor's previously-used expression vector (p104).
Importantly, the final production cell lines reported here express
a different allotype (Gm(f+)) of the TNV mAbs than the original,
hybridoma-derived TNV mAbs (Glm(z)). This is because the 12B75
heavy chain gene derived from the GenPharm mice encodes an Arg
residue at the C-terminal end of the CH1 domain whereas Centocor's
IgG1 expression vector p104 encodes a Lys residue at that position.
Other heavy chain expression plasmids (e.g. p1786 and p1788) were
prepared in which the J-C intron, complete constant region coding
sequence and 3' flanking sequence were derived from the 12B75 heavy
chain gene, but cell lines transfected with those genes were not
selected as the production cell lines. Vectors were carefully
designed to permit one-step cloning of future PCR-amplified V
regions that would result in final expression plasmids.
[0287] PCR-amplified variable region cDNAs were transferred from
L28 or pBC vectors to intermediate-stage, 12B75-based vectors that
provided the promoter region and part of the J-C intron (see Table
2 for plasmid identification numbers). Restriction fragments that
contained the 5' half of the antibody genes were then transferred
from these intermediate-stage vectors to the final expression
vectors that provided the 3' half of the respective genes to form
the final expression plasmids (see Table 2 for plasmid
identification numbers).
[0288] Cell Transfections and Subcloning. Expression plasmids were
either linearized by restriction digest or the antibody gene
inserts in each plasmid were purified away from the plasmid
backbones. Sp2/0 and 653 mouse myeloma cells were transfected with
the heavy and light chain DNA by electroporation. Fifteen different
transfections were done, most of which were unique as defined by
the Ab, specific characteristics of the Ab genes, whether the genes
were on linearized whole plasmids or purified gene inserts, and the
host cell line (summarized in Table 5). Cell supernatants from
clones resistant to mycophenolic acid were assayed for the presence
of human IgG by ELISA and quantitated using purified rTNV148B as a
reference standard curve.
Highest-Producing rTNV148B Cell Lines
[0289] Ten of the best-producing 653 parental lines from rTNV148B
transfection 2 (produced 5-10 :g/ml in spent 24-well cultures) were
subcloned to screen for higher-producing cell lines and to prepare
a more homogeneous cell population. Two of the subclones of the
parental line 2.320, 2.320-17 and 2.320-20, produced approximately
50 :g/ml in spent 24-well cultures, which was a 5-fold increase
over their parental line. A second round of subcloning of subcloned
lines 2.320-17 and 2.320-20 led
[0290] The identification numbers of the heavy and light chain
plasmids that encode each mAb are shown. In the case of
transfections done with purified mAb gene inserts, plasmid p13
(pSV2gpt) was included as a source of the gpt selectable marker.
The heavy chain constant regions were encoded either by the same
human IgG1 expression vector used to encode Remicade ('old') or by
the constant regions contained within the 12B75 (GenPharm/Medarex)
heavy chain gene ('new'). H1/L2 refers to a "novel" mAb made up of
the TNV14 heavy chain and the TNV148 light chain. Plasmids p1783
and p1801 differ only by how much of the J-C intron their heavy
chain genes contain. The transfection numbers, which define the
first number of the generic names for cell clones, are shown on the
right. The rTNV148B-producing cell lines C466 (A, B, C, D) and
C467A described here derived from transfection number 2 and 1,
respectively. The rTNV14-producing cell line C476A derived from
transfection number 3.
TABLE-US-00005 TABLE 5 Summary of Cell Transfections. Plasmids HC
DNA Transfection no. mAb HC/LC/gpt vector format Sp2/0 653 rTNV148B
1783/1776 old linear 1 2 rTNV14 1781/1775 old linear 3 -- rTNV148B
1788/1776/13 new insert 4, 6 5, 7 rTNV14 1786/1775/13 new insert 8,
10 9, 11 rTNV148 1787/1776/13 new insert 12 17 rH1/L2 1786/1776/13
new insert 13 14 rTNV148B 1801/1776 old linear 16
[0291] ELISA assays on spent 24-well culture supernatants indicated
that these second-round subclones all produced between 98 and 124
:g/ml, which was at least a 2-fold increase over the first-round
subclones. These 653 cell lines were assigned C code designations
as shown in Table 6.
[0292] Three of the best-producing Sp2/0 parental lines from
rTNV148B transfection 1 were subcloned. Two rounds of subcloning of
parental line 1.73 led to the identification of a clone that
produced 25 :g/ml in spent 24-well cultures. This Sp2/0 cell line
was designated C467A (Table 6).
[0293] Highest-Producing rTNV14 Cell Lines
[0294] Three of the best-producing Sp2/0 parental lines from rTNV14
transfection 3 were subcloned once. Subclone 3.27-1 was found to be
the highest-producer in spent 24-well cultures with a production of
19 :g/ml. This cell line was designated C476A (Table 6).
TABLE-US-00006 TABLE 6 Summary of Selected Production Cell Lines
and their C codes. The first digit of the original clone names
indicates which transfection the cell line derived from. All of the
C-coded cell lines reported here were derived from transfections
with heavy and light chain whole plasmids that had been linearized
with restriction enzymes. Original Spent 24-well mAb Clone Name C
code Host Cell Production rTNV148B 2.320-17-36 C466A 653 103:g/ml
2.320-20-111 C466B 653 102:g/ml 2.320-17-4 C466C 653 98:g/ml
2.320-20-99 C466D 653 124:g/ml 1.73-12-122 C467A Sp2/0 25:g/ml
rTNV14 3.27-1 C476A Sp2/0 19:g/ml
Characterization of Subcloned Cell Lines
[0295] To more carefully characterize cell line growth
characteristics and determine mAb-production levels on a larger
scale, growth curves analyses were performed using T75 cultures.
The results showed that each of the four C466 series of cell lines
reached peak cell density between 1.0.times.10.sup.6 and
1.25.times.10.sup.6 cells/ml and maximal mAb accumulation levels of
between 110 and 140 :g/ml (FIG. 7). In contrast, the best-producing
Sp2/0 subclone, C467A, reached peak cell density of
2.0.times.10.sup.6 cells/ml and maximal mAb accumulation levels of
25 :g/ml (FIG. 7). A growth curve analysis was not done on the
rTNV14-producing cell line, C476A.
[0296] An additional growth curve analysis was done to compare the
growth rates in different concentrations of MHX selection. This
comparison was prompted by recent observations that C466 cells
cultured in the absence of MHX seemed to be growing faster than the
same cells cultured in the normal amount of MHX (1.times.). Because
the cytotoxic concentrations of compounds such as mycophenolic acid
tend to be measured over orders of magnitude, it was considered
possible that the use of a lower concentration of MHX might result
in significantly faster cell doubling times without sacrificing
stability of mAb production. Cell lines C466A and C466B were
cultured either in: no MHX, 0.2.times.MHX, or 1.times.MHX. Live
cell counts were taken at 24-hour intervals for 7 days. The results
did reveal an MHX concentration-dependent rate of cell growth (FIG.
8). Cell line C466A showed a doubling time of 25.0 hours in
1.times.MHX but only 20.7 hours in no MHX. Similarly, cell line
C466B showed a doubling time of 32.4 hours in 1.times.MHX but only
22.9 hours in no MHX. Importantly, the doubling times for both cell
lines in 0.2.times.MHX were more similar to what was observed in no
MHX than in 1.times.MHX (FIG. 8). This observation raises the
possibility than enhanced cell performance in bioreactors, for
which doubling times are an important parameter, could be realized
by using less MHX. However, although stability test results (see
below) suggest that cell line C466D is capable of stably producing
rTNV148B for at least 60 days even with no MHX present, the
stability test also showed higher mAb production levels when the
cells were cultured in the presence of MHX compared to the absence
of MHX.
[0297] To evaluate mAb production from the various cell lines over
a period of approximately 60 days, stability tests were performed
on cultures that either contained, or did not contain, MHX
selection. Not all of the cell lines maintained high mAb
production. After just two weeks of culture, clone C466A was
producing approximately 45% less than at the beginning of the
study. Production from clone C466B also appeared to drop
significantly. However, clones C466C and C466D maintained fairly
stable production, with C466D showing the highest absolute
production levels (FIG. 9).
Conclusion
[0298] From an initial panel of eight human mAbs against human
TNF.alpha., TNV148B was selected as preferred based on several
criteria that included protein sequence and TNF neutralization
potency, as well as TNV14. Cell lines were prepared that produce
greater than 100 :g/ml of rTNV148B and 19 :g/ml rTNV14.
EXAMPLE 5
Arthritic Mice Study Using Anti-TNF Antibodies and Controls Using
Single Bolus Injection
[0299] At approximately 4 weeks of age the Tg197 study mice were
assigned, based on gender and body weight, to one of 9 treatment
groups and treated with a single intraperitoneal bolus dose of
Dulbecco's PBS (D-PBS) or an anti-TNF anatibody of the present
invention (TNV14, TNV148 or TNV196) at either 1 mg/kg or 10
mg/kg.
[0300] RESULTS: When the weights were analyzed as a change from
pre-dose, the animals treated with 10 mg/kg cA2 showed consistently
higher weight gain than the D-PBS-treated animals throughout the
study. This weight gain was significant at weeks 3-7. The animals
treated with 10 mg/kg TNV148 also achieved significant weight gain
at week 7 of the study. (See FIG. 10).
[0301] FIGS. 11A-C represent the progression of disease severity
based on the arthritic index. The 10 mg/kg cA2-treated group's
arthritic index was lower then the D-PBS control group starting at
week 3 and continuing throughout the remainder of the study (week
7). The animals treated with 1 mg/kg TNV14 and the animals treated
with 1 mg/kg cA2 failed to show significant reduction in AI after
week 3 when compared to the D-PBS-treated Group. There were no
significant differences between the 10 mg/kg treatment groups when
each was compared to the others of similar dose (10 mg/kg cA2
compared to 10 mg/kg TNV14, 148 and 196). When the 1 mg/kg
treatment groups were compared, the 1 mg/kg TNV148 showed a
significantly lower AI than 1 mg/kg cA2 at 3, 4 and 7 weeks. The 1
mg/kg TNV148 was also significantly lower than the 1 mg/kg
TNV14-treated Group at 3 and 4 weeks. Although TNV196 showed
significant reduction in AI up to week 6 of the study (when
compared to the D-PBS-treated Group), TNV148 was the only 1 mg/kg
treatment that remained significant at the conclusion of the
study.
EXAMPLE 6
Arthritic Mice Study Using Anti-TNF Antibodies and Controls as
Multiple Bolus Doses
[0302] At approximately 4 weeks of age the Tg197 study mice were
assigned, based on body weight, to one of 8 treatment groups and
treated with a intraperitoneal bolus dose of control article
(D-PBS) or antibody (TNV14, TNV148) at 3 mg/kg (week 0).
[0303] Injections were repeated in all animals at weeks 1, 2, 3,
and 4. Groups 1-6 were evaluated for test article efficacy. Serum
samples, obtained from animals in Groups 7 and 8 were evaluated for
immune response induction and pharmacokinetic clearance of TNV14 or
TNV148 at weeks 2, 3 and 4.
[0304] RESULTS: No significant differences were noted when the
weights were analyzed as a change from pre-dose. The animals
treated with 10 mg/kg cA2 showed consistently higher weight gain
than the D-PBS-treated animals throughout the study. (See FIG.
12).
[0305] FIGS. 13A-C represent the progression of disease severity
based on the arthritic index. The 10 mg/kg cA2-treated group's
arthritic index was significantly lower then the D-PBS control
group starting at week 2 and continuing throughout the remainder of
the study (week 5). The animals treated with 1 mg/kg or 3 mg/kg of
cA2 and the animals treated with 3 mg/kg TNV14 failed to achieve
any significant reduction in AI at any time throughout the study
when compared to the d-PBS control group. The animals treated with
3 mg/kg TNV148 showed a significant reduction when compared to the
d-PBS-treated group starting at week 3 and continuing through week
5. The 10 mg/kg cA2-treated animals showed a significant reduction
in AI when compared to both the lower doses (1 mg/kg and 3 mg/kg)
of cA2 at weeks 4 and 5 of the study and was also significantly
lower than the TNV14-treated animals at weeks 3-5. Although there
appeared to be no significant differences between any of the 3mg/kg
treatment groups, the AI for the animals treated with 3 mg/kg TNV14
were significantly higher at some time points than the 10 mg/kg
whereas the animals treated with TNV148 were not significantly
different from the animals treated with 10 mg/kg of cA2.
EXAMPLE 7
Arthritic Mice Study using Anti-TNF Antibodies and Controls as
Single Intraperitoneal Bolus Dose
[0306] At approximately 4 weeks of age the Tg197 study mice were
assigned, based on gender and body weight, to one of 6 treatment
groups and treated with a single intraperitoneal bolus dose of
antibody (cA2, or TNV148) at either 3 mg/kg or 5 mg/kg. This study
utilized the D-PBS and 10 mg/kg cA2 control Groups.
[0307] When the weights were analyzed as a change from pre-dose,
all treatments achieved similar weight gains. The animals treated
with either 3 or 5 mg/kg TNV148 or 5 mg/kg cA2 gained a significant
amount of weight early in the study (at weeks 2 and 3). Only the
animals treated with TNV148 maintained significant weight gain in
the later time points. Both the 3 and 5 mg/kg TNV148-treated
animals showed significance at 7 weeks and the 3 mg/kg TNV148
animals were still significantly elevated at 8 weeks post
injection. (See FIG. 14).
[0308] FIG. 15 represents the progression of disease severity based
on the arthritic index. All treatment groups showed some protection
at the earlier time points, with the 5 mg/kg cA2 and the 5 mg/kg
TNV148 showing significant reductions in AI at weeks 1-3 and all
treatment groups showing a significant reduction at week 2. Later
in the study the animals treated with 5 mg/kg cA2 showed some
protection, with significant reductions at weeks 4, 6 and 7. The
low dose (3 mg/kg) of both the cA2 and the TNV148 showed
significant reductions at 6 and all treatment groups showed
significant reductions at week 7. None of the treatment groups were
able to maintain a significant reduction at the conclusion of the
study (week 8). There were no significant differences between any
of the treatment groups (excluding the saline control group) at any
time point.
EXAMPLE 8
Arthritic Mice Study Using Anti-TNF Antibodies and Controls as
Single Intraperitoneal Bolus Dose Between Anti-TNF Antibody and
Modified Anti-TNF Antibody
[0309] To compare the efficacy of a single intraperitoneal dose of
TNV148 (derived from hybridoma cells) and rTNV148B (derived from
transfected cells). At approximately 4 weeks of age the Tg197 study
mice were assigned, based on gender and body weight, to one of 9
treatment groups and treated with a single intraperitoneal bolus
dose of Dulbecco=S PBS (D-PBS) or antibody (TNV148, rTNV148B) at 1
mg/kg.
[0310] When the weights were analyzed as a change from pre-dose,
the animals treated with 10 mg/kg cA2 showed a consistently higher
weight gain than the D-PBS-treated animals throughout the study.
This weight gain was significant at weeks 1 and weeks 3-8. The
animals treated with 1 mg/kg TNV148 also achieved significant
weight gain at weeks 5, 6 and 8 of the study. (See FIG. 16).
[0311] FIG. 17 represents the progression of disease severity based
on the arthritic index. The 10 mg/kg cA2-treated group's arthritic
index was lower then the D-PBS control group starting at week 4 and
continuing throughout the remainder of the study (week 8). Both of
the TNV148-treated Groups and the 1 mg/kg cA2-treated Group showed
a significant reduction in AI at week 4. Although a previous study
(P-099-017) showed that TNV148 was slightly more effective at
reducing the Arthritic Index following a single 1 mg/kg
intraperitoneal bolus, this study showed that the AI from both
versions of the TNV antibody-treated groups was slightly higher.
Although (with the exception of week 6) the 1 mg/kg cA2--treated
Group was not significantly increased when compared to the 10 mg/kg
cA2 group and the TNV148-treated Groups were significantly higher
at weeks 7 and 8, there were no significant differences in AI
between the 1 mg/kg cA2, 1 mg/kg TNV148 and 1 mg/kg TNV148B at any
point in the study.
