Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF AUTOIMMUNE DISORDERS
RELATED APPLICATIONS
[001] This application claims priority to US Provisional Appln. No. 60/918518,
filed
on March 15, 2007, which is incorporated in its entirety herein.
BACKGROUND
[002] Protein heterogeneity can be caused by a range of post-translational
modifications. Protein heterogeneity often results from different types of
post-
translational modifications, including carboxylation, hydroxylation,
proteolytic
processing, suflation, and glycosylation, the latter of which is the most
common
modification (Walsh and Jefferis (2006) Nat Biotech 24(10): 1241). Post-
translational
modifications can potentially affect product production levels (by
influencing, for
example, the degree of proper protein folding), stability, and a range of
pharmacokinetic and pharmacodynamic parameters, as well as safety and
immunogenicity. Post-translational modifications of therapeutic biologics, or
protein-
based biopharmaceuticals, may affect protein properties relevant to their
therapeutic
application.
[003] N and/or C-terminal heterogeneity is an example of protein heterogeneity
which must be considered in the manufacture of protein-based
biopharmaceuticals.
N-terminal heterogeneity results from proteolytic processing at the amino
terminal
portion of the protein, where such processing may result in a population of
proteins
having different sizes. Variations in N-terminal proteolysis may occur in
proteins
comprising a signal sequence. In addition, N-terminal glutamine residues can
undergo spontaneous cyclization to form pyroglutamic acid. Thus, obtaining a
homogenous population of proteins which can be used for therapeutic purposes
often
presents a challenge.
[004] Lymphotoxin beta receptor (LTBR) is a member of the tumor necrosis
factor
receptor (TNFR) family. The receptor is expressed on the surface of cells in
the
parenchyma and stroma of most lymphoid organs but is absent on T- and B-
lymphocytes. Signaling through LTBR by the LTa/B heterotrimer (LT) is
important
during lymphoid development. LTBR is also known to bind the ligand LIGHT
(homologous to lymphotoxins, exhibits inducible expression, and competes with
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herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T
lymphocytes), which has been implicated in T-cell driven events, both in the
periphery
and in the thymus. LT and LIGHT are expressed on the surface of activated
lymphocytes. Blocking the LT pathway with a soluble decoy LTBR has been shown
to be effective to treat autoimmune disease in various animal models.
[005] The development of soluble forms of LTBR having reduced heterogeneity
and
optimal dosing regimens for administration of these molecules would be of
great
benefit.
SUMMARY OF THE INVENTION
[006] In one aspect the invention pertains to a composition comprising a
population
of lymphotoxin-(3 receptor (LT-(3-R)-Ig-fusion proteins which comprise a
variant LT-
(3-R extracellular domain of 193 or 194 amino acids in length and a variant Ig
portion
of 227 amino acids in length, wherein at least 90% of the LT-(3-R-Ig-fusion
proteins
are missing no more than 5 amino acids from the N-terminus of the mature form
of
the wild type LT-(3-R extracellular domain set forth in SEQ ID NO:21 and
wherein
the LT-(3-R-Ig-fusion proteins lack N-terminal pyroglutamic acid.
[007] In one embodiment, the N-terminal amino acid of the variant LT-[i-R-Ig
fusion protein is a non-polar amino acid.
[008] In one embodiment, the non polar amino acid is either a valine (amino
acid six
of the mature form of the wild type LT-(3-R extracellular domain of SEQ ID
NO:21)
or an alanine (amino acid five of the mature form of the wild type LT-(3-R
extracellular domain of SEQ ID NO:21).
[009] In one embodiment, the N-terminal amino acid of at least 95% of the LT-
[3-R-
Ig-fusion proteins is either a valine (amino acid six of the mature form of
the wild
type LT-(3-R extracellular domain of SEQ ID NO:21) or an alanine (amino acid
five
of the mature form of the wild type LT-(3-R extracellular domain of SEQ ID
NO:21).
[0010] In one embodiment, the composition is made by expressing a nucleic acid
molecule comprising a nucleotide sequence encoding the extracellular domain of
LTBR set forth in SEQ ID NO:4 in a mammalian cell.
[0011] In one embodiment, the nucleic acid molecule comprises the sequence set
forth in SEQ ID NO:3.
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[0012] In one embodiment, variant Ig portion comprises Fc regions of an IgGI
isotype.
[0013] In one embodiment, the variant Ig portion comprises the amino acid
sequence
set forth in SEQ ID NO:2.
[0014] In one embodiment, the Ig portion is non-glycosylated.
[0015] In one embodiment, the composition is made by expressing a nucleic acid
molecule encoding the LT-(3-R- Ig fusion protein set forth in SEQ ID NO:5 in a
mammalian cell.
[0016] In one embodiment, the nucleic acid molecule comprises the sequence set
forth in SEQ ID NO:7.
[0017] In one embodiment, the step of expressing is done at manufacturing
scale.
[0018] In one aspect, the invention pertain to a composition comprising a
population
of lymphotoxin-(3 receptor-immunoglobulin (LT-(3-R-Ig)-fusion proteins
comprising a
variant LT-P-R extracellular domain and a variant Ig portion, wherein the
variant LT-
(3-R extracellular domain is aglycosylated.
[0019] In one embodiment, the aglycosylated extracellular domain of LTBR
comprises amino acids 1 to 194 of SEQ ID NO: 10.
[0020] In another aspect, the invention pertains to a composition comprising a
population of lymphotoxin-[i receptor-immunoglobulin (LT-[3-R-Ig)-fusion
proteins,
the fusion proteins comprising a variant LT-P-R extracellular domain of 193 or
194
amino acids in length and a variant Ig portion, wherein the population has
reduced N-
terminal pyroglutamic acid formation, and reduced C-terminal heterogeneity
compared to wild-type LT-(3-R-Ig fusion proteins.
[0021] In one embodiment, at least 90% of the LT-[i-R-Ig-fusion proteins
comprise a
variant LT-P-R extracellular domain as set forth the amino acid sequence of
SEQ ID
NO: 4 or SEQ ID NO: 23.
[0022] In one embodiment, the variant Ig portion comprises a mutation in the
hinge
region.
[0023] In one aspect, the invention pertains to a pharmaceutical composition
comprising a population of lymphotoxin-[3 receptor (LT-[3-R)-Ig-fusion
proteins
which comprise a variant LT-0-R extracellular domain of 193 or 194 amino acids
in
length and a variant Ig portion of 227 amino acids in length, wherein at least
90% of
the LT-(3-R-Ig-fusion proteins are missing no more than 5 amino acids from the
N-
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terminus of the mature form of the wild type LT-P-R extracellular domain set
forth in
SEQ ID NO:21 and wherein the LT-(3-R-Ig-fusion proteins lack N-terminal
pyroglutamic acid and a pharmaceutically acceptable carrier.
[00241 In one embodiment, the N-terminal amino acid of the variant LT-(3-R-Ig
fusion protein is a non-polar amino acid.
[0025] In one embodiment, the non polar amino acid is either a valine (amino
acid six
of the mature form of the wild type LT-P-R extracellular domain of SEQ ID
NO:21)
or an alanine (amino acid five of the mature form of the wild type LT-P-R
extracellular domain of SEQ ID NO:21).
[0026] In one embodiment, the N-terminal amino acid of at least 95% of the LT-
(3-R-
Ig-fusion proteins is either a valine (amino acid six of the mature form of
the wild
type LT-P-R extracellular domain of SEQ ID NO:21) or an alanine (amino acid
five
of the mature form of the wild type LT-P-R extracellular domain of SEQ ID
NO:21).
[0027] In one embodiment, the composition is made by expressing a nucleic acid
molecule comprising a nucleotide sequence encoding the extracellular domain of
LTBR set forth in SEQ ID NO:4 in a mammalian cell.
[0028] In one aspect, the invention pertains to a method of treating an
autoimmune
disorder comprising administering the pharmaceutical composition of claim 23
to a
subject in need thereof.
[0029] In one embodiment, the autoimmune disorder is selected from the group
consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus
erythematosus
(SLE).