EXAMPLE 9
Anti-TNF Antibody for the Treatment or Prevention of Type 1
Diabetes (T1D)
Introduction
[0312] The Sponsor is requesting a pre-IND Type B meeting to
discuss development plans for SIMPONI.RTM. (golimumab) administered
subcutaneously (SC) in the treatment of Type 1 diabetes (T1D) to
determine whether benefit with SIMPONI.RTM. can be established in
newly diagnosed T1D patients in a Phase 2a efficacy and safety
study. This study in a newly diagnosed population would be an
initial step in the development program to understand whether
SIMPONI.RTM. would have benefit in pre-T1D patients to stabilize
endogenous insulin production to prevent or delay disease
progression.
Product Name and Application Number
[0313] SIMPONI.RTM. (golimumab)
Chemical Name and Structure
[0314] SIMPONI.RTM. (golimumab) is a human monoclonal antibody
(mAb) with an immunoglobulin G (IgG) 1 heavy chain isotype (Glm [z]
allotype) and a kappa light chain isotype. Golimumab has a heavy
chain (HC) comprising SEQ ID NO:36 and a light chain (LC)
comprising SEQ ID NO:37. Golimumab binds with high affinity to both
soluble and transmembrane forms of tumor necrosis factor alpha
(TNF.alpha.) and inhibits TNF.alpha. bioactivity. Golimumab is
classified according to the Anatomical Therapeutic Chemical (ATC)
Classification System as a TNF.alpha. inhibitor (ATC code:
LO4AB06).
[0315] Golimumab is approved under the trade name of SIMPONI.RTM.
in the United States (US) and a total of 89 countries worldwide as
of 6 Oct. 2015 for the treatment of rheumatoid arthritis (RA),
psoriatic arthritis (PsA), and ankylosing spondylitis (AS) as a 50
mg SC injection administered once a month as well as in 67
countries for the treatment of ulcerative colitis (UC) as a 200 mg
SC injection at Week 0, followed by 100 mg at Week 2 induction
regimen, and maintenance therapy with 100 mg every 4 weeks
thereafter.
[0316] Specifically in the US, SIMPONI.RTM. is approved for the
following indications: [0317] in combination with methotrexate
(MTX) for the treatment of adult patients with moderately to
severely active RA [0318] alone or in combination with MTX for the
treatment of adult patients with active PsA [0319] for the
treatment of adult patients with active AS [0320] for the treatment
of adult patients with ulcerative colitis
Dosage Form, Route of Administration, and Dosing
[0321] The Sponsor intends to use 2 dosage forms in the initial
Phase 2a study to support the proposed doses across the target
population. These 2 dosage forms are described below:
[0322] Prefilled Syringe [0323] Each 50 mg single dose prefilled
glass syringe (27 gauge 1/2 inch needle) contains 50 mg of
SIMPONI.RTM. per 0.5 mL of solution.
[0324] VarioJect [0325] A new pediatric presentation known as the
VarioJect is being developed by the Sponsor as a platform device
intended for multiple pediatric indications that provides
tiered-fixed dosing in pediatric patients who are dosed by body
surface area (BSA). The VarioJect is a manual injector capable of
delivering doses of 10 mg through 45 mg in 5 mg increments. [0326]
Additional details are provided throught the specification below
and in the figures.
Disease Background
Disease Etiology and Prevalence
[0327] In the United States, T1D is one of the three most prevalent
chronic diseases of childhood and approximately 3 million people
have this disease (Daneman 2006, Stanescu, Lord et al. 2012). The
annual incidence is highest in children and adolescents, at
.about.20 cases/100,000/year in those younger than 20 years old,
accounting for nearly 15,000 new cases yearly.(Maahs, West et al.
2010, Stanescu, Lord et al. 2012) In the U.S. and worldwide, the
incidence of T1D is on the rise, with increases of .about.3-5% per
year in most areas (Daneman 2006, Stanescu, Lord et al. 2012). In
the US, between 2001 and 2009, the prevalence of T1D in children
and adolescents rose by 23%.(JDRF 2013) The diagnosis of T1D
increases during childhood and peaks in adolescence: in those 0-4,
5-9, 10-14, and 15-19 years old annual rates are 14, 22, 26, and 13
cases/100,000, respectively (Stanescu, Lord et al. 2012).
Comparatively, T1D rates are much lower in adults, with the
incidence only up to .about.8/100,000 (Molbak, Christau et al.
1994). In children, the clinical course leading to diagnosis is
relatively fulminant, with symptoms (ie, polydipsia, polyuria, and
weight loss) usually apparent only days or weeks before diagnosis,
whereas in adults the course is usually much more indolent, with
milder symptoms occurring over months or longer. Children with
new-onset and existing T1D frequently suffer the severe metabolic
derangement of diabetic ketoacidosis (DKA) during which they are at
high risk of cerebral edema, herniation and death.
[0328] Data from the past 4 decades demonstrate that T1D results
from an autoimmune attack on the pancreatic beta cells (Mordes,
Borten et al. 1996, Eisenbarth 2004, Han, Donelan et al. 2013). A
proinflammatory combination of innate and adaptive and cellular and
humoral responses is responsible for T1D (Bluestone, Herold et al.
2010). Similar to other autoimmune diseases, T1D is hypothesized to
occur in predisposed individuals who encounter a diabetogenic
environmental trigger.(Atkinson, Bluestone et al. 2011) A number of
HLA- and other immune system-associated genes are linked to T1D
susceptibility (van Belle, Coppieters et al. 2011). Those with T1D
appear to have abnormalities in both central and peripheral
tolerance mechanisms that involve .beta.-cell-reactive T cells
(Eisenbarth 2004). Although both dietary and infectious factors
have been implicated, none have been found to be causally
associated with T1D. It is hypothesized that some environmental
encounter activates an inflammatory response, that in predisposed
individuals, results in the activation and recruitment of
macrophages, dendritic cells, CD4 and CD8 T cells, and B cells to
islets (Bluestone, Herold et al. 2010, Atkinson, Bluestone et al.
2011). Soluble factors, including proinflammatory cytokines and
beta cell autoantibodies, participate in and amplify this response,
and the end result is terminal destruction of beta cells.
[0329] Current Treatment Paradigm
[0330] Currently the only therapy available for those with T1D is
exogenous insulin which must be given as multiple subcutaneous
shots every day or by continuous plus pulse insulin via a
subcutaneous pump. Insulin, as well as numerous blood glucose
checks, are required every day in those with T1D and a careful
balance of current blood glucose levels, diet, and exercise must be
taken into consideration with every insulin administration to
maintain the careful balance between hyper- and hypoglycemia, both
of which can be life threatening. There are no adjunctive therapies
or disease-modifying therapies available for those with T1D, and
glycemic management with exogenous insulin is required for life for
those who develop this disease. The positive effect of intensive
insulin treatment on the outcome of microvascular complications was
demonstrated by the landmark study Diabetes Control and
Complications Trial (DCCT) in which subjects enrolled in the
intensive treatment cohort (aiming at fasting and premeal blood
glucose levels of 70 to 120 mg/dL, postmeal blood glucose levels of
180 mg/dL, and a hemoglobin A.sub.1c [HbA.sub.1c; glycosylated
hemoglobin] in the non-diabetic range) had a reduction in the onset
and/or progression of nephropathy, retinopathy, and neuropathy
relative to subjects enrolled in the conventional treatment
cohort.
[0331] However, despite the development over the past decades of
short- and long-acting insulin analogues that have a more favorable
pharmacokinetic and pharmacodynamic profile compared to older
generation types of insulin formulations, and the improved
technology in insulin delivery devices, many subjects still fail to
achieve the recommended target of HbA.sub.1c of less than 7%. A
recent analysis using data from regional or national registries
from 19 countries in Europe, North America, and Western Australia
comparing glycemic control in >320,000 subjects with T1DM,
showed that overall less than 30% of patients had an HbA.sub.1c of
<7.5% (McKnight 2015). Limitations associated with T1DM
treatments include fear of hypoglycemia, excessive glucose
fluctuations, and body weight increases (Cryer 2003, Larger
2005).
[0332] The reported incidence of hypoglycemia varies considerably
among studies, with greater incidence in both symptomatic and
severe hypoglycemia observed in subjects (Cryer 1989). Furthermore,
studies using continuous blood glucose measurements over prolonged
periods have generally found that the frequency and duration of
hypoglycemia, especially the nocturnal hypoglycemia, is even
greater than what was previously thought (Guillod 2007, Wentholt
2007). Another factor that prevents patients with T1DM from
achieving adequate glycemic is undesirable weight gain which often
occurs as a consequence of intensive insulin treatment, with a
subsequent reduction of glycosuria. It is estimated that the
calories lost in the urine due to the prior poor glycemic control
can account for 70% to 100% of the body weight gained when glucose
control is improved by an intensified insulin regimen (Carlson
1993). Furthermore, by reversing the metabolic state from
catabolism to anabolism, insulin has a lipogenic effect, leading to
increase in body fat (Jacob 2006, Nair 1983), and an anti-catabolic
effect on proteins leading to increases in lean body mass (Torbay
1985).
[0333] Differently from Type 2 Diabetes Mellitus (T2DM) for which
many treatment options are available, insulin remains the mainstay
of therapy for patients with T1DM. Since the discovery of the
therapeutic use of animal insulin in 1922, various types of insulin
have been developed, ranging from the traditional insulins to the
more modern insulin analogues, with insulin lispro being the first
short acting analogue approved by the FDA in 1996, and glargine the
first long-acting analogue approved in 2000. Since then, other
short and long-acting analogues have been approved and their use
has become increasingly common. This is in part due to the need for
achieving near euglycemia in subjects with T1DM and the marked
increase in risk for severe hypoglycemia seen with non-analogue
insulins. Indeed, the vast majority of clinical studies involving
long-acting insulins that have compared their efficacy and
tolerability to Neutral Protamine Hagedorn (NPH) insulin have shown
that generally their once-daily dosing regimen appears to be
similar to NPH in terms of glycemic control and are associated with
a significantly lower rate of hypoglycemia (particularly at night),
and less glucose fluctuation (Ratner 2000, Porcellati 2004,
Hermansen 2004, Raskin 2007). With respect to short-acting
analogues, these are generally similar to regular insulin in
lowering HbA.sub.1c, while they show other important advantages
such as better control in post-prandial hyperglycemia, improved
inter- and intra-patient variability, and reduced risk of
hypoglycemia. Importantly, due to their improved pharmacokinetics
these analogues provide greater flexibility and convenience to the
patients, along with better compliance, as the short-acting
analogues may be injected immediately before or even after a meal
(compared with 30 minutes prior to meals for regular insulin) and
the long-acting analogues once-daily (compared to twice daily for
NPH).
[0334] Given that pramlintide is currently the only non-insulin
agent approved as adjunctive therapy in T1DM and the shortcomings
of insulin monotherapy, an unmet need exists for novel therapies in
T1DM.
Rationale for Studies to Preserve .beta.-cell Function in New Onset
T1D
[0335] As described above, the DCCT was a large study conducted in
subjects with T1D to evaluate the potential benefits of intensive
insulin therapy compared with standard insulin therapy on the
development and progression of long-term complications (DCCT 1993).
In an important analysis of results from the intensive treatment
group from this study, subjects who retained significant residual
.beta.-cell function (as measured by C-peptide .gtoreq.0.2 pmol/mL)
were compared with subjects who did not retain significant
.beta.-cell function (C-peptide <0.2 pmol/mL). Those with
residual .beta.-cell function, relative to those without residual
.beta.-cell function, were found to have a significantly reduced
rate of severe hypoglycemic events and a reduced rate of
progression of microvascular complications (DCCT 1987; DCCT 1998;
Steffes 2003; Palmer 2004). Additionally, recent evaluation by of
data from the DCCT study (Lachin et al) supports short- and
long-term clinical benefits in those with stimulated C-peptide of
.gtoreq.0.2 pmol/mL, and there does appear to be clinical benefits
of maintenance of C-peptide even below the 0.2 pmol/mL
threshold.
[0336] The notion that maintaining endogenous insulin production
has important short- and long-term benefits in those with T1D has
been some of the most important justification for numerous
interventional trials in new onset T1D in the past 1 to 2 decades.
Although an ultimate goal in this field may be "full" remission of
T1D (and thus insulin independence), a more realistic, and likely a
more achievable goal is to prevent destruction of .beta.-cells
present at diagnosis (often considered to be .about.10-20% of
baseline numbers). Even though individuals may still require some
exogenous insulin, metabolic control would be improved and
T1D-associated complications would be lessened. Recent data in T1D
have supported this concept. Specifically, maintaining some graft
function in T1D subjects receiving islet cell transplantation, even
in those who have not achieved or lost full insulin independence,
severe hypoglycemic events are lessened and glycemic control
improved (Agarwal and Brayman 2012, Blau, Abegg et al. 2015). In
the interventional, new onset trial of alefacept in T1D,
individuals in the treatment arm maintained higher levels of
C-peptide production than those in the placebo arm and had
approximately half the incidences of major hypoglycemia (defined by
BG levels of .gtoreq.55 mg/dL), even though insulin independence
was not achieved (Rigby, JCI, 2015). Thus in addition to the
well-accepted notion that preservation of some endogenous insulin
production has important benefits on many long-term, in particular
T1D-associated microvascular damage, there are important immediate
benefits of lessening hypoglycemia in T1D, a condition which poses
the most short-term morbidity and mortality in this disease.
Development Landscape in Type 1 Diabetes
[0337] With the elucidation of the autoimmune basis of T1D and
following successful studies in rodent models, several small-scale
clinical trials were conducted in the 1980s and `90s to evaluate
potent, non-specific immune suppressants in patients recently
diagnosed with T1D (Bougneres, Carel et al. 1988, Glandt and Herold
2004, Herold, Gitelman et al. 2009). At the time of clinical
diagnosis, it is believed a substantial number (perhaps up to 20%)
of .beta.-cells remain but are dysfunctional due to exhaustion and
local inflammation. These residual cells are likely responsible for
the nadir in exogenous insulin often seen in the weeks to months
following diagnosis, known as the "honeymoon" period. Early
clinical trials used non-selective immune suppressants, including
cyclosporine, azathioprine and/or prednisone (Harrison, Colman et
al. 1985, Bougneres, Carel et al. 1988, Cook, Hudson et al. 1989,
Bougneres, Landais et al. 1990). Although insulin independence was
observed in some trials, this effect was lost when immune
suppression was stopped. These studies demonstrated that if the
inflammatory response to .beta.-cells and the immune attack could
be mitigated early in disease, functional beta cells could be
spared.
[0338] A number of trials using other approaches to reverse
diabetes autoimmunity (eg, nicotinamide, anti-oxidants) have been
conducted which, taken together, have not shown efficacy (Chase,
Butler-Simon et al. 1990, Ludvigsson, Samuelsson et al. 2001).
Thus, the only consistent approaches to reverse diabetes
autoimmunity have been in studies using potent immune suppressants
proven in other conditions, which directly impact the activity of
immune cells or their processes. Because of the at-risk population
(ie, children) and therapeutic alternative (ie, insulin), an ideal
therapy would have low immune to non-immune side effects, and
preferably an extended off-therapy effect.
[0339] Over the past 2 decades biologic agents have been developed
that target specific inflammatory mediators and cells and have
significant efficacy in other human autoimmune and inflammatory
conditions. A number of these agents have been able to reverse
autoimmune disease in rodent models, which has provided the
rationale for evaluation in T1D in clinical trials. Studies using
anti-CD3, anti-CD20, LFA-3Ig, and CTLA4-Ig have shown the ability
to slow, but not halt, beta cell loss (Pescovitz, Greenbaum et al.
2009, Gottlieb, Quinlan et al. 2010, Orban, Bundy et al. 2011,
Sherry, Hagopian et al. 2011, Tolerx 2011, Gitelman, Gottlieb et
al. 2013, Moran, Bundy et al. 2013); while IL1-beta blockade,
anti-CD25, and anti-thymocyte globulin have no effect on
.beta.-cell loss (Gottlieb, Quinlan et al. 2010, Gitelman, Gottlieb
et al. 2013, Moran, Bundy et al. 2013). As discussed in more detail
below, only one class of agent, anti-TNF.alpha.s, namely
etanercept, was shown in a small Phase I trial to not only preserve
endogenous insulin production but actually increase it in newly
diagnosed diabetics. Thus, compared to most other autoimmune
conditions, no immunomodulatory agent (biologic or otherwise) has
been shown to be consistently effective in T1D with a safety
profile acceptable for routine use in children, the population that
has the most to benefit from a T1D-disease modifying therapy.