100301 In one embodiment, the invention pertains to a pharmaceutical
composition
comprising a population of lymphotoxin-[3 receptor-immunoglobulin (LT-(3-R-Ig)-
fusion proteins, the fusion proteins comprising a variant LT-P-R extracellular
domain
of 193 or 194 amino acids in length and a variant Ig portion, wherein the
population
has reduced N-terminal pyroglutamic acid formation and reduced C-terminal
heterogeneity compared to wild-type LT-(3-R-Ig fusion proteins and a
pharmaceutically acceptable carrier.
[0031] In one embodiment, at least 90% of the LT-[i-R-Ig-fusion proteins
comprise a
variant LT-P-R extracellular domain as set forth the amino acid sequence of
SEQ ID
NO: 4 or SEQ ID NO: 23.
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[0032] In one embodiment, the variant Ig portion comprises a mutation in the
hinge
region.
[0033] In one embodiment, the amino acid sequence set forth in SEQ ID NO:5.
[0034] In one aspect, the invention pertains to a method of treating an
autoimmune
disorder comprising administering the pharmaceutical composition of claim 29
to a
subject in need thereof.
[0035] In one embodiment, the autoimmune disorder is selected from the group
consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus
erythematosus
(SLE).
[0036] In one embodiment, the autoimmune disorder is rheumatoid arthritis.
[0037] In one embodiment, the pharmaceutical composition is administered to
the
subject at a dose of from about 0.6 to 3 mg/kg biweekly.
[0038] In one embodiment, the pharmaceutical composition is administered
subcutaneously.
[0039] In one aspect, the invention pertains to an isolated polypeptide
comprising a
variant LT-P-R extracellular domain of 193 or 194 amino acids in length and a
variant
Ig portion of 227 amino acids in length, wherein the polypeptide is missing no
more
than 5 amino acids from the N-terminus of the mature form of the wild type LT-
P-R
extracellular domain set forth in SEQ ID NO:21 and wherein the polypeptide
lacks N-
terminal pyroglutamic acid.
[0040] In one embodiment, the N-terminal amino acid is a non-polar amino acid.
[0041] In one embodiment, the non polar amino acid is either a valine (amino
acid six
of the mature form of the wild type LT-P-R extracellular domain of SEQ ID
NO:21)
or an alanine (amino acid five of the mature form of the wild type LT-P-R
extracellular domain of SEQ ID NO:21).
100421 In one embodiment, the polypeptide is made by expressing a nucleic acid
molecule comprising a nucleotide sequence encoding the extracellular domain of
LTBR set forth in SEQ ID NO:4 in a mammalian cell.
[0043] In one embodiment, the invention pertains to an isolated nucleic acid
molecule
encoding the polypeptide of the invention.
[0044] In one embodiment, the nucleic acid molecule comprises of the
nucleotide
sequence set forth in SEQ ID NO:7.
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[0045] In one embodiment, the inention pertains to a vector comprising the
nucleic
acid molecule of the invention
[0046] In one embodiment, the invention pertains to a host cell expressing the
vector
of the invention.
[0047] In one embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.
[0048] In one embodiment, the invention pertains to a process for making a
composition comprising a population of lymphotoxin-[3 receptor (LT-(3-R)-Ig-
fusion
proteins which comprise a variant LT-P-R extracellular domain and a variant Ig
portion, wherein at least 90% of the LT-(3-R-Ig-fusion proteins are missing no
more
than 5 amino acids from the N-terminus of the mature form of the wild type LT-
P-R
extracellular domain set forth in SEQ ID NO:21, the process comprising,
expressing a
nucleic acid molecule encoding the LT-(3-R- Ig fusion protein set forth in SEQ
ID
NO:8 in a mammalian cell, obtaining the population from the culture
supernatant,
and, optionally, purifying the supernatant, to thereby obtain a composition
comprising
a population of lymphotoxin-(3 receptor (LT-(3-R)-Ig-fusion proteins which
comprise a
variant LT-P-R extracellular domain and a variant Ig portion, wherein at least
90% of
the LT-[3-R-Ig-fusion proteins are missing no more than 5 amino acids from the
N-
terminus of the mature form of the wild type LT-P-R portion set forth in SEQ
ID
NO:21.
[0049] In one embodiment, the nucleic acid molecule comprises the nucleotide
sequence set forth in SEQ ID NO:7.
[0050] In one embodiment, the nucleic acid molecule consists of the nucleotide
sequence set forth in SEQ ID NO:7.