Anti-Tumor Necrosis Factor Alpha Inhibitors
[0340] Tumor necrosis factor alpha (TNF.alpha.) is a principal
proinflammatory cytokine produced primarily by macrophages and T
cells in response to a variety of stimuli and mediates a wide range
of biological activities. It is expressed as a 26 kilodalton (kDa)
type II membrane protein that, upon proteolysis, is released as a
soluble 17 kDa monomer that self-associates into the biologically
active trimeric form. Tumor necrosis factor.alpha. is part of the
TNF ligand superfamily, a group of related cytokines with
overlapping functions that influence cell proliferation and cell
death in processes ranging from development to immune response. As
indicated by its name, TNF.alpha. was initially described as an
inducer of apoptosis with murine tumor cell lines, but more recent
studies suggest chronic inflammation due to TNF.alpha. can lead to
tumor promotion and metastasis.
[0341] The functional activities modulated by TNF.alpha. can
include cell activation leading to proliferation, differentiation,
induction of cytokine and chemokine production and induction of
adhesion proteins, or initiation of programmed cell death. The 2
receptors that engage TNF.alpha. are found on virtually all cell
types. TNF receptor 1 (TNF-R1) (or p55) contains an intracellular
death domain and can signal cytotoxic events, while TNF receptor 2
(TNF-R2) (or p75) appears to be involved in TNF.alpha. signaling in
lymphocytes. The reasons for the complexity of the physiological
response to TNF.alpha. production is multifactorial and include the
biological form of TNF.alpha. present (soluble or transmembrane),
the functional receptor (TNF-R1 and/or TNF-R2), accessory proteins,
and signaling pathways available in the target cell, the tissue in
which it is produced, and the timing and duration of
expression.
[0342] While limited, local, expression of TNF.alpha. is important
in the host inflammatory and protective immune response to injury
and infectious pathogens, chronic expression of TNF.alpha. in
specific organs can lead to significant pathology. High levels of
TNF.alpha. have been implicated in the pathophysiology of diseases
such as rheumatoid arthritis (RA), psoriatic arthritis (PsA),
ankylosing spondylitis (AS), inflammatory bowel disease and T1D.
Inhibition of TNF has been shown to mitigate disease activity in
animal models of arthritis and colitis, and this has led to the
successful clinical and commercial development of anti-TNF.alpha.
agents, including the monoclonal antibodies (mAbs) golimumab,
infliximab, adalimumab, and certolizumab pegol; and the soluble TNF
receptor p75 fragment crystallizable (Fc) fusion protein,
etanercept, for the treatment of immune-mediated inflammatory
diseases such as RA, inflammatory bowel disease, and adjacent
indications.
Mechanism of TNF Blockade in Type 1 Diabetes
[0343] Studies have shown that TNF.alpha. is a proinflammatory
cytokine is critical in the autoimmune process and beta cell
destruction in T1D.(Cavallo, Pozzilli et al. 1991, Rabinovitch
1998) Tumor necrosis factor-a produced by activated macrophages,
dendritic cells, and CD4+T cells promotes inflammation via its
participation in the acute phase response, pro-proliferative
effects, and activation and recruitment of other cells in the
innate and adaptive immune response. Tumor necrosis factor-a,
dendritic cells (DCs), monocytes, and CD4+ T cells are all found in
the insulitic lesion in T1D, and all are implicated in beta cell
inflammation (insulitis) and beta cell killing.(Cavallo, Pozzilli
et al. 1991, Rabinovitch 1998) In preclinical studies, systemic
treatment of non-obese diabetic (NOD) mice (a model of spontaneous
autoimmune diabetes) with TNF.alpha. early in life increases the
frequency and accelerates diabetes onset. Treatment with antibodies
to TNF.alpha. slows the onset, decreases the frequency, and in some
cases fully prevents diabetes.(Rabinovitch 1998) In some
non-diabetes-prone mouse strains (eg, C57BL/6), systemic treatment
with TNF.alpha. results in insulitis.(Rabinovitch 1998) Transgenic
expression of TNF.alpha. in NOD mice islets (NF-.alpha.-NOD), which
express TNF.alpha. early in life, accelerates disease
progression.(Koulmanda, Bhasin et al. 2012) No studies have
specifically used a TNF.alpha. inhibitor to prevent or reverse
diabetes in rodent models of T1D.
[0344] Tumor necrosis factor-.alpha. appears to promote diabetes
autoimmunity by enhancing the recruitment of inflammatory cells to
the islets, activating cells and enhancing autoantigen
presentation.(Rabinovitch 1998, Kodama, Davis et al. 2005) Tumor
necrosis factor-.alpha. activates vascular endothelium,
upregulating MHC I and adhesion molecules.(Argiles, Lopez-Soriano
et al. 1994) In murine models of T1DM, some of the first cells to
infiltrate islets are dendritic cells (DCs).(Argiles, Lopez-Soriano
et al. 1994, Rabinovitch 1998) Dendritic cells and other
antigen-presenting cells (APCs), which are critical for .beta.-cell
antigen presentation to T cells, are activated by TNF.alpha. by up
regulation of MHC I and II and costimulatory molecules.
(Rabinovitch 1998, Kleijwegt, Laban et al. 2010) Tumor necrosis
factor-.alpha. also directly increases MHC I, and synergizes with
IFN.gamma. to upregulate MHC II, on beta cells, both of which
appear to increase their susceptibility to T cell
killing.(Atkinson, Bluestone et al. 2011)
[0345] In addition, there are important non-immune effects of
TNF.alpha. that make blocking it attractive in T1D. Tumor necrosis
factor-.alpha. has direct cytostatic effects and impairs insulin
production and secretion, and it has cytocidal activity, killing
beta cells directly.(Mandrup-Poulsen, Bendtzen et al. 1987,
Kawahara and Kenney 1991, Dunger, Schroder et al. 1995, Rabinovitch
1998) Tumor necrosis factor-.alpha. also impairs insulin signaling
and increases peripheral insulin resistance, which in experimental
models can be reversed by blocking TNF.alpha..(Rabinovitch 1998,
Koulmanda, Bhasin et al. 2012) Patients with new-onset T1D have
elevated serum TNF.alpha. levels compared with those with
long-standing disease or healthy controls.(Cavallo, Pozzilli et al.
1991) There are case reports of patients with T1D who were started
on TNF.alpha. blockers for other autoimmune diseases in whom
insulin requirements dropped due to an apparent increase in insulin
sensitivity.(Yazdani-Biuki, Stelzlet al. 2004, Boulton and Bourne
2007, van Eijk, Peters et al. 2007, Arif, Cox et al. 2010)
Therefore, TNF.alpha. also has potent metabolic effects that may
contribute to diabetes by increasing beta cell stress and
death.
[0346] Data to support a role of TNF.alpha. in T1D, and that
blocking it may have the ability to modify the course of disease in
humans, comes from a report of a small, pilot clinical trial of
etanercept in newly diagnosed T1D conducted by Mastrandrea et
al.(Mastrandrea, Yu et al. 2009) Eligibility included patients aged
3-18 years, .about.6 weeks from diagnosis, who were diabetes
autoantibody (i.e., GAD-65 and/or islet cell antibody) positive,
had normal WBC and platelet counts, and normal liver and renal
function. Patients received etanercept 0.4 mg/kg (maximum 25 mg)
dosed subcutaneously twice weekly for 24 weeks or placebo. The
study enrolled 18 subjects (mean age .about.12.5 years), 10 into
the etanercept arm (0.4 mg/kg SQ twice weekly.times.24 weeks) and 8
into the placebo arm. Patients were followed for 6 months.
Etanercept-treated participants showed lower insulin requirements
and lower HbA1c than placebo participants, and, importantly, a 39%
increase in C-peptide production from baseline assessed by 2-hour
area under the curve (AUC) C-peptide level in response to a
mixed-meal tolerance test (MMTT) was seen. In contrast, placebo
group had a 20% decrease in C-peptide production. There were no
severe adverse events in either group. Three etanercept-treated
patients reported mild, transient paresthesias, but otherwise the
frequency of adverse events (all mild) was similar in the two
groups. Although there has been no follow up on these very
promising initial observations, the Sponsor contends that the data
generated in this study will provides vital rationale for a
potential benefit of TNF.alpha. blockers in T1D and justify further
study in this population.
[0347] The above evidence from preclinical and clinical studies
demonstrates that TNF.alpha. has a critical role in T1D development
and progression. There are immune, direct toxic, and metabolic
effects of TNF.alpha. that suggest that blocking this cytokine is a
very attractive approach to further investigate clinically. The
fact that there are no disease modifying therapies available for
those with T1D, and that there continues to be severe short and
long-term morbidity and mortality even with the best glycemic
control with insulin, supports the notion that there is a
significant unmet need to find therapies to improve the course,
glycemic control, and lives of those with T1D.
[0348] As discussed above, in T1D, like many other autoimmune
disorders, TNF.alpha. appears to play a key role in disease
initiation and progression. The clinical utility of TNF.alpha.
blockade in other autoimmune disorders is well established. There
are a number of FDA-approved agents to block TNF.alpha., including
adalimumab, golimumab, infliximab, certolizumab pegol and
etanercept, and this class of biologic therapies has been the most
extensively evaluated, prescribed and, have been successful in the
treatment of a spectrum of autoimmune diseases in adults and
children such as rheumatoid arthritis, psoriasis, psoriatic
arthritis, ankylosing spondylitis, ulcerative colitis and Crohn's
disease. There are a number of off-label and experimental uses for
TNF.alpha. inhibitors. This includes allogeneic islet transplant
regimens, where adding TNF.alpha. inhibitors have shown to improve
graft survival, perhaps reflecting both beneficial immunologic and
metabolic effects of blocking TNF.alpha. on beta cell survival in
this indication. Golimumab, infliximab, certolizumab pegol and
adalimumab are monoclonal antibodies that bind TNF.alpha.; whereas
etanercept is a fusion protein consisting of the TNF receptor bound
to an IgG tail and also binds lymphotoxin alpha (LT.alpha.).
Lymphotoxin alpha appears to have a role in rodent autoimmune
diabetes, but has not been shown to be directly involved in human
T1D. With respect to clinical use in children, etanercept has been
FDA-approved in children 2 years old and above with JIA since 1999,
infliximab was approved in children 6 years old and above with
Crohn's disease and ulcerative colitis in 2009 and 2011,
respectively and adalimumab was approved for JIA in those 2 years
old and above in 2008 and Crohn's disease in those 6 years old and
above in 2014. Golimumab has been studied in over 200 children age
2 years old and above with JIA and pediatric UC. In addition to
efficacy, the safety profiles of these agents in children are well
documented and similar to the safety profile in adults across
numerous indications.
[0349] In summary, there is strong pre-clinical and clinical data
implicating TNF.alpha. in the immune and metabolic pathogenesis of
T1D, and that blocking TNF.alpha. has the ability to interfere with
diabetes autoimmunity and preserve beta cells. There is almost 2
decades of successful clinical experience of TNF.alpha.-blockers
with in a variety of human autoimmune diseases, including in
children as young as 2 years old. T1D continues to be a significant
burden on individuals, their families and society and there is a
significant unmet need for a disease modifying therapy in T1D which
can assist in maintaining endogenous beta cell function and lessen
the short- and long-term sequelae of this disease.
Clinical Experience with Golimumab
Adult Program
Description of Overall Development Program Across Indications
[0350] SIMPONI.RTM. (golimumab) administered as a SC injection is
currently approved in the US for adults for the treatment of
moderately to severely active rheumatoid arthritis (RA) in
combination with methotrexate (MTX); active psoriatic arthritis
(PsA), alone or in combination with MTX; active ankylosing
spondylitis (AS); and moderately to severely active UC in patients
with an inadequate response to or intolerance of prior treatments
or requiring continuous steroid therapy.
[0351] The approved dosage in the adult rheumatology indications is
50 mg administered by SC injection once a month. The approved
dosage for adults with UC is a 200 mg SC injection at Week 0
followed by 100 mg SC at Week 2 as induction (200 mg.fwdarw.100
mg), followed by maintenance therapy of 100 mg SC q4w.
Established Safety Profile
[0352] Long-term safety (safety extensions for up to 5 years) has
been evaluated in .studies for RA, AS, and PsA, and a long-term
extension of approximately 4 years of follow-up has been completed
in UC. More than 11,000 subjects have been exposed to golimumab in
clinical studies, and since the product launch, another estimated
393,000 patients have been exposed to golimumab worldwide as of 06
Oct. 2015. (A listing of the all clinical studies in the program,
along with the number of subjects exposed in each study is provided
in Table 8).
[0353] The safety profile of golimumab is consistent with drug
products in the TNF.alpha. inhibitor class.
[0354] The ADRs observed for golimumab in clinical studies are
presented in Table 7.
[0355] The ADR frequencies in Table 7 are based on data from 5,717
golimumab-treated subjects in the Phase 2 and 3 clinical studies:
3,090 subjects in RA studies (C0524T02, C0524T05, C0524T06,
C0524T11, C0524T28, C0524T12, and CNTO148ART3001), 394 subjects in
the PsA study (C0524T08), 564 subjects in the AS studies (C0524T09,
C0524T29), 1,245 subjects in UC studies (C0524T16, C0524T17, and
C0524T18), 231 subjects in the severe, persistent asthma study
(C0524T03), and 193 subjects with active nr-axial SpA (P07642
[MK-8259-006]).
[0356] The ADRs listed in the table below are classified according
to frequency and SOC. The frequency categories are defined in the
footnote of the table as Very common, Common, Uncommon, Rare, Very
rare, and Not Known.
TABLE-US-00007 TABLE 7 Summary of golimumab adverse reactions in
clinical studies Infections and infestations Very common: Upper
respiratory tract infection (nasopharyngitis, pharyngitis,
laryngitis, and rhinitis) Common: Bacterial infections (such as
cellulitis), lower respiratory tract infection (pneumonia), viral
infections (such as influenza and herpes), bronchitis, sinusitis,
superficial fungal infections, abscess Uncommon: Sepsis including
septic shock, opportunistic infections (invasive fungal infections,
bacterial, atypical mycobacterial and protozoal), arthritis
bacterial, pyelonephritis Rare: Hepatitis B reactivation,
histoplasmosis, coccidioidomycosis, pneumocystosis, tuberculosis,
bursitis infective Neoplasm benign and malignant Rare: Lymphoma,
leukemia Not known: Pediatric malignancy* Investigations Common:
Alanine aminotransferase increased, aspartate aminotransferase
increased Uncommon: Neutrophil count decreased Blood and lymphatic
system disorders Common: Anemia Uncommon: Leukopenia,
thrombocytopenia, pancytopenia Immune system disorders Common:
Autoantibody positive, non-serious allergic reactions Nervous
system disorders Common: Dizziness, paraesthesia Rare:
Demyelinating disorders (central and peripheral) Cardiac disorders
Rare: Congestive heart failure (new onset or worsening) Vascular
disorders Common: Hypertension Rare: Vasculitis (systemic)
Respiratory, thoracic and mediastinal disorders Uncommon:
Interstitial lung disease Gastrointestinal disorders Uncommon:
Constipation Skin and subcutaneous tissue disorders Common: Rash,
alopecia Uncommon: Psoriasis: new onset, palmar/plantar, and
pustular Rare: Vasculitis (cutaneous) Musculoskeletal and
connective tissue disorders Rare Lupus-like syndrome General
disorders and administration site conditions Common: Pyrexia,
injection site reaction (injection site erythema, urticaria,
induration, pain, bruising, pruritus, irritation, paraesthesia)
*Observed with other TNF.alpha. blockers, but not observed in
clinical studies with golimumab
Pediatric Program
Description of Overall Pediatric Development Program Across
Indications
[0357] Golimumab is not currently approved for the use in pediatric
patients. However, a study of SC golimumab in children with pJIA
(CNTO148JIA3001) was conducted and a clinical development program
with subcutaneously administered golimumab in pediatric UC is
currently ongoing (CNTO148UCO1001).
[0358] CNTO148JIA3001 was a Phase 3, multicenter, double-blind,
randomized withdrawal study with the primary objective to assess
the clinical efficacy of SC administration of golimumab in
pediatric subjects (ages 2 to less than 18 years) with pJIA
manifested by .gtoreq.5 joints with active arthritis despite
methotrexate (MTX) therapy for .gtoreq.3 months. The CNTO148JIA3001
study consisted of an open label phase where patients received SC
golimumab 30 mg/m.sup.2 q4w +MTX for 16 weeks, followed by a
randomized withdrawal phase where patients who achieved an American
College of Rheumatology (ACR) pediatric (Ped) 30 response at Week
16 received either golimumab 30 mg/m.sup.2 +MTX or placebo +MTX q4w
through Week 48. There was also a long-term extension in which the
median follow-up was approximately 2 years. A total of 173 subjects
(75.7% female; 24.3% male) were enrolled in the study, and 154
subjects entered the randomized withdrawal phase at Week 16. The
mean age was 11.2 years (52.0% aged 12 to 17 years; 48.0% aged 2 to
11 years) and the mean weight was 43.1 kg.
[0359] CNTO148UCO1001 was a Phase 1b, multicenter, open-label study
to assess the PK, safety, and efficacy of golimumab in children
with pediatric UC. This multicenter, open-label study enrolled
pediatric subjects aged 2 through 17 years with moderately to
severely active UC who demonstrated an inadequate response to,
failed to tolerate, or had medical contraindication to conventional
therapies (ie, IV or oral corticosteroids or the immunomodulators
AZA or 6-MP), and were naive to anti-TNF.alpha. agents. Subjects
received weight-based dose regimens of SC golimumab as follows:
[0360] Subjects with body weight <45 kg: 90 mg/m.sup.2 at Week 0
and 45 mg/m.sup.2 at Week 2, and 45 mg/m.sup.2 q4w starting at Week
6 among Week 6 responders [0361] Subjects with body weight >45
kg: 200 mg at Week 0 and 100 mg at Week 2, and 100 mg q4w starting
at Week 6 among Week 6 responders
[0362] The study was divided into 2 parts: the PK portion through
Week 14, and the study extension through Week 126. The 14-Week PK
portion of Study 1 (CNTO148UCO1001) has been completed and the
study extension is ongoing. A total of 35 subjects (51.4% female;
48.6% male) were enrolled in study. The mean age was 13.4 years
(71.4% aged 12 to 17 years; 28.6% aged 2 to 11 years) and the mean
weight was 51.7 kg.
Established Safety Profile
[0363] Golimumab was well tolerated in pediatric subjects of 2 to
<18 years of age. In general, the safety profile of golimumab in
the pJIA and pediatric UC studies including the type and frequency
of the adverse reactions seen was consistent with the known safety
profile for the adult populations studied and consistent with other
TNF.alpha. inhibitors. No new safety signals were observed.
Proposed Phase 2A New Onset T1D Study--Cnto148Dml2001
[0364] The Sponsor is planning a Phase 2a new onset T1D study in
newly diagnosed T1D patients to evaluate the effect of SIMPONI.RTM.
on the preservation of .beta.-cell s for maintenance of endogenous
insulin. The results of this study will provide key information on
whether SIMPONI.RTM. via TNF.alpha. blockade impacts T1D disease
progression and support further development in new onset disease as
well as in a "pre-disease" state, prior to onset of symptoms and
requirement for exogenous insulin. The key aspects of this study
are outlined below.
Study Design
[0365] This is a Phase 2a, randomized, double-blind,
placebo-controlled, parallel-group, multicenter study of golimumab
in subjects with T1D. Approximately 81 subjects of 6 to 21 years of
age will be randomly assigned in a 2:1 ratio to receive golimumab
or placebo, administered subcutaneously (SC). Study subjects who
weigh <45 kg who are randomized to the golimumab treatment group
will receive an induction dose of SC golimumab 60 mg/m.sup.2 at
Weeks 0 and 2 followed by a maintenance dose of 30 mg/m2 at Week 4
and q2w through Week 52. Study subjects who weigh .gtoreq.45 kg who
are randomized to the golimumab treatment group will receive an
induction dose of SC golimumab 100 mg at Weeks 0 and 2 followed by
a maintenance dose of SC golimumab 50 mg at Week 4 and q2w through
Week 52 (Section 0). Subjects randomized to the placebo treatment
group will receive a SC placebo injection q2w through Week 52 (FIG.
18). To facilitate recruitment and retention of subjects, the
Sponsor will allow self-administration of study agent at home.
[0366] During the study, all subjects will receive intensive
management of their diabetes with exogenous insulin. Subjects, and
when applicable their caretakers, must agree to follow the current
recommendations of tight glycemic control with specific HbA1c
targets as defined by the American Diabetes Association. These
current recommendations are intended to achieve glucose levels that
appear to decrease some of the short- and long-term sequela of T1D.
Specific to this study, this will include a HbA1c target of less
than 8% for those aged 6 to 12 years, less than 7.5% for those
between from age 13 through 18 years, and less than 7% for those 19
years old and above. The subject's glycemic control will be
monitored by the subject's primary care physician. In addition
HbA1c and other parameters of glycemic control will be assessed at
screening, and during the study.
Study Population
[0367] Males and females 6 through 21 years of age with newly
diagnosed T1D will be enrolled in this study. Participants will
meet accepted criteria for enrollment in new onset T1D studies
including meeting the current ADA definition of T1D, being positive
for at least 1 of 5 recognized T1D autoantibodies, showing evidence
of residual .beta.-cell function (defined by a peak c-peptide of at
least 0.2 pmol/mL following a mixed-meal tolerance test), and
randomization in the trial within 100 days of T1D diagnosis.
Exclusion criteria will focus on identifying individuals who may be
at any particular risk due to immune or infectious risks if
included in the trial.
[0368] The age range was chosen for the following reasons:
Unique attributes of TID in children and young adults compared to
older adults:
[0369] Recent data have confirmed that there are important
differences in T1D disease in children and younger adults (ie,
through .about.age 21) and older adults. It is accepted that there
are a number of clinical differences in the presentation and course
of disease in younger and older individuals with newly diagnosed
T1D, including what can be described as a more aggressive
presentation (ie, more rapid need for full insulin replacement) and
initial course in the younger group. In a recent report by
Greenbaum et al that conducted a thorough evaluation of the natural
history of c-peptide decline in placebo subjects in a number of
recent new onset T1D studies, the findings showed similar rates of
c-peptide decline in those .about.7-21 years old with a more rapid
decline than in those >21 years old, strongly suggesting that
there is a different immunopathology in these "younger" and "older"
individuals. In addition, to support this contention, in some
immunotherapeutic trials in T1D there is different efficacy in the
younger and older individuals. For example, alefacept (LFA3-Ig) and
abatacept (CTLA4-Ig) appear to have preferential effects in younger
individuals (<18 to 21 years of age) while thymoglobulin appears
to have a beneficial effect only in older individuals (>21 years
of age; (Gitelman, Gottlieb et al. 2013, Orban, Bundy et al. 2014,
Rigby, Harris et al. 2015). Due to the nature and course of T1D and
the lifelong and cumulative morbidity and mortality associated with
this disease, those that will be most positively impacted by a
therapy that slows or stabilizes disease progression are children
and young adults. Because of the aforementioned differences in
disease in this population compared with older adult individuals,
the direct study of children and young adults with T1D is essential
to most appropriately develop a disease modifying therapy that will
be most impactful in this disease.
Data Supporting a Prospect of Benefit of TNF.alpha. Blockers in New
Onset T1D in Children:
[0370] The Sponsor recognizes that demonstrating a clinical benefit
with a therapeutic intervention in an adult population is sometimes
desired prior to evaluation in a pediatric population. However,
from the small etanercept pilot study (also referenced above),
conducted by investigators at the University of Buffalo showed that
there were beneficial effects on c-peptide production, HbA1c and
exogenous insulin use in children 7-18 years old (protocol allowed
for children as low as 3 years of age) with a 24 week course of the
TNF.alpha.-blocker etanercept. Although this was a small trial (18
total participants with 10 receiving etanercept), the results were
extremely promising, showing that there was on average an increase
in endogenous insulin production with etanercept at 24 weeks versus
at time of enrollment, during a time when there was loss in
c-peptide in the placebo group. Given these results, and
considering the extensive experience with golimumab and other
TNF.alpha. blockers in both adult and pediatric indications, The
Sponsor contends that the prospect of benefit for
TNF.alpha.-blockade in children with newly diagnosed T1D has been
demonstrated and therefore it is appropriate to evaluate golimumab
in this particular population as a disease-modifying therapy.
Extensive Safety and Efficacy Experience of TNF.alpha. Blockers
Including Golimumab
[0371] The Sponsor recognizes that some of the intended study
population, in terms of age, may be considered a particularly
susceptible population to evaluate immune therapies. We believe
that in the case of golimumab itself and as a member of the class
of TNF.alpha.-blockers, there is robust safety (and efficacy)
experience in children down to the age of 2 in other autoimmune
diseases. Etanercept has been FDA-approved in children 2 years old
and above with JIA since 1999. Infliximab was approved in children
6 years old and above with Crohn's disease and ulcerative colitis
in 2009 and 2011, respectively. Adalimumab was approved for JIA in
those 2 years old and above in 2008 and Crohn's disease in those 6
years old and above in 2014. Golimumab has 1been studied in over
200 children ages 2 years old and above with JIA and is currently
under registration for this indication in Europe. In addition all
of the above agents are approved for a wide variety of autoimmune
conditions in adults, including rheumatoid arthritis, Crohn's
disease, ulcerative colitis and psoriasis. Thus, the safety and
side effect profiles of TNF.alpha.-blocking agents, including
golimumab, are well established not only in adults, but
specifically the pediatric population, likely much more than any
other immunetherapy that has been evaluated in T1D.
[0372] Taken together, there is strong rationale to focus the
golimumab study in new-onset T1D on younger individuals and include
only children and young adults 6 to 21 years old. Not only is there
robust specific efficacy and safety data from golimumab and the
class of TNF.alpha.-blockers in pediatric autoimmunity, but there
has been extremely promising data in this younger age range that
TNF.alpha.-blockade has the ability to slow, if not reverse, the
loss of .beta.-cells in those newly diagnosed T1D, thus meeting a
critical bar of a "prospect of benefit" of TNF.alpha.-blockade in
this age group. It is well agreed, and shown, that T1D diabetes is
different in younger versus older individuals and that there can be
a significantly different responses to immune therapy in these
groups, where efficacy (or lack thereof) in one group may actually
be misinformative to the other. The Sponsor is choosing the upper
age "cut-off" as age 21, as many of these studies appears to show a
differentiating break in the "younger" and "older" disease at this
age. Age 6 has been an accepted lower age of enrollment in other
new onset T1D studies (ie, abatacept and canakinumab) and is also
the age cutoff for approved UC in children and inclusive of the JIA
age range for the class of approved TNF.alpha.-blockers.
Dose Selection
[0373] The proposed dosing of golimumab in this trial integrates
knowledge regarding disease-specific considerations in T1D, an
understanding of the comparative efficacy of TNF.alpha. inhibition
of etanercept in the aforementioned pilot trial in children with
T1D, and the sponsor's experience with golimumab. In T1D, the
destruction of .beta.-cells is irreversible and appears to be rapid
at the onset of clinical disease. For more inflammatory
immune-mediated diseases, higher or more frequent induction doses
followed by lower maintenance doses are often used, such as with
the use of anti-TNF.alpha. agents for Crohn's disease and
ulcerative colitis..sup.7,8,9 For T1D, there is also a need to
quickly suppress disease activity to prevent further destruction of
.beta.-cell present at enrollment in study. Given that golimumab
steady-state concentrations are generally established after 12
weeks, induction doses should be employed followed by a maintenance
dosing regimen in order to achieve steady-state concentrations
earlier to offset further .beta.-cell loss. Data from previous
adult and pediatric studies were evaluated along with population PK
and mechanistic PK/target engagement (TE) modeling to determine the
proposed dosing regimen for this Phase 2a new onset T1D study.
[0374] Considering the anticipated need for induction and
maintenance dosing in T1D, various BSA-adjusted dose regimens were
explored via simulation with a population PK model and a
mechanistic PK/TE model that assesses free TNF.alpha. suppression.
The population PK model for golimumab was based on an established
polyarticular JIA model in which all subjects were on concomitant
methotrexate (MTX). Since MTX has been previously shown to affect
golimumab exposure.sup.5 and patients with T1D are not expected to
be concomitantly treated with methotrexate, a 36% increase in
golimumab clearance was accounted for in the simulations (FIG. 19).
Analysis of subjects 6 to 21 years old using the CDC growth charts
for height and weight were performed comparing the proposed T1D
dosing regimen to JIA (FIG. 19 Panel A) and ped UC (FIG. 19 Panel
B) with dosing regimens that have already been studied in the
pediatric population. The simulations were based on the established
JIA population PK model with faster clearance of golimumab (36%)
due to non-co-administration of MTX in the T1D population. For a
child, golimumab 30 mg/m.sup.2 (50 mg/1.67 m.sup.2) would be
approximately equivalent to 50 mg dose for an adult subject
weighing 60 kg (with BSA of 1.67 m.sup.2). Thus the 30 mg/m.sup.2
dose is designed to be similar to the 50 mg dose in adults and the
60 mg/m.sup.2 dose to be similar to the 100 mg dose in adults.
Based on these simulations, PK exposure for the proposed T1D dosing
regimen is expected to be between the JIA and ped UC exposure (both
simulated without MTX), though the q2w maintenance dosing interval
will result in slightly higher trough concentrations. In adults,
golimumab 50 mg q4w was the minimum effective dosing regimen for
the treatment of RA, PsA, or AS. Due to the absence of concomitant
MTX in T1D subjects, it is expected that the 30 mg/m.sup.2 q4w
dosing regimen that is the pediatric equivalent to 1the adult 50 mg
q4w dose may not result in sufficient systemic exposure for
suppressing TNF.alpha.; therefore the Sponsor contends a higher
dose or more frequent dosing interval should be studied.
[0375] The mechanistic PK/TE model incorporated PK exposure from
the above population PK model (with 36% higher clearance without
concomitant MTX) paired with a target-mediated drug disposition
(TMDD) model was used to assess the interaction between drug and
target and to simulate the suppression of TNF.alpha. after
anti-TNF.alpha. administration (FIG. 20). The PK/TE model was
developed based on the assumption that the etanercept dosing
regimen tested in T1D results in adequate TNF.alpha. suppression
given the positive results previously observed..sup.6 The
TNF.alpha. kinetic parameters were obtained from preclinical
studies and allometric scaling, and the same set of TNF.alpha.
kinetics parameters were used to compare the effect of golimumab
and etanercept. TNF.alpha. suppression in the systemic circulation
was also assumed to be representative of that in the pancreas. The
golimumab dosing regimen was designed to approximate the extent of
TNF.alpha. suppression following the etanercept dosing regimen in
the pilot trial in children with new onset TID,.sup.6 (Mastrandrea,
2009), after accounting for the differences in PK and TNF.alpha.
binding affinity between golimumab and etanercept. The PK/TE model
suggested that an induction dose regimen of 60 mg/m.sup.2 SC (to a
maximum of 100 mg) at Week 0 and Week 2 followed by a maintenance
dose regimen of 30 mg/m.sup.2 SC (to a maximum of 50 mg) q2w or 60
mg/m.sup.2 SC (to a maximum of 100 mg) q4w allows suppression of
TNF.alpha. to a level closer to approximating that of etanercept,
in contrast to the 30 mg/m.sup.2 SC q4w maintenance dose (FIG. 20).
The 30 mg/m.sup.2 q2w and 60 mg/m.sup.2 q4w maintenance dose
regimens would have the same overall exposure (AUC) and similar
TNF.alpha. suppression; however, the 60 mg/m.sup.2 q4w regimen
would have higher peak/trough concentration fluctuation and thus
more fluctuation on suppression of TNFa. It is known that
TNF.alpha. has direct cytostatic and cytocidal effects on beta
cells. Thus the TNF.alpha. elevations that would be expected to
occur during the troughs with the higher, but less frequent dosing,
may be damaging to residual beta cells. As the golimumab exposure
of these dosing regimens may be considered equivalent, the 30
mg/m.sup.2 SC (to a maximum of 50 mg) q2w dose regimen was
preferred as the simulations have shown the q2w maintenance dosing
interval will result in slightly higher trough concentrations
resulting in a smaller peak/trough fluctuation in an attempt to
optimally protect .beta.-cells from the direct effects of
TNF.alpha..