[0051] In one embodiment, the invention pertains to a method of treating
rheumatoid
arthritis in a human subject, the method comprising administering to the
subject a
dose of LT-[3-R-Ig fusion protein, wherein the dose is sufficient to maintain
an
average concentration of from about 0.14 ug/ml to about 3.5 ug/ml in the serum
of the
subject.
[0052] In another aspect, the invention pertains to a method of treating
rheumatoid
arthritis in a human subject, the method comprising administering to the
subject a
dose of LT-(3-R-Ig fusion protein, wherein the dose is sufficient to maintain
an
average a minimal average concentration of about 0.6 ug/ml in the serum of the
subject.
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[0053] In one embodiment, the LTBR-Ig fusion protein comprises the amino acid
sequence set forth in SEQ ID NO:5.
[0054] In one embodiment, the concentration is achieved by administering LT-(3-
R-Ig
fusion protein at a dose of from about 0.01 to about 5 mg/kg once every 7-60
days.
[0055] In one embodiment, the invention pertains to a method of treating
rheumatoid
arthritis in a human subject, the method comprising administering to the
subject a
dose of LT-(3-R-Ig fusion protein of from about 0.6 to 3 mg/kg not more than
twice
every 7-30 days.
[0056] In one embodiment, method comprising administering to the subject a
dose of
LT-(3-R-Ig fusion protein of from about 0.6 to 3 mg/kg once every 7-14 days.
[0057] In one embodiment, administration is once every 14-30 days.
[0058] In one embodiment, administration is once every 28-60 days.
[0059] In one embodiment, administration is once every 7-30 days.
[0060] In one embodiment, the invention pertains to a method of treating an
autoimmune disorder in a human subject, the method comprising administering to
the
subject a dose of a pharmaceutical composition comprising a population of LT-
(3-R-Ig
fusion proteins comprising a variant LT-[i-R extracellular domain of 193 or
194
amino acids in length, wherein at least 90% of the LT-(3-R-Ig-fusion proteins
are
missing no more than 5 amino acids from the N-terminus of the mature form of
the
wild type LT-0-R extracellular domain set forth in SEQ ID NO:21.and wherein
the
dose is sufficient to maintain a minimal average concentration of about 0.6
ug/ml in
the serum of the subject.
[0061] In one embodiment, the LT-(3-R-Ig fusion protein further comprises a
variant
Ig portion.
[0062] In one embodiment, the autoimmune disorder is selected from the group
consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus
erythematosus
(SLE).
[0063] In one embodiment, the pharmaceutical composition comprises the amino
acid
sequence set forth in SEQ ID NO:5.
[0064] In one embodiment, administration is twice monthly.
[0065] In one embodiment, administration once monthly.
[0066] In one embodiment, administration is subcutaneous.
[0067] In one embodiment, the dose is about 1 mg/kg.
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[0068] In one embodiment, the dose is about 3 mg/kg.
[0069] In one embodiment, the dose is about 1 mg/kg administered about every 7
to
20 days.
[0070] In one embodiment, the dose is about 3 mg/kg administered about every
14 to
30 days.
[0071] In one embodiment, the dose is about 1 mg/kg administered about every
14
days.
[0072] In one embodiment, the autoimmune disorder is rheumatoid arthritis and
the
subject has been treated with a rheumatoid arthritis drug after being
diagnosed with
rheumatoid arthritis and prior to administration of the LT-(3-R-Ig fusion
protein.
[0073] In one embodiment, the rheumatoid arthritis drug is chosen from the
group
consisting of a DMARD, an NSAID, and a corticosteroid.
[0074] In one embodiment, the human is a DMARD-inadequate responder.
[0075] In one embodiment, the rheumatoid arthritis drug is a TNF inhibitor.
[0076] In one embodiment, the rheumatoid arthritis drug is adalimumab (Humira
),
etanercept (Enbrel ), or infliximab (Remicade(g).
[0077] In one embodiment, LT-[3-R-Ig is administered in combination with the
rheumatoid arthritis drug.
[0078] In one embodiment, the human is evaluated to determine if the response
to the
rheumatoid arthritis drug is inadequate prior to administration of LT-[3-R-Ig.
[0079] In one embodiment, the human is determined to have an inadequate
response
to the rheumatoid arthritis drug, and then the human is administered LT-(3-R-
Ig.
[0080] In one embodiment, the human is asymptomatic for a first manifestation
of
rheumatoid arthritis and is symptomatic for a second manifestation of
rheumatoid
arthritis.
[0081] In one embodiment, LT-[3-R-Ig is administered in place of the
rheumatoid
arthritis drug.
[0082] In one embodiment, administration is in combination with a tumor
necrosis
factor (TNF) inhibitor.
[0083] In one embodiment, the TNF inhibitor is adalimumab (Humira ),
etanercept
(Enbrel ), or infliximab (Remicade ).
[0084] In one embodiment, the human is an anti-TNF-inadequate responder.
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[0085] In one embodiment, administration is in combination with a non-
steroidal anti-
inflammatory agent (NSAID), a corticosteroid, or a disease modifying
antirheumatic
drug (DMARD).
[0086] In one embodiment, administration is in combination with methotrexate.
[0087] In one embodiment, the human is a DMARD-inadequate responder.
BRIEF DESCRIPTION OF DRAWINGS
[0088] Figure 1 describes the changes made from LTBR01 to LTBR06 to improve
heterogeneity. The bold letters indicate the secretion sequence, and the
italicized /
underlines letters indicate the amino acids which are removed in LTBR06
relative to
LTBRO 1, i.e., amino acids 1-4 and the last amino acid (lysine). Three
consensus sites
for N-linked glycosylation are located at Asnl3, 150 and 276. Amino acid
positions
refer to full length LTBR, i.e., amino acids 1-4 are those which are removed
in
LTBR06.
[0089] Figure 2 provides an alignment of LTBROI, LTBR05, LTBR06, and LTBR09.
The secretion sequence is omitted from the LTBR sequences.
[0090] Figure 3 describes LTBR06 in comparison to wild type and provides a
schematic of the protein.
[0091] Figure 4 describes the amino acid sequence of the LTBR06 construct
(mature
form of LTBR06 shown in SEQ ID NO: 5). LTBR06 is a disulfide-linked,
glycosylated, dimeric protein. There are 28 cysteine residues and 6
glycosylation
sites, the latter of which is indicated in bold.
[0092] Figure 5 describes aglycosylated hLT[iR hIgGl(mature form of protein).
Asparagine to glutamine mutations in LT[iR extracellular domain are shown in
bold.
The huLT[3R is residues 1-204, the huIgGl Fc is residues 205-431 above (the
Fc's
glycosylation site is intact).
[0093] Figure 6 describes hinges of which may be used in the LTBR IgG fusion
proteins of the invention.
[0094] Figure 7 describes a table which describes a summary of results from a
hinge
expression analysis.
[0095] Figure 8 is a graph depicting the percent improvement in RA symptom
scores
(tender joint counts (TJC) and swollen joint counts (SJC)) in patients
following
treatment with LTBR-Fc.
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100961 Figure 9 provides a graph which depicts a decrease in Tender Joint
Counts
(TJC) in patients following treatment with LTBR-Fc (LTBR06).
[0097] Figure 10 provides a graph which depicts a decrease in Swollen Joint
Counts n
(SJC) in patients following treatment with LTBR-Fc (LTBR06).
[0098] Figure 11 provides a graph which depicts the median % change in Swollen
Joint Counts n (SJC) in patients following treatment with LTBR-Fc (LTBR06).
-[0099] Figure 12 provides a graph which depicts ACR20 improvements in
patients
following treatment with LTBR-Fc (LTBR06).
[00100] Figure 13 graphically depicts serum levels of differentially
sialylated
LFA3TIP and hu-LTBR-Ig (LTBR05) in mice 24 hours post administration with 100
ug/animal.
[00101] Figure 14 illustrates a comparison of LTBR-Fc and a typical antibody
and shows that at identical dosing significant differences in efficacy are
observed for
prolonged time frames. Arrows A and B indicate the typical alpha and beta
phases for
an antibody or Fc-fusion protein, respectively. For an antibody the gray line
indicated
by arrow C shows typical lower limit concentration for efficacy, whereas arrow
D
shows LTBR-Fc has efficacy at significantly lower concentrations.