[0376] The safety and efficacy of golimumab has been extensively
characterized in subjects with RA, PsA, AS and UC. In a Phase 2
study in subjects with RA, 4 different dosing regimens were
evaluated (50 mg q2w or q4w and 100 mg q2w or q4w) of which all
doses tested were generally well tolerated and effective in
maintaining clinical response through Week 52. In pJIA, a 30
mg/m.sup.2 q4w dosing regimen was studied up to a maximum of 50 mg
(the approved adult RA dose). In ped UC, subjects below 45 kg
received SC induction doses of 90 mg/m.sup.2 (to a maximum of 200
mg) at Week 0 and 45 mg/m.sup.2 (to a maximum of 100 mg) at Week 2
followed by a maintenance dosing regimen of 45 mg/m.sup.2 (to a
maximum of 100 mg) SC q4w while subjects 45 kg and above received
induction doses of 200 mg at Week 0 and 100 mg at Week 2 followed
by a maintenance dose of 100 mg q4w. To date, the dosing regimens
studied were well tolerated overall with no new adverse drug
reactions identified with frequency, type and seventies similar to
those observed in the adult rheumatology and IBD studies. In
conclusion, considering disease specific issues of T1D,
pharmacologic comparisons of golimumab and etanercept and specific
clinical experience with golimumab, the 60 mg/m.sup.2 SC (to a
maximum of 100 mg) at Week 0 and Week 2 induction followed by a 30
mg/m.sup.2 SC (to a maximum of 50 mg) q2w was selected as the
recommended dose regimen. A weight cut-off (45 kg) will also be
studied such that patients over the weight cut-off will receive
golimumab from the already approved adult PFS presentations. The
Sponsor used both disease- and therapy-specific considerations to
develop this dosing regimen in an attempt to give this
proof-of-concept trial the best opportunity for success. Although
not a regimen specifically used in the past, the proposed T1D
dosing regimen will achieve golimumab exposures observed between
the aforementioned HA and UC dosing in children (which the Agency
has supported for study in overlapping age ranges) and thus
mitigate safety concerns with this specific approach. If this trial
is successful, considerations for dose ranging will be explored in
subsequent clinical studies.
Duration of Treatment
[0377] Subjects will receive SC golimumab or placebo through Week
52. In part, a goal of this trial is to reproduce and extend
findings of the 24 week pilot clinical trial of etanercept in T1D
showing the .beta.-cell sparing by neutralizing TNF.alpha.. Due to
the natural history of .beta.-cell loss following diagnosis
documenting a statistically significant and clinically meaningful
positive effect of a .beta.-cell sparing agent in a trial of a
reasonable size is most probable at approximately 1 year, and an
accepted major (including primary) endpoint is the provoked
c-peptide production at 12 months. In approved indications,
golimumab is used as a chronic therapy and thus the proposed length
of treatment is consistent with clinical use of this agent. Through
a year of on-therapy evaluation, with an additional off-therapy
evaluation, we believe we will obtain important efficacy and safety
data to help guide further clinical development of this therapy in
T1D.
Rationale and Selection of Major Endpoints
[0378] The major trial endpoints in study CNTO148DML2001 will be
consistent with those used and accepted by leading T1D research
networks (including T1D TrialNet and the Immune Tolerance Network)
and cited by major health authority guidelines (including those of
the FDA and EMA). Specifically, the primary endpoint is to
stimulate c-peptide response (4h AUC) following a mixed-meal
tolerance test at Week 52, as an objective measure of endogenous
insulin production. As this will be a placebo-controlled trial, a
positive study will be defined as showing a statistically
significant difference in the c-peptide AUC in the active versus
placebo treatment groups at Week 52. Provoked c-peptide evaluations
will also be conducted at approximately Weeks 13, 26, 39, 78, and
104 as secondary endpoints. The goal of these evaluations is to
obtain insight on the time course of the effect of golimumab on
.beta.-cell preservation (Weeks 12, 26, and 38) and the off-therapy
durability of response (Weeks 78 and 104 evaluations). Major
secondary endpoints will evaluate other potential positive effects
on glycemic control (HbAlc), insulin use (in U/kg/day), and rates
of hypoglycemia (including levels .ltoreq.70, 55, and 35
mg/dL).
Safety Monitoring
[0379] In addition to evaluating the efficacy of golimumab in
preventing the continued loss of .beta.-cell function, there will
be extensive safety evaluation. As an immune modulator, the focus
of these evaluations will be to determine if there are increase
risks of infection or untoward effects on immune status. This will
include careful documentation of signs or symptoms of local or
systemic infections during study visits and at home (using patient
reported outcomes). The Sponsor will also be monitoring white blood
cell counts and for indications of dampened immune response via
regular evaluation of EBV and CMV status. Due to the experience of
golimumab and other TNF.alpha. blockers, the Sponsor anticipates
the likelihood of non-immune side effects to be low, but we will be
monitoring for any clinical or chemical evidence of such effects
via physical examinations and laboratory evaluations including
renal function tests, liver function, and hematologic tests. The
goal of this project is to determine if there is a beneficial
effect on the progression of T1D, and although not expected, the
Sponsor will be able to use the evaluations for this to determine
if there are endocrinologic adverse effects, such as increased
rates of hypoglycemia, poorer glycemic control or more rapid loss
of endogenous beta cell function.
[0380] An independent, external Data Monitoring Committee (DMC)
will be established to monitor data on an ongoing basis to ensure
the continuing safety of the subjects enrolled in this study. The
committee will meet periodically to review interim data. After the
review, the DMC will make recommendations regarding the
continuation of the study. The safety reviews will focus on
particular AEs, SAES, and mortality.
[0381] SAE reports will be provided to the DMC members on an
ongoing basis. The DMC will have access to unblinded data and
review tabulated safety summaries (if appropriate) and any
additional data that the DMC may request during the conduct of the
study. No formal statistical hypothesis testing is planned. In
addition, during the study, the Sponsor's study responsible
physician (or designee) will regularly review blinded safety data
from the sites and notify the DMC and appropriate Sponsor personnel
of any issues. The protocol to be submitted with the
Investigational New Drug (IND) Application to the Agency will
describe all safety assessments and monitoring to be performed in
this study and the makeup and roles and responsibilities of the DMC
including specific stopping rules.
[0382] One important safety measure also includes exclusions of
subjects who have immune suppression due to concurrent disorders or
therapies, and exclude individuals with existing or history of
significant infections, including tuberculosis.
Methods of Administration
[0383] Given the proposed regimen of q2w dosing during the
maintenance portion of the Phase 2a study, the option for at-home
administration is expected to aid in a patient's routine (ie, not
needing to visit the site of care for every dose) and also increase
study enrollment and retention. If a patient or caregiver is to
perform at home administration, he/she should be instructed in
injection techniques, and their ability to inject subcutaneously
should be assessed to ensure the proper administration. In
addition, it is recommended that the first self-injection or
caregiver injection be performed under the supervision of a
qualified healthcare professional.
Presentation Proposed for Use in the Phase 2a New Onset T1D
Study
Ultrasafe
[0384] For pediatric subjects with body weight kg, golimumab will
be administered subcutaneously using the 50 mg PFS-U device, which
is already approved for use in adults. For additional details, see
below and FIG. 21.
SIMPONI.RTM. UltraSafe Passive.RTM. Needle Guard (PFS-U)
[0385] The UltraSafe is a manually-operated, single-use, disposable
needle guard system that is an accessory to a prefilled syringe and
is intended for use as a safety mechanism to reduce the occurrence
of accidental needlesticks to healthcare professionals and patients
or their caregivers after administration and during disposal of a
used prefilled syringe. The UltraSafe accepts either a 0.5 mL or a
1.0 mL PFS. There is no direct drug product contact with the device
whatsoever, either during assembly or use.
[0386] The device's clear plastic construction and the design of
the viewing slot permit visualization of the syringe. The passive
nature of the device permits normal needle insertion and when the
plunger stopper of the syringe is fully advanced and the drug dose
is delivered, the spring-aided guard is released. The guard
automatically advances over the syringe and needle as the user
relaxes their grip until it latches in a locked position.
[0387] The features of the UltraSafe are illustrated in FIG. 21
VarioJect
[0388] The Sponsor is developing a pediatric presentation known as
the VarioJect as a platform device across multiple pediatric
programs to facilitate BSA-dosing, and is planning to utilize the
VarioJect device for this study. The Sponsor has previously
discussed the data required to support the registration of the
VarioJect device in other pediatric programs for SIMPONI.RTM. which
include actual use and label comprehension assessments, data from a
human factor study, and additional device performance data.
[0389] Dosage and device selection charts (Table 9) will be
developed to allow healthcare providers, caregivers, and/or
pediatric subjects (as applicable) to determine the corresponding
absolute milligram (mg) dose and the combination of injection
devices to be used.
SIMPONI.RTM. VarioJect
[0390] The VarioJect device would be intended for delivery of a
single dose of drug, based on the BSA dose regimen, ranging from
doses of 10 mg to 45 mg, in 5 mg increments. The VarioJect device
is designed to be assembled with the same 1 mL Becton Dickson Hypak
syringe containing 0.5 mL of SIMPONI.RTM. drug product (PFS) that
has been used in the already approved SIMPONI SmartJect
autoinjector and PFS-U. Note that the VarioJect device has no
contact with the drug product, and therefore, the VarioJect device
has no effect on the biochemical properties or stability of the
drug product.
[0391] The VarioJect device has been developed as a platform
technology by Ypsomed, Holding A G, Switzerland, an experienced
supplier of prefilled pens for other indications. The device has
been designed in accordance with the design control requirements of
the Quality System regulation, 21 CFR Part 820.
[0392] The overall configuration of the VarioJect device and its
features are depicted in FIG. 22.
[0393] FIG. 23 shows the device at the different stages of use: the
device is primed, dose settings are selected by turning the plunger
to the desired fixed dose, and the dose is administered by pushing
the plunger.
[0394] To use the device, the user first removes the cap (FIG. 24,
Step B), then primes the VarioJect by tapping the bubbles (visible
though the viewing window as shown in FIG. 24, Step C) to the top
of the syringe and pressing on the plunger to remove the air (FIG.
24, Step C). The user then dials the plunger to select the
appropriate dose (FIG. 24, Step D). In the next step, the user
presses the device against the skin at approximately a 90 degree
angle, causing the needle guard to retract and the needle to be
inserted into the selected SC injection site (FIG. 24, Step E), and
then pushes the plunger to deliver the dose (FIG. 24, Step F).
Following delivery, the user removes the device, allowing the
needle guard to passively extend and lock over the needle, offering
protection against accidental needle sticks (FIG. 24, Step F).
After the dose is administered, the plunger locks in the down
position, preventing reuse of the device.
[0395] The pen is designed to deliver between 0.10 mL to 0.45 mL in
0.05 mL increments. The requirement on dosage accuracy was
established based on the ISO 11608-1 2012 Needle-based injection
systems for medical use, Requirements and test methods, Part 1:
Needle-based injection systems, and USP 31 General
Requirements/Injections. The technical design requirement for
delivery accuracy is that the pen must deliver the dialed dose
-0.00/+0.05 mL, where 0.05 mL is the minimum increment. The needle
protrusion distance of a nominal 4.5 mm limits the injection depth
to subcutaneous tissue.
[0396] The device has a number of features to help ensure that it
is used properly and safely. An orange priming band and white arrow
indicating that the plunger should be pushed serve to remind the
user that the device must be primed before use. The orange priming
band disappears post-priming, indicating that this step has been
completed, and the dose cannot be selected until the device has
been primed. Graphics on the plunger align with the dose selection
notch, clearly indicating the dose that is being selected, and
detents provide tactile feedback to the user that the device is
properly aligned. After dose administration, the plunger locks in
the down position, and the dose that was delivered locks into the
dose notch, both confirming and providing a record of the dose that
was delivered. Additionally, the lock safeguards against the
potential for unauthorized reuse of leftover product. When the pen
is removed from the injection site, the needle guard automatically
extends and locks out. This passive needle safety feature aids in
reducing the potential for accidental needle sticks and also
minimizes the visual exposure of the needle to some patients and
caregivers who may have a fear of needles.
Development of the VarioJect
[0397] Design and development of the device were guided by ISO
11608-1 2012 Needle-based injection systems for medical use,
Requirements and test methods, Part 1: Needle-based injection
systems as well as FDA Draft Guidance: Technical Considerations for
Pen, Jet, and Related Injectors Intended for Use with Drugs and
Biological Products.
[0398] Testing will include bench tests ensuring accurate delivery
of the drug product as well as other suitability and Human Factors
studies.
[0399] Early studies have been conducted on form, features, and
general usability of the device. One round of ethnographic research
was conducted, in which the Sponsor observed and interviewed
parents and children, where the child was taking insulin or growth
hormone injections. In addition, 5 formative human factors studies
including parents of pediatric subjects, caregivers, and children
were conducted to test and refine the design. A draft picture-based
IFU was developed and tested along with the device concepts.
Results from the testing were positive, generally confirming the
overall form of the selected device design and the IFU. Some design
enhancements were incorporated to reduce use errors, and the
updated design was retested with users. For example, the thumb rest
was added to the plunger to help users understand the orientation
in which the device should be held, and the flange geometry was
adjusted to allow for preferred grips during use.
Future Development of the VarioJect
[0400] In preparation for clinical studies, the Sponsor has
completed all verification and validation testing to assess safety,
usability, and performance of the VarioJect device. Although the
Sponsor does not anticipate any significant design changes to the
device used for clinical studies, the Sponsor does intend to
enhance the robustness of the VarioJect design based on findings
during routine development efforts. During the Simulated Use Safety
study, although the study successfully demonstrated the
functionality of the sharps injury prevention feature of the
device, it was noted that there were rare instances where select
users, using very high forces exceeding typical delivery forces
(>70 N), were capable of overcoming the maximum push-through
forces during priming and dosing the device. Participants in the
Simulated Use Study performed 26 to 33 VarioJect injections each
(557 devices total among 18 participants) into a pad at a rapid
rate, which is not considered representative use when injecting
patients where users would take their time and excessive force
would not be used. Note that this failure had not been observed
previously in any of the multiple human factors studies performed
with the device. The Sponsor intends to pursue minor design
enhancements to further strengthen the priming and dosing end stop
features by increasing the overlapping area between the contacting
end stop surfaces to mitigate the risk of administering the
incorrect dose to the patient.
[0401] The current specification for the priming end stop is 46 N
and the specification for the dosing end stop is 69 N. The dosing
end stop specification is 1.5.times. the priming end stop
specification due to risk of an overdose resulting from failure of
the dosing end stop. Verification tests of these features show that
the current design exceeds the specifications by about 50% for
priming and 35% for dosing. Based on results from the Simulated Use
Safety study, the Sponsor is increasing the specifications and
implementing minor design changes to increase the force required to
push past the priming and dosing end stops. The proposed
modifications will have no impact to the user interface (all forces
to operate and use steps remain unchanged) and therefore, the
device used clinically will be representative of the commercial
device. The commercial device that includes these minor changes
will be fully verified via bench testing to confirm that there is
no impact to device performance and to ensure the specification has
been increased and is appropriate for potential excessive force
during use. Only minor changes will be made to the external
features of the device. An overview of the changes is presented in
FIG. 24.
[0402] External Features Accommodating the Modifications
[0403] 1 Dose button will increase in diameter by 0.5-1.0 mm.
[0404] 2 End cap will increase in diameter by 0.5-1.0 mm.
[0405] The improvements will increase the robustness of the
device's performance while mitigating risks of incorrect dosing to
the patient, without changing the user interface. For the clinical
study, appropriate training will be provided to minimize the
potential for pushing past the end stop.
Suitability of the VarioJect for Pediatric Use
[0406] The injection characteristics of the VarioJect are similar
in injection depth and duration to those performed by a healthcare
provider using a manually injected subcutaneous hypodermic needle.
Additionally, as described above, this manual injector has been
designed with a number of features to help ensure that it is used
properly and safely in pediatric patients and includes a passive
needle safety feature. This important safety feature aids in
reducing the potential for accidental needle sticks and also
minimizes the exposure of the needle to some young patients who may
have a fear of needles. The needle insertion depth of this device
is only 4.5 mm, designed to limit the injection to subcutaneous
tissue in pediatric patients. Additionally, similar to an insulin
pen, this device is suitable for at-home administration by
caregivers and patients, including self-administration by
appropriately trained pediatric patients capable of
self-administration.
[0407] An overview of the risk management activities related to
in-home-use and needle length, are discussed below.
In-Home-Use
[0408] In early development of the VarioJect device, the Sponsor
conducted ethnographic research in the homes of children who
require injections, primarily insulin and human growth hormone.
Insights were gleaned from this research that led to design
improvements to help ensure safe and effective use of VarioJect in
the home environment, and these improvements were subsequently
confirmed to be effective in human factors studies.
Examples Include
[0409] Clear dose labeling and detents on the dose selection knob
to provide assurance of correct dose selection, a primary concern
for parent caregivers [0410] Recording of the dose in the dose
selection notch after use to allow users to confirm the correct
dose was delivered [0411] Sequencing the use steps to allow parents
to prepare the dose, then go to their child to perform the
injection, a common practice for at-home injections of children
[0412] Structuring the instructions to allow users to easily find
where they are in the injection process, in case they are
interrupted during the procedure
[0413] In addition, risks associated with at-home injections were
captured in the Application FMEA to ensure proper mitigation.
Examples Include
[0414] Improper transportation or storage of the device by the
user, or expired product, leading to degraded drug. Clear labeling
was added to help ensure proper refrigeration and inspection of the
product prior to use. [0415] Choking hazard associated with the
cap. Holes were added to the cap to allow for breathing in the
event of airway blockage, and warnings added to keep out of reach
of children. [0416] Unintentional access to the device by a child.
Users instructed to keep device inside package when warming to
limit unintentional access.
[0417] Furthermore, an analysis was performed on the potential for
abuse of the device that might occur in the home environment. Novo
Nordisk Engineering, an expert on drug delivery devices used in a
home setting, including insulin delivery pens prescribed for
children, was hired by the Sponsor to investigate abuse potential
for the VarioJect device. Analysis of different abuse scenarios was
included in the Application FMEA, along with appropriate
mitigations, including specifications to make it difficult for
users to separate the syringe from the device or extract a second
dose from the syringe.
[0418] The VarioJect and its associated instructions for use also
have several safety features, as well as features to improve
overall usability that facilitate in-home-use. To confirm that this
device is appropriate for in-home-use in the intended population,
the Sponsor will conduct a pediatric Human Factors validation study
designed to evaluate in-home-use by subjects (both pediatric
subjects and caregivers) considered representative of the intended
user population. During the human factors validation study, all
injections will be performed in a room designed to represent a
home-like setting for patients and caregivers. Prescribing a
self-injectable to pediatrics is a serious matter to the health
care community and training is always provided as a requirement
before caregivers can inject their children at home or to allow
patients to self-inject. The device and IFU was developed within
this context. Furthermore, the Sponsor has evaluated the injection
naive patient population in formative testing. Based on this
testing, the VarioJect is considered reasonably intuitive to users.
The Sponsor has taken steps to improve the design based on prior
formative testing. As an example, formative testing has shown that
untrained users are inclined to skip steps related to priming
(ensuring proper device orientation and tapping bubbles to the
top). In order to mitigate the risk of improper priming, which can
cause under dosing which is classified as a low risk to users, the
Sponsor has incorporated colored graphics on the device and clear
step-by-step instructions. Although there has been significant
focus and effort to improve the usability of the device and
associated IFU, the required training has proven to be the best way
to ensure proper use. As such, for the human factors study, all
patients and family caregivers will be provided with
representative, though minimal, training thus mimicking the
real-world context in which these devices will be deployed. The
Sponsor views the simulated human factors study as pivotal to
demonstrating that the device is safe for in-home-use and is
sufficient to address the clinical risks associated with this
device. However, in order to address regulatory requirements, the
Sponsor intends to conduct an actual use study on the VarioJect (in
pediatric UC patients) designed to capture and document real-life
VarioJect handling and use experience data from injections
administered by subjects and caregivers in the home-setting,
including complaints and failures in use. The actual use study is
considered as additional support of the pivotal human factors
data.
Needle Length
[0419] A 4.5mm needle length was chosen for VarioJect based on
published literature.sup.1,2 pertaining to needle lengths for
injecting subcutaneously in children. This needle length was chosen
to account for both manufacturing tolerances on glass syringes as
well as tolerances associated with assembly of the syringe in the
VarioJect in order to appropriately balance the small risks of
intramuscular and intradermal delivery. Based on data provided in
the published literature, the risk of intramuscular injections in
the 7-17 year age group is low, and although the risk of
intramuscular injections is slightly higher for the 2-6 year age
group, the risk is still low. Likewise, the risk of intradermal
injections in the 2-6 year age group is low, and although slightly
higher for the 7-17 year age group, the risk is still low. A pig
bio-distribution study was conducted to further evaluate the depth
of injection with the VarioJect. This study demonstrated that the
depth of injection using VarioJect falls within the range expected
for a needle and syringe. This conclusion supports the anticipated
result as depth of injection with a needle and syringe is impacted
by the person administering the injection and depends on the angle
of insertion and how far the needle is inserted, whereas the
proposed delivery method with VarioJect would have a fixed needle
insertion angle and depth. Accounting for the manufacturing
dimensional variation on glass syringes as well as variation
associated with assembly of the syringe in the VarioJect, the
tolerance for depth of injection is .+-.1.25 mm. The commercially
anticipated tolerance is much less as demonstrated by historical
needle protrusion measurements with the SIMPONI.RTM. SmartJect
autoinjector (7.4-8.6 mm) which uses the same BD Hypak syringe.
Furthermore, there is generally a wide variation in syringe and
needle selection and administration technique used to administer
many liquid drugs subcutaneously, yet published studies have not
found significant clinical concerns related to subcutaneous
injection variability. And, as a class, mAbs do not appear to
require precise or device-specific administration into a particular
location in the subcutaneous space to be safe and effective, and
small variations in PK (although not expected) are not likely to
impact efficacy outcomes. Nonetheless, safety assessments will be
collected as part of the proposed study.
Overview of Development Goals in Type 1 Diabetes Leading to
Evaluation in "Pre-T1D"
[0420] As noted above, progression of T1D is associated with a
significant impact on quality of life in early stages and eventual
morbidity and mortality. To date, a number of compounds have been
explored in those newly diagnosed with T1D to maintain residual
beta cell function, which in turn will improve glycemic control and
reduce short- and long-term complications of disease. However, the
Sponsor believes there is an unmet medical need and important
opportunity to delay or prevent T1D in those who are at high risk.
It is well established that the autoimmune-mediated .beta.-cell
loss, was initiated many years before the clinical diagnosis. The
autoimmune process that is occurring at the time of clinical
diagnosis, and thus being targeted in "new onset" studies, is
likely very similar to that which had been occurring in the
preceding months or years, and thus agents that show (even modest)
efficacy in newly diagnosed T1D can be considered as candidates to
examine in those at high risk of developing T1D (ie, "Pre-T1D").
This is evidenced through the support/approval of the Agency for
studies of abatacept (NCT01773707) and teplizumab (NCT01030861) in
those with serologic evidence of T1D autoimmunity but has not yet
met diabetes mellitus clinical criteria. In high-risk patients who
can be identified to progress towards disease (ie, auto-antibody
positive and dysglycemia), treating earlier to intercept or delay
onset of disease may have both near and long-term benefits. In the
near term, young children/adolescents may avoid the requirement of
multiple daily injections of exogenous insulin. Further, it is
expected that patients diagnosed with T1D at younger ages may have
more aggressive disease. Therefore, delaying onset by 2 or more
years may avoid more rapid progression of .beta.-cell destruction.
In the long-term, early preservation of .beta.-cell mass and good
glycemic control may mitigate severe complications later in life
such as cardiovascular disease or hospitalization which are
associated with significant morbidity and mortality, and
socioeconomic burden.
[0421] As the Sponsor believes there is a continuum of disease
progression in T1D, if positive results are observed in the trial
outlined herein, in addition to consideration for developing as a
therapy in new-onset disease, the Sponsor would anticipate further
evaluation in T1D pre-disease or "interception". The Sponsor
considers that children/adolescent patients 6 to 21 years of age
with .gtoreq.2 auto-antibodies for T1D with dysglycemia who are not
yet insulin dependent are patients greatest at-risk for progression
to clinical disease and would most benefit from treatment in the
pre-disease state. The Sponsor proposes to take a staged approach
in determining whether golimumab is an effective treatment that
provides benefit in the target population described above.
[0422] The Sponsor's first step in the staged approach is to
establish the benefit-risk of golimumab in new onset T1D study
described in this Briefing Document. The results of this study will
provide key information on the effect of golimumab on preservation
of .beta.-cell mass as well as the safety profile in this patient
population. In the early planning stages of a pilot study in 6 to
21 year olds who have not yet met the formal clinical diagnosis of
T1D but who have a first degree relative with T1D, and are double
auto-antibody positive for T1D with slightly elevated HbA1c (ie,
5.6 to 6.4%). The goal of this pilot study is to obtain early
safety and efficacy information to establish the mechanism of
benefit with golimumab in the pre-disease state. This trial would
start after the study in new-onset individuals discussed above.
These two initial studies will be complementary and provide key
data to inform planning for a larger, more formal,
"proof-of-concept" clinical trial in those at high risk for
developing T1D study to assess the benefit-risk of treatment with
golimumab in the target pre-T1D population.
[0423] The Sponsor intends to communicate the results from these
two initial studies in the context of preparations for the
proof-of-concept study in those at risk for T1D to align with the
Agency on a robust development plan for golimumab in the treatment
of pre-T1 D. [0424] Table 8: Studies in Support of the Simponi
Pediatric Presentations [0425] Cross-reference will be made to data
previously submitted as this presentation is already approved for
use in adults. [0426] Abbreviations: FDA=Food and Drug
Administration; HCP=Health Care Professional; [0427] HFS=Human
Factors Study; IFU=Instructions for use; PFS=prefilled syringe;
PFS-U=prefilled syringe in the UltraSafe Passive.RTM. needle guard;
PK=pharmacokinetic(s); [0428] sBLA=Supplemental Biologics License
Application; UC=ulcerative colitis.
A--VarioJect Actual Use Study
[0428] [0429] Study: [0430] VarioJect Actual Use Study (includes
assessment of labeling comprehension, safety, and device
durability/robustness). This study will be conducted as a substudy
of CNTO148UCO1002 which will be conducted to provide PK data in
pediatric with body weight <45 kg to support an extrapolation
based approach to the pediatric UC indication, and to demonstrate
that the VarioJect device can achieve the expected drug exposure in
the intended pediatric population. [0431] Objectives: [0432] To
provide supportive data that the VarioJect as designed, together
with the appropriate training and written Instructions for Use, is
suitable for at-home administration by subjects or their
caregivers. To provide supportive safety data demonstrating that
the VarioJect is suitable for use in the pediatric patient
population. To provide supportive VarioJect durability and
robustness data. [0433] Description: All study participants
performing the injection will be asked to complete a questionnaire
regarding their experience using the VarioJect following the second
at-home administration. All device complaints and device-related
AEs will be captured and investigated including the return of the
device for inspection. A visual assessment of a random sampling of
approximately 50-100 used VarioJect devices will be performed to
assess device durability and robustness. [0434] Comments: Study and
questionnaire are designed to capture and document real-life
VarioJect handling and use experience data from injections
administered by subjects and caregivers in the home-setting,
including complaints and failures in use.
B--PFS-U Actual Use Study
[0434] [0435] Study: [0436] PFS-U Actual Use Study (includes
assessment of labeling comprehension and safety). This study will
be conducted as a substudy of CNTO148UCO1002. [0437] Objectives:
[0438] To provide supportive data that the PFS-U as designed,
together with the appropriate training and written Instructions for
Use, is suitable for at-home administration by pediatric subjects
or their caregivers. To provide supportive safety data
demonstrating that the PFS-U is suitable for use in the pediatric
patient population. [0439] Description: [0440] All study
participants performing the injection will be asked to complete a
questionnaire regarding their experience using the PFS-U following
the second at-home administration. All device complaints and
device-related AEs will be captured and investigated including the
return of the device for inspection. [0441] Comments: [0442] Study
and questionnaire are designed to capture and document real-life
PFS-U handling and use experience data from injections administered
by subjects and caregivers in the home-setting, including
complaints and failures in use.
C--Pediatric Summative Human Factors Study
[0442] [0443] Study: [0444] Pediatric Summative Human Factors
(includes assessment of label comprehension) [0445] Objectives:
[0446] The objectives of this study are to provide pivotal device
usability data indicating that the VarioJect and PFS-U can be used
safely in the intended population (by caregivers, Health Care
Professionals [HCP], or self-administration) under realistic
conditions and to validate the device instructions for use. [0447]
Description: [0448] Simulated device use study in the target
population, in a representative use environment conducted by human
factors experts, as guided by FDA guidance: Applying Human Factors
and Usability Engineering to Medical Devices to Optimize Safety and
Effectiveness in Design (2011). In this study, approximately 45
subjects will be divided into 3 groups. Group 1 will be a mix of
family caregivers and HCP (VarioJect injection). Group 2 will be
pediatric subjects capable of self-injection (VarioJect injection).
Group 3 will be pediatric subjects capable of self-injection (PFS-U
injection). Overall performance success is achieved when the user
delivers the complete dose without making any use errors that could
result in serious harm. The proposed study assigns pass/fail
criteria at the individual task level, and behaviors such as
errors, close calls, and/or difficulties will be recorded at the
individual task level. Subjective participant feedback will be
collected in narrative form, and participants will be asked
open-ended questions about the procedure and device design.
Participants will be asked probe questions to evaluate their
knowledge and understanding of the instructions provided in the
IFU. [0449] Comments: [0450] Protocol, along with the instructions
for use, has been submitted to the FDA for review and comment (eCTD
IND 100181; Sequence No. 0307). Included with this submission was a
summary of the use errors seen thus far in formative studies and a
discussion of how these studies informed product design and
labeling. The results of this human factors study will be
summarized in the sBLA, along with the final HFS report, and is
intended to support the registration of the new VarioJect and to
extend the use of the PFS-U for pediatric use.
D--VarioJect Performance Testing
[0450] [0451] Study: [0452] 1.) Verification testing [0453] 2.)
Functional stability testing [0454] 3.) Accelerated and real-time
aging of device components [0455] 4.) Assembly process validation
[0456] 5.) Simulated use (safety) study [0457] 6.) Biocompatibility
testing [0458] 7.) Shipping testing [0459] 8.) Biochemical testing
following VarioJect delivery [0460] Objectives: [0461] The
objective of these studies is to demonstrate that the VarioJect
device meets its design requirements. [0462] Description: [0463] 1.
Verification testing is conducted according to the following.
[0464] ISO 11608:2012 Needle-based injection systems for medical
use--Requirements and test methods. VarioJect is designated
D2--integrated, single dose, non-replaceable container, whereby a
portion of the deliverable volume is expelled. [0465] FDA Draft
Guidance Technical Considerations for Pen, Jet, and Related
Injectors Intended for Use with Drugs and Biological Products.
[0466] Key elements of the development and test program include:
[0467] Design and verification that doses selected and dialed meet
accuracy criteria throughout the shelf life of the product [0468]
Design and verification that needle extension is limited and
appropriate for subcutaneous administration [0469] Verification of
design and manufacturing durability in expected use [0470]
Verification that device safety features intended to protect
against accidental needle sticks operate reliably [0471] 2.
Functional stability testing evaluates aging of the drug-device
combination product followed by device testing to ensure device
functionality. The assembled product is stored at the recommended
temperature of 2-8.degree. C., as well as accelerated (25.degree.
C.) conditions. [0472] 3. For accelerated aging testing, device
sub-assemblies are exposed to elevated temperature aging followed
by assembly with the drug-filled PFS and device testing to ensure
device functionality. The accelerated aging data is supported by
real time aging testing of device sub-assemblies stored at room
temperature. [0473] 4. Assembly process validation involves
assembly of drug-device combination product using equipment that
will be used to assemble the VarioJect for commercial launch,
followed by device testing. [0474] 5. The simulated use (safety)
study will include 500+ mock injections demonstrating successful
operation of needle safety feature following ISO 23908:2011 Sharps
injury protection--Requirements and test methods--Sharps protection
features for single-use hypodermic needles, introducers for
catheters and needles used for blood sampling and Guidance for
Industry and FDA Staff Medical Devices with Sharps Injury
Prevention Features. [0475] 6. Biocompatibility testing is
performed in accordance with ISO-10993-1: 2009 Biological
evaluation of medical devices--Part 1: Evaluation and testing
within a risk management process for skin contacting surface device
having limited contact duration (.ltoreq.24 hours). [0476] 7.
Shipping testing is performed in accordance with ASTM D4169:
Standard Practice for Performance Testing of Shipping Containers
and Systems, and includes an assessment of container closure
integrity. [0477] 8. Biochemical testing following VarioJect
delivery is conducted to determine if the shear forces on SIMPONI
generated during delivery through the VarioJect had an adverse
effect on the biochemical attributes of SIMPONI.