DETAILED DESCRIPTION OF INVENTION
1001021 In order that the present invention may be more readily understood,
certain terms are first defined.
[00103] The term "fusion protein" refers to a molecule comprising two or more
proteins or fragments thereof linked by a covalent bond via their individual
peptide
backbones, most preferably generated through genetic expression of a
polynucleotide
molecule encoding those proteins. In a preferred embodiment, the fusion
protein
includes an immunoglobulin domain.
[00104] The generic term "immunoglobulin" comprises five distinct classes of
antibody that can be distinguished biochemically. All five classes of
antibodies are
clearly within the scope of the present invention, the following discussion
will
generally be directed to the IgG class of immunoglobulin molecules.
1001051 The term "immunoglobulin fusion protein" refers to a fusion of a
functional portion of a polypeptide (generally comprising the extracellular
domain of
a cell surface protein) with one or more portions of an immunoglobulin
constant
region, e.g. the hinge, CH1, CH2 or CH3 domains or portions or combinations
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thereof. In one embodiment, the polypeptide is a member of the TNF family of
receptors. The portions of the Ig molecule may derive from any of the various
immunoglobulin isotypes, including, for example, IgGI, IgG2, IgM, IgA etc.
Immunoglobulin fusion proteins are referred to herein as Ig for Fc fusion
proteins.
1001061 In a preferred embodiment, the protein used in the methods and
compositions of the invention is an immunoglobulin fusion protein. For
example, the
fusion protein may comprise a receptor, or ligand binding portion thereof, and
a
dimerization domain, e.g., an Fc domain.
[00107] As used herein, the term "variant LTBR extracellular domain" refers
the mature form of a polypeptide (or protein) having an amino acid sequence
that
differs from the sequence presented in SEQ ID NO: 1 and SEQ ID NO: 21 (wild
type
hLTBR) at one or more amino acid positions.
[00108] As used herein, the term "variant Ig portion" refers to a polypeptide
(or
protein) having an amino acid sequence that differs from Fc regions known in
the art,
including the sequence provided herein as SEQ ID NO: 22, at one or more amino
acid
positions.
[00109] The term "reduced N-terminal heterogeneity" refers to a decrease in
the number of proteins in a population having different N-terminal amino acid
residues or that occur in different forms relative to a control protein
population. For
example, expression of a control protein may result in a protein population
comprising proteins with missing N-terminal amino acid residues ranging from
one
amino acid to three amino acids (relative to the predicted translated
protein), also
described as N-1, N-2, and N-3. In this instance, the population of proteins
would
include three different types, i.e., N-1, N-2, and N-3. A population of
proteins having
reduced N-terminal heterogeneity, therefore, would include a population of
proteins
having less than three different N-terminal amino acid residues, e.g., N-1 and
N-2, N-
2 and N-3, or N-1 and N-3 types of proteins or reduced percentage of one or
more of
these types of variant molecules. It should be noted that the N-termial amino
acids
themselves may not be different, e.g., for a protein having an N-terminus of N-
1
relative to the wild-type form, the N-terminal amino acid may be an alanine
and for a
protein having an N-terminus of N-3 relative to the wild-type form, the N-
terminal
amino acid may be also an alanine. Accordingly, in one embodiment, N-terminal
heterogeneity has to do with the overall difference in lengths among the
proteins
within a population (expressed in relative terms to the predicted translated
protein).
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As such, the term "N-#" refers to the number of amino acids deleted from the
amino
terminus of the wild-type translated protein. For example, if a wild-type
predicted
protein.sequence was AAGTY, an N-1 protein would not comprise the initial A
and
would instead begin with AGTY, although both proteins begin with A. Similarly,
an
N-2 protein would begin with GTY.
[00110] In another embodiment, N-terminal heterogeneity can be reduced by
reducing the potential number of variant forms in a population of proteins.
For
example, N-terminal glutamine residues can undergo spontaneous cyclization to
form
pyroglutamic acid which can lead to further heterogeneity. In one embodiment,
the
formation of pyroglutamic acid is reduced or eliminated compared to that
present in
wild-type proteins.