TABLE-US-00008 [0477] TABLE 9 Dose chart for study CNTO148DML2001
in pediatric subjects with Type 1 Diabetes with body weight < 45
Dose (mg) from VarioJect and/or PFS-U Body weight rounded to the
nearest 5 kg and height rounded to the nearest 10 cm See device
selection table for additional administration instructions Height
Weight (kg) (cm) 10 15 20 25 30 35 40 45 First Induction Dose (at
Week 0) 70 55 65 75 85 80 55 70 80 90 90 60 75 85 95 105 105 100 65
75 90 100 110 110 125 200 110 65 80 95 105 115 115 135 200 120 70
85 100 110 120 120 140 200 130 90 100 115 125 125 145 200 140 90
105 120 130 130 150 200 150 110 120 135 135 155 200 160 115 125 140
140 160 200 170 130 145 145 165 200 180 145 145 170 200 Second
Induction Dose (at Week 2) and Maintenance Dose (q4w) 70 25 30 35
40 80 30 35 40 45 90 30 35 40 45 50 55 100 30 40 45 50 55 60 65 100
110 35 40 45 50 55 60 65 100 120 35 40 50 55 60 65 70 100 130 45 50
55 60 65 70 100 140 45 55 60 65 70 75 100 150 55 60 65 70 75 100
160 55 65 70 75 80 100 170 65 70 75 80 100 180 75 80 85 100 Device
selection DOSE (mg) DEVICES.sup.a 25 1 VarioJect* 30 1 VarioJect*
35 1 VarioJect* 40 1 VarioJect* 45 1 VarioJect* 50 1 PFS-U-50** 55
2 VarioJect* 60 2 VarioJect* 65 2 VarioJect* 70 2 VarioJect* 75 2
VarioJect* 80 2 VarioJect* 85 2 VarioJect* 90 2 VarioJect* 95 1
PFS-U-50, 1 VarioJect*** 100 1 PFS-U-100** 105 1 PFS-U -50, 2
VarioJect*** 110 1 PFS-U -100, 1 VarioJect*** 115 1 PFS-U -100, 1
VarioJect*** 120 1 PFS-U -100, 1 VarioJect*** 125 1 PFS-U -100, 1
VarioJect*** 130 1 PFS-U -100, 1 VarioJect*** 135 1 PFS-U -100, 1
VarioJect*** 140 1 PFS-U -100, 1 VarioJect*** 145 1 PFS-U -100, 1
VarioJect*** 150 1 PFS-U -100, 1 PFS-U -50** 155 1 PFS-U -100, 2
VarioJect*** 160 1 PFS-U -100, 1 PFS-U -50, 1 VarioJect*** 165 1
PFS-U -100, 1 PFS-U -50, 1 VarioJect*** 170 1 PFS-U -100, 1 PFS-U
-50, 1 VarioJect*** 175 1 PFS-U -100, 1 PFS-U -50, 1 VarioJect***
180 1 PFS-U -100, 1 PFS-U -50, 1 VarioJect*** KEY *VarioJect Only
**PFS-U Only ***Combinations of VarioJect and PFS-U .sup.aThe
VarioJect is designed to administer SC doses of golimumab in 5 mg
increments from 10 mg to 45 mg.
REGULATORY CONSIDERATIONS
Plans to Open IND for T1D
[0478] The Sponsor is planning to open an IND for the study of
golimumab for the treatment of T1D by 2Q 2016. SIMPONI.RTM. has
been approved in the US for the treatment of Rheumatoid Arthritis,
Psoriatic Arthritis, Ankylosing Spondylitis and Ulcerative
Colitis.
[0479] The Sponsor has the following active BLAs with the Division
of Pulmonary, Allergy, and Rheumatology Products (DPARP) or the
Division of Gastroenterology and In-Born Error Products DGIEP).
[0480] BLA125289: For golimumab (SIMPONI.RTM.) approved on 24 Apr.
2009 with the following indications: [0481] SIMPONI.RTM., in
combination with methotrexate, is indicated for the treatment of
adult patients with moderately to severely active rheumatoid
arthritis. [0482] SIMPONI.RTM., alone or in combination with
methotrexate, is indicated for the treatment of adult patients with
active psoriatic arthritis. [0483] SIMIPONI.RTM. is indicated for
the treatment of adult patients with active ankylosing spondylitis.
[0484] SIMPONI.RTM. is indicated in adult patients with moderately
to severely active ulcerative colitis who have demonstrated
corticosteroid dependence or who have had an inadequate response to
or failed to tolerate oral aminosalicylates, oral corticosteroids,
azathioprine, or 6-mercaptopurine for: [0485] inducing and
maintaining clinical response [0486] improving endoscopic
appearance of the mucosa during induction [0487] inducing clinical
remission [0488] achieving and sustaining clinical remission in
induction responders
[0489] The Sponsor has 4 active INDs in support of the golimumab
development program with DPARP or DGIEP [0490] IND 09925 for the
study of CNTO148 (golimumab) for the treatment of moderately to
severely active rheumatoid arthritis (including polyarticular JIA)
[0491] IND 12723 for the study of CNTO148 (golimumab) for the
treatment of active psoriatic arthritis [0492] IND 12729 for the
study of CNTO148 (golimumab) for the treatment of active ankylosing
spondylitis [0493] IND 100181 for the study of CNTO148 (golimumab)
for the treatment of ulcerative colitis (including pediatric
UC)
[0494] As noted in the IND requirements table below (Table 10), the
Sponsor proposes to either submit items to the new IND or
cross-refer to IND 09925 or BLA125289. These will be text
cross-references only (no electronic hyperlinks).
TABLE-US-00009 TABLE 10 IND requirements Table IND Requirement
IND/BLA Reference Introductory Statement and General To be included
in new IND Investigational Plan [21 CR 312.23(a)(3)] Investigator's
Brochure [21 CFR To be included in new IND 312.23(a)(5)] Protocols
[21 CFR 312.23(a)(6)] To be included in new IND Chemistry,
Manufacturing, and Control Information [21 CFR 312.23(a)(7) 1.
Chemistry and Manufacturing To be included in new IND Introduction
2. Drug Substance [21 CFR Cross-reference to IND 09925
312.23(a)(7)(iv)(a)] 3. Drug Product [21 CFR 312.23 Cross-reference
to IND 09925 or IND (a)(7)(iv)(b)] 100181 and if applicable,
include new and unique information to clinical trials conducted
under the new IND 4. A brief general description of the
Cross-reference to IND 09925 or IND composition, manufacture, and
control of 100181 and if applicable, include new any placebo to be
used in the proposed and unique information to clinical trials
clinical trial(s) [21 CFR conducted under the new IND
312.23(a)(7)(iv)(c)] 5. a copy of all labels and labeling to be To
be included in the new IND provided to each investigator [21 CFR
312.23(a)(7)(iv)(d)] Pharmacology and Toxicology Information [21
CFR 312.23(a)(8)] 1. Pharmacology and Drug Distribution Cross
reference to BLA 125289 [21 CFR 312.23(a)(8)(i)] 2. Toxicology:
Integrated Summary [21 Cross reference to BLA 125289 CFR
312.23(a)(8)(ii)(a)] 3. Toxicology: Full Data Tabulation [21 Cross
reference to BLA 125289 CFR 312.23(a)(8)(ii)(b)] 4. Toxicology: GLP
Certification [21 Cross reference to BLA 125289 CFR
312.23(a)(8)(iii) Previous Human Experience with the Cross
reference to BLA 125289 Investigational Drug [21 CFR 312.23(a)(9)]
21 CFR 312.23(a)(10) (i) Drug dependence and abuse potential
Cross-reference to BLA 125289 (ii) Radioactive drugs N/A (iii)
Pediatric studies Status of pediatric program to be included in new
IND
[0495] It will be clear that the invention can be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0496] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
REFERENCES
[0497] Agarwal, A. and K. L. Brayman (2012). "Update on islet cell
transplantation for type 1 diabetes." Semin Intervent Radiol 29(2):
90-98. [0498] Argiles, J. M., J. Lopez-Soriano and F. J.
Lopez-Soriano (1994). "Cytokines and diabetes: the final step?
Involvement of TNF-alpha in both type I and II diabetes mellitus."
Horm Metab Res 26(10): 447-449. [0499] Arif, S., P. Cox, B. Afzali,
G. Lombardi, R. I. Lechler, M. Peakman and V. Mirenda (2010).
"Anti-TNFalpha therapy--killing two birds with one stone?" Lancet
375(9733): 2278. [0500] Atkinson, M. A., J. A. Bluestone, G. S.
Eisenbarth, M. Hebrok, K. C. Herold, D. Accili, M. Pietropaolo, P.
R. Aryan, M. Von Herrath, D. S. Markel and C. J. Rhodes (2011).
"How does type 1 diabetes develop?: the notion of homicide or
beta-cell suicide revisited." Diabetes 60(5): 1370-1379. [0501]
Blau, J. E., M. R. Abegg, W. A. Flegel, X. Zhao, D. M. Harlan and
K. I. Rother (2015). "Long-term immunosuppression after solitary
islet transplantation is associated with preserved C-peptide
secretion for more than a decade." Am J Transplant 15(11):
2995-3001. [0502] Bluestone, J. A., K. Herold and G. Eisenbarth
(2010). "Genetics, pathogenesis and clinical interventions in type
1 diabetes." Nature 464(7293): 1293-1300. [0503] Bougneres, P. F.,
J. C. Carel, L. Castano, C. Boitard, J. P. Gardin, P. Landais, J.
Hors, M. J. Mihatsch, M. Paillard, J. L. Chaussain and et al.
(1988). "Factors associated with early remission of type I diabetes
in children treated with cyclosporine." N Engl J Med 318(11):
663-670. [0504] Bougneres, P. F., P. Landais, C. Boisson, J. C.
Carel, N. Frament, C. Boitard, J. L. Chaussain and J. F. Bach
(1990). "Limited duration of remission of insulin dependency in
children with recent overt type I diabetes treated with low-dose
cyclosporin." Diabetes 39(10): 1264-1272. [0505] Boulton, J. G. and
J. T. Bourne (2007). "Unstable diabetes in a patient receiving
anti-TNF-alpha for rheumatoid arthritis." Rheumatology (Oxford)
46(1): 178-179. [0506] Cavallo, M. G., P. Pozzilli, C. Bird, M.
Wadhwa, A. Meager, N. Visalli, A. J. Gearing, D. Andreani and R.
Thorpe (1991). "Cytokines in sera from insulin-dependent diabetic
patients at diagnosis." Clin Exp Immunol 86(2): 256-259. [0507]
Chase, H. P., N. Butler-Simon, S. Garg, M. McDuffie, S. L. Hoops
and D. O'Brien (1990). "A trial of nicotinamide in newly diagnosed
patients with type 1 (insulin-dependent) diabetes mellitus."
Diabetologia 33(7): 444-446. [0508] Cook, J. J., I. Hudson, L. C.
Harrison, B. Dean, P. G. Colman, G. A. Werther, G. L. Warne and J.
M. Court (1989). "Double-blind controlled trial of azathioprine in
children with newly diagnosed type I diabetes." Diabetes 38(6):
779-783. [0509] Daneman, D. (2006). "Type 1 diabetes." Lancet
367(9513): 847-858. [0510] Dunger, A., D. Schroder, P. Augstein, T.
Witstruck, G. Wachlin, L. Vogt, B. Ziegler and S. Schmidt (1995).
"Impact of metabolic activity of beta cells on cytokine-induced
damage and recovery of rat pancreatic islets." Acta Diabetol 32(4):
217-224. [0511] Eisenbarth, G. S. (2004). "Type 1 diabetes:
molecular, cellular and clinical immunology." Adv Exp Med Biol 552:
306-310. [0512] Gitelman, S. E., P. A. Gottlieb, M. R. Rigby, E. I.
Felner, S. M. Willi, L. K. Fisher, A. Moran, M. Gottschalk, W. V.
Moore, A. Pinckney, L. Keyes-Elstein, S. Aggarwal, D. Phippard, P.
H. Sayre, L. Ding, J. A. Bluestone, M. R. Ehlers and S. S. Team
(2013). "Antithymocyte globulin treatment for patients with
recent-onset type 1 diabetes: 12-month results of a randomised,
placebo-controlled, phase 2 trial." Lancet Diabetes Endocrinol
1(4): 306-316. [0513] Gitelman, S. E., P. A. Gottlieb, M. R. Rigby,
E. I. Felner, S. M. Willi, L. K. Fisher, A. Moran, M. Gottschalk,
W. V. Moore, A. Pinckney, L. Keyes-Elstein, S. Aggarwal, D.
Phippard, P. H. Sayre, L. Ding, J. A. Bluestone, M. R. Ehlers and
t. S. S. Team (2013). "Antithymocyte globulin therapy for patients
with recent-onset type 1 diabetes: a randomized double-blind phase
2 trial." The Lancet Diabetes and Endocrinology In Press. [0514]
Glandt, M. and K. C. Herold (2004). "Treatment of type 1 diabetes
with anti-T-cell agents: from T-cell depletion to T-cell
regulation." Curr Diab Rep 4(4): 291-297. [0515] Gottlieb, P. A.,
S. Quinlan, H. Krause-Steinrauf, C. J. Greenbaum, D. M. Wilson, H.
Rodriguez, D. A. Schatz, A. M. Moran, J. M. Lachin, J. S. Skyler
and M. M. F. D. Z. B. S. G. Type 1 Diabetes TrialNet (2010).
"Failure to preserve beta-cell function with mycophenolate mofetil
and daclizumab combined therapy in patients with new-onset type 1
diabetes." Diabetes Care 33(4): 826-832. [0516] Han, S., W.
Donelan, H. Wang, W. Reeves and L. J. Yang (2013). "Novel
autoantigens in type 1 diabetes." Am J Transl Res 5(4): 379-392.
[0517] Harrison, L. C., P. G. Colman, B. Dean, R. Baxter and F. I.
Martin (1985). "Increase in remission rate in newly diagnosed type
I diabetic subjects treated with azathioprine." Diabetes 34(12):
1306-1308. [0518] Herold, K. C., S. Gitelman, C. Greenbaum, J.
Puck, W. Hagopian, P. Gottlieb, P. Sayre, P. Bianchine, E. Wong, V.
Seyfert-Margolis, K. Bourcier, J. A. Bluestone and I. T. N. A. I.
S. G. Immune Tolerance Network (2009). "Treatment of patients with
new onset Type 1 diabetes with a single course of anti-CD3 mAb
Teplizumab preserves insulin production for up to 5 years." Clin
Immunol 132(2): 166-173. [0519] JDRF. (2013). "Type 1 diabetes
facts;
http://jdrforg/about-jdrf/fact-sheets/type-1-diabetes-facts/."
2013. [0520] Kawahara, D. J. and J. S. Kenney (1991). "Species
differences in human and rat islet sensitivity to human cytokines.
Monoclonal anti-interleukin-1 (IL-1) influences on direct and
indirect IL-1-mediated islet effects." Cytokine 3(2): 117-124.
[0521] Kleijwegt, F. S., S. Laban, G. Duinkerken, A. M. Joosten, A.
Zaldumbide, T. Nikolic and B. O. Roep (2010). "Critical role for
TNF in the induction of human antigen-specific regulatory T cells
by tolerogenic dendritic cells." J Immunol 185(3): 1412-1418.
[0522] Kodama, S., M. Davis and D. L. Faustman (2005). "The
therapeutic potential of tumor necrosis factor for autoimmune
disease: a mechanistically based hypothesis." Cell Mol Life Sci
62(16): 1850-1862. [0523] Koulmanda, M., M. Bhasin, Z. Awdeh, A.
Qipo, Z. Fan, D. Hanidziar, P. Putheti, H. Shi, E. Csizuadia, T. A.
Libermann and T. B. Strom (2012). "The role of TNF-alpha in mice
with type 1- and 2-diabetes." PLoS One 7(5): e33254. [0524]
Ludvigsson, J., U. Samuelsson, C. Johansson and L. Stenhammar
(2001). "Treatment with antioxidants at onset of type 1 diabetes in
children: a randomized, double-blind placebo-controlled study."
Diabetes Metab Res Rev 17(2): 131-136. [0525] Maahs, D. M., N. A.
West, J. M. Lawrence and E. J. Mayer-Davis (2010). "Epidemiology of
type 1 diabetes." Endocrinol Metab Clin North Am 39(3): 481-497.
[0526] Mandrup-Poulsen, T., K. Bendtzen, C. A. Dinarello and J.
Nerup (1987). "Human tumor necrosis factor potentiates human
interleukin 1-mediated rat pancreatic beta-cell cytotoxicity." J
Immunol 139(12): 4077-4082. [0527] Mastrandrea, L., J. Yu, T.
Behrens, J. Buchlis, C. Albini, S. Fourtner and T. Quattrin (2009).
"Etanercept treatment in children with new-onset type 1 diabetes:
pilot randomized, placebo-controlled, double-blind study." Diabetes
Care 32(7): 1244-1249. [0528] Molbak, A. G., B. Christau, B.
Marner, K. Borch-Johnsen and J. Nerup (1994). "Incidence of
insulin-dependent diabetes mellitus in age groups over 30 years in
Denmark." Diabet Med 11(7): 650-655. [0529] Moran, A., B. Bundy, D.
J. Becker, L. A. DiMeglio, S. E. Gitelman, R. Goland, C. J.
Greenbaum, K. C. Herold, J. B. Marks, P. Raskin, S. Sanda, D.
Schatz, D. K. Wherrett, D. M. Wilson, J. P. Krischer, J. S. Skyler,
G. Type 1 Diabetes TrialNet Canakinumab Study, L. Pickersgill, E.
de Koning, A. G. Ziegler, B. Boehm, K. Badenhoop, N. Schloot, J. F.
Bak, P. Pozzilli, D. Mauricio, M. Y. Donath, L. Castano, A. Wagner,
H. H. Lervang, H. Perrild, T. Mandrup-Poulsen, A. S. Group, F.
Pociot and C. A. Dinarello (2013). "Interleukin-1 antagonism in
type 1 diabetes of recent onset: two multicentre, randomised,
double-blind, placebo-controlled trials." Lancet 381(9881):
1905-1915. [0530] Mordes, J. P., R. Borten, J. Doukas, M. Rigby, B.
Whalen, D. Zipris, D. L. Greiner and A. A. Rossini (1996). "The
BB/Wor rat and the balance hypothesis of autoimmunity." Diabetes
Metab Rev 12(2): 103-109. [0531] Orban, T., B. Bundy, D. J. Becker,
L. A. Dimeglio, S. E. Gitelman, R. Goland, P. A. Gottlieb, C. J.
Greenbaum, J. B. Marks, R. Monzavi, A. Moran, M. Peakman, P.
Raskin, W. E. Russell, D. Schatz, D. K. Wherrett, D. M. Wilson, J.
P. Krischer, J. S. Skyler and G. Type 1 Diabetes TrialNet Abatacept
Study (2014). "Costimulation modulation with abatacept in patients
with recent-onset type 1 diabetes: follow-up 1 year after cessation
of treatment." Diabetes Care 37(4): 1069-1075. [0532] Orban, T., B.
Bundy, D. J. Becker, L. A. DiMeglio, S. E. Gitelman, R. Goland, P.
A. Gottlieb, C. J. Greenbaum, J. B. Marks, R. Monzavi, A. Moran, P.
Raskin, H. Rodriguez, W. E. Russell, D. Schatz, D. Wherrett, D. M.
Wilson, J. P. Krischer, J. S. Skyler and G. Type 1 Diabetes
TrialNet Abatacept Study (2011). "Co-stimulation modulation with
abatacept in patients with recent-onset type 1 diabetes: a
randomised, double-blind, placebo-controlled trial." Lancet
378(9789): 412-419. [0533] Pescovitz, M. D., C. J. Greenbaum, H.
Krause-Steinrauf, D. J. Becker, S. E. Gitelman, R. Goland, P. A.
Gottlieb, J. B. Marks, P. F. McGee, A. M. Moran, P. Raskin, H.
Rodriguez, D. A. Schatz, D. Wherrett, D. M. Wilson, J. M. Lachin,
J. S. Skyler and C. D. S. G. Type 1 Diabetes TrialNet Anti (2009).
"Rituximab, B-lymphocyte depletion, and preservation of beta-cell
function." N Engl J Med 361(22): 2143-2152. [0534] Rabinovitch, A.
(1998). "An update on cytokines in the pathogenesis of
insulin-dependent diabetes mellitus." Diabetes Metab Rev 14(2):
129-151. [0535] Rigby, M. R., K. M. Harris, A. Pinckney, L. A.
DiMeglio, M. S. Rendell, E. I. Felner, J. M. Dostou, S. E.
Gitelman, K. J. Griffin, E. Tsalikian, P. A. Gottlieb, C. J.
Greenbaum, N. A. Sherry, W. V. Moore, R. Monzavi, S. M. Willi, P.
Raskin, L. Keyes-Elstein, S. A. Long, S. Kanaparthi, N. Lim, D.
Phippard, C. L. Soppe, M. L. Fitzgibbon, J. McNamara, G. T. Nepom
and M. R. Ehlers (2015). "Alefacept provides sustained clinical and
immunological effects in new-onset type 1 diabetes patients." J
Clin Invest 125(8): 3285-3296. [0536] Sherry, N., W. Hagopian, J.
Ludvigsson, S. M. Jain, J. Wahlen, R. J. Ferry, Jr., B. Bode, S.
Aronoff, C. Holland, D. Carlin, K. L. King, R. L. Wilder, S.
Pillemer, E. Bonvini, S. Johnson, K. E. Stein, S. Koenig, K. C.
Herold, A. G. Daifotis and I. Protege Trial (2011). "Teplizumab for
treatment of type 1 diabetes (Protege study): 1-year results from a
randomised, placebo-controlled trial." Lancet 378(9790): 487-497.
[0537] Stanescu, D. E., K. Lord and T. H. Lipman (2012). "The
epidemiology of type 1 diabetes in children." Endocrinol Metab Clin
North Am 41(4): 679-694. [0538] Tolerx, G. a. (2011).
"GlaxoSmithKline and Tolerx announce phase III DEFEND-1 study of
otelixizumab in type 1 diabetes did not meet its primary endpoint."
Retrieved Mar. 11, 2011. [0539] van Belle, T. L., K. T. Coppieters
and M. G. von Herrath (2011). "Type 1 diabetes: etiology,
immunology, and therapeutic strategies." Physiol Rev 91(1): 79-118.
[0540] van Eijk, I. C., M. J. Peters, M. T. Nurmohamed, A. W. van
Deutekom, B. A. Dijkmans and S. Simsek (2007). "Decrease of
fructosamine levels during treatment with adalimumab in patients
with both diabetes and rheumatoid arthritis." Eur J Endocrinol
156(3): 291-293. [0541] Yazdani-Biuki, B., H. Stelzl, H. P.
Brezinschek, J. Hermann, T. Mueller, P. Krippl, W. Graninger and T.
C. Wascher (2004). "Improvement of insulin sensitivity in insulin
resistant subjects during prolonged treatment with the
anti-TNF-alpha antibody infliximab." Eur J Clin Invest 34(9):
641-642.
Sequence CWU 1
1
3715PRTHomo sapiensMISC_FEATURE(1)..(5)Heavy Chain complementarity
determining region 1 (CDR1). 1Ser Tyr Ala Met His 1 5 217PRTHomo
sapiensMISC_FEATURE(1)..(17)Heavy Chain complementarity determining
region 2 (CDR2).MISC_FEATURE(1)..(1)Xaa at position 1 is selected
from Ile, Phe or Val.MISC_FEATURE(2)..(2)Xaa at position 2 is
selected from Ile or Met.MISC_FEATURE(3)..(3)Xaa at position 3 is
selected from Ser or Leu.MISC_FEATURE(4)..(4)Xaa at position 4 is
selected from Tyr or Phe.MISC_FEATURE(10)..(10)Xaa at position 10
is selected from Lys or Tyr.MISC_FEATURE(11)..(11)Xaa at position
11 is selected from Ser or Tyr.MISC_FEATURE(17)..(17)Xaa at
position 17 is selected from Asp or Gly. 2Xaa Xaa Xaa Xaa Asp Gly
Ser Asn Lys Xaa Xaa Ala Asp Ser Val Lys 1 5 10 15 Xaa 317PRTHomo
sapiensMISC_FEATURE(1)..(17)Heavy Chain complementarity determining
region 3 (CDR3).MISC_FEATURE(4)..(4)Xaa at position 4 is selected
from Ile or Val.MISC_FEATURE(5)..(5)Xaa at position 5 is selected
from Ser, Ala or Gly.MISC_FEATURE(9)..(9)Xaa at position 9 is
selected from Asn or Tyr. 3Asp Arg Gly Xaa Xaa Ala Gly Gly Xaa Tyr
Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 411PRTHomo
sapiensMISC_FEATURE(1)..(11)Light Chain complementarity determining
region 1 (CDR1).MISC_FEATURE(7)..(7)Xaa at position 7 is selected
from Ser or Tyr. 4Arg Ala Ser Gln Ser Val Xaa Ser Tyr Leu Ala 1 5
10 57PRTHomo sapiensMISC_FEATURE(1)..(7)Light Chain complementarity
determining region 2 (CDR2). 5Asp Ala Ser Asn Arg Ala Thr 1 5
610PRTHomo sapiensMISC_FEATURE(1)..(10)Light Chain complementarity
determining region 3 (CDR3). 6Gln Gln Arg Ser Asn Trp Pro Pro Phe
Thr 1 5 10 7126PRTHomo sapiensMISC_FEATURE(1)..(126)heavy chain
variable region sequences as presented in original Figure
4MISC_FEATURE(1)..(30)framework 1MISC_FEATURE(28)..(28)Xaa at
position 28 is selected from Ile or
Thr.MISC_FEATURE(31)..(35)complementarity determining region 1
(CDR1).MISC_FEATURE(36)..(49)framework 2MISC_FEATURE(43)..(43)Xaa
at position 43 is selected from Lys or
Asn.MISC_FEATURE(50)..(66)complementarity determining region 2
(CDR2).MISC_FEATURE(50)..(50)Xaa at position 50 is selected from
Ile, Phe or Val.MISC_FEATURE(51)..(51)Xaa at position 51 is
selected from Ile or Met.MISC_FEATURE(52)..(52)Xaa at position 52
is selected from Ser or Leu.MISC_FEATURE(53)..(53)Xaa at position
53 is selected from Tyr or Phe.MISC_FEATURE(59)..(59)Xaa at
position 59 is selected from Lys or Tyr.MISC_FEATURE(60)..(60)Xaa
at position 60 is selected from Ser or
Tyr.MISC_FEATURE(66)..(66)Xaa at position 66 is selected from Asp
or Gly.MISC_FEATURE(67)..(98)framework 3MISC_FEATURE(70)..(70)Xaa
at position 70 is selected from Val or
Ile.MISC_FEATURE(75)..(75)Xaa at position 75 is selected from Ser
or Pro.MISC_FEATURE(78)..(78)Xaa at position 78 is selected from
Thr or Ala.MISC_FEATURE(80)..(80)Xaa at position 80 is selected
from Tyr or Phe.MISC_FEATURE(94)..(94)Xaa at position 94 is
selected from Tyr or Phe.MISC_FEATURE(99)..(115)complementarity
determining region 3 (CDR3).MISC_FEATURE(102)..(102)Xaa at position
102 is selected from Ile or Val.MISC_FEATURE(116)..(126)J6 region
7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Xaa Phe Ser Ser
Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Xaa Gly Leu
Glu Trp Val 35 40 45 Ala Xaa Xaa Xaa Xaa Asp Gly Ser Asn Lys Xaa
Xaa Ala Asp Ser Val 50 55 60 Lys Xaa Arg Phe Thr Xaa Ser Arg Asp
Asn Xaa Lys Asn Xaa Leu Xaa 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Xaa Tyr Cys 85 90 95 Ala Arg Asp Arg Gly
Xaa Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 8108PRTHomo
sapiensMISC_FEATURE(1)..(108)light chain variable region sequences
as presented in original Figure 5MISC_FEATURE(1)..(23)framework
1MISC_FEATURE(24)..(34)complementarity determining region 1
(CDR1).MISC_FEATURE(35)..(49)framework
2MISC_FEATURE(50)..(56)complementarity determining region 2
(CDR2).MISC_FEATURE(57)..(88)framework
3MISC_FEATURE(89)..(98)complementarity determining region 3
(CDR3).MISC_FEATURE(99)..(108)J3 region 8Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 105 9157PRTHomo sapiensMISC_FEATURE(1)..(157)human TNF
alpha monomer sequence 9Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly Gln
Leu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn
Gly Val Glu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu
Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln
Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile 65 70 75 80 Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90
95 Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys
100 105 110 Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu
Glu Lys 115 120 125 Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp
Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile
Ile Ala Leu 145 150 155 1018DNAHomo sapiens 10ttggtccagt cggactgg
181118DNAHomo sapiens 11cacctgcact cggtgctt 181230DNAHomo sapiens
12cactgttttg agtgtgtacg ggcttaagtt 301318DNAHomo sapiens
13gccgcacgtg tggaaggg 181425DNAHomo sapiens 14agtcaaggtc ggactggctt
aagtt 251528DNAHomo sapiens 15gttgtcccct ctcacaatct tcgaattt
281618DNAHomo sapiens 16ggcggtagac tactcgtc 18177PRTHomo sapiens
17Met Asp Trp Thr Trp Ser Ile 1 5 1835DNAHomo sapiens 18tttcgtacgc
caccatggac tggacctgga gcatc 351934DNAHomo sapiens 19tttcgtacgc
caccatgggg tttgggctga gctg 342035DNAHomo sapiens 20tttcgtacgc
caccatggag tttgggctga gcatg 352135DNAHomo sapiens 21tttcgtacgc
caccatgaaa cacctgtggt tcttc 352235DNAHomo sapiens 22tttcgtacgc
caccatgggg tcaaccgcca tcctc 35236PRTHomo sapiens 23Thr Val Thr Val
Ser Ser 1 5 2436DNAHomo sapiens 24gtgccagtgg cagaggagtc cattcaagct
taagtt 36255PRTHomo sapiens 25Met Asp Met Arg Val 1 5 2631DNAHomo
sapiens 26tttgtcgaca ccatggacat gagggtcctc c 312728DNAHomo sapiens
27tttgtcgaca ccatggaagc cccagctc 28286PRTHomo sapiens 28Thr Lys Val
Asp Ile Lys 1 5 2941DNAHomo sapiens 29ctggtttcac ctatagtttg
cattcagaat tcggcgcctt t 413035DNAHomo sapiens 30catctccaga
gacaattcca agaacacgct gtatc 353135DNAHomo sapiens 31gtagaggtct
ctgttaaggt tcttgtgcga catag 353219PRTHomo
sapiensMISC_FEATURE(1)..(19)Signal sequence for heavy chain
variable region sequences as presented in original Figure 4 32Met
Gly Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10
15 Val Gln Cys 3320PRTHomo sapiensMISC_FEATURE(1)..(20)Signal
sequence for light chain variable region sequences as presented in
original Figure 5 33Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu
Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly 20 34428DNAHomo
sapiensMISC_FEATUREheavy chain variable region DNA sequences as
presented in original Figure 2A-2B with coding sequence 1 to 421
34atggggtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag
60gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc
120tgtgcagcct ctggttcacc ttcagtagct atgctatgca ctgggtccgc
caggctccgg 180caaggggctg gagtgggtgg cagttatatc atatgatgga
aaataaatac tacgcagact 240ccgtgaaggg ccgattcacc atctagagac
aattccaaga acacgctgta tctgcaaatg 300aacagccaga gctgaggaca
cggctgtgta ttactgtgcg agagatcgag gtatatcagc 360aggtggaata
ctactactac tacggtatgg acgtctgggg gcaagggacc acggtcaccg 420tctcctca
42835387DNAHomo sapiensMISC_FEATURElight chain variable region DNA
sequences as presented in original Figure 3 with coding sequence 1
to 387 35atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga
taccaccgga 60gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga
aagagccacc 120ctctcctgca gggccagtca gagtgttagc agctacttag
cctggtacca acagaaacct 180ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 240aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 300gaagattttg
cagtttatta ctgtcagcag cgtagcaact ggcctccatt cactttcggc
360cctgggacca aagtggatat caaacgt 38736456PRTHomo
sapiensMISC_FEATURE(1)..(456)Golimumab Heavy Chain (HC) 36Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 20
25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp
Val 35 40 45 Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ile Ala
Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140 Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150
155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile 195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275
280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln 290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 355 360 365 Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395
400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys 450 455
37215PRTHomo sapiensMISC_FEATURE(1)..(215)Golimumab Light Chain
(LC) 37Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Phe Thr Phe
Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 100 105 110 Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120
125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly
Glu Cys 210 215
* * * * *
References