[00111] The term "reduced C terminal heterogeneity" likewise refers to a
decrease in the number of proteins in a population having different C-terminal
amino
acid residues relative to a control protein population. In addition, the term
"C-#"
refers to the number of amino acids deleted from the carboxy terminus of a
protein.
[00112] The term "glycosylation" refers to the covalent linking of one or more
carbohydrates to a polypeptide. Typically, glycosylation is a
posttranslational event
which can occur within the intracellular milieu of a cell or extract
therefrom. The term
glycosylation includes, for example, N-linked glycosylation (where one or more
sugars are linked to an asparagine residue) and/or 0-linked glycosylation
(where one
or more sugars are linked to an amino acid residue having a hydroxyl group
(e.g.,
serine or threonine).
[00113] The phrase "TNF family of receptors" refers to any receptor, whether
naturally membrane bound or secreted (as in the case of osteoprotegerin),
which has
the canonical TNF family cysteine bridging patterns or any receptor which
binds to a
defined member of the TNF family of ligands (e.g. Banner et al 1993). The
claimed
invention in other embodiments relates to TNF family receptor-Ig fusions
obtained by
the methods discussed herein, as well as to pharmaceutical preparations
comprising
them.
1001141 A "signal peptide" or "signal sequence" is a peptide sequence that
directs a newly synthesized polypeptide to which the signal peptide is
attached to the
endoplasmic reticulum (ER) for further post-translational processing and
distribution.
The mature form of a protein refers to the protein without the signal
sequence.
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[00115] As used herein, a "soluble LTBR" is a polypeptide that includes all or
a
portion of the extracellular domain of human LTBR (e.g., an LTBR
immunoglobulin
fusion). Preferred soluble LTBRs are soluble molecules which include
sufficient
sequence from the extracellular region of LTBR that they can bind to a ligand,
e.g., LT
or LIGHT, with at least 10% and preferably at least 50% of the affinity of the
molecule of SEQ ID NO: 1.
[00116] The term "ligand binding domain" or "ligand binding portion" as used
herein refers to any native receptor (e.g., cell surface receptor) or any
region or
derivative thereof retaining at least a qualitative ligand binding ability,
and preferably
the biological activity of a corresponding native receptor.
[00117] The terms "approximately" and "about", as used herein in reference to
a number generally includes numbers that fall within a range of 10% in either
direction of the number (greater than or less than the number) unless
otherwise stated
or otherwise evident from the context (except where such number would exceed
100% of a possible value).
[00118] As defined herein, the term "conservative substitutions" denotes the
replacement of an amino acid residue by another, biologically similar residue.
For
example, one would expect conservative amino acid substitutions to have little
or no
effect on the biological activity, particularly if they represent less than
10% of the
total number of residues in the polypeptide or protein. Preferably,
conservative amino
acids substitutions represent changes in less than 5% of the polypeptide or
protein,
most preferably less than 2% of the polypeptide or protein (e.g., when
calculated in
accordance with SEQ ID NO 5, most preferred conservative substitutions would
represent fewer than 9 amino acid substitutions in the wild type amino acid
sequence). In a particularly preferred embodiment, there is a single amino
acid
substitution in the sequence, wherein the both the substituted and replacement
amino
acid are non-cyclic.
[00119] Other examples of particularly conservative substitutions include the
substitution of one hydrophobic residue such as isoleucine, valine, leucine or
methionine for one another, or the substitution of one polar residue for
another, such
as the substitution of arginine for lysine, glutamic for aspartic acid, or
glutamine for
asparagine, and the like.
Genetically encoded amino acids generally may be divided into four families:
(1)
acidic: aspartate, glutamate; (2) basic: lysine, arginine, histidine; (3)
nonpolar:
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alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan;
and (4) uncharged polar: glycine, asparagine, glutamine, cysteine, serine,
threonine,
tyrosine. Phenylalanine, tryptophan, and tyrosine may be classified jointly as
aromatic
amino acids. In one embodiment, the amino acid at the N-terminal of the
polypeptide
of the invention is a nonpolar, amino acid (i.e., alanine, valine, leucine,
isoleucine,
phenylalanine, metionine, tryptophan, glycine, and cysteine), excluding imino
acids,
i.e., proline.
[00120] The term "treating" refers to administering a therapy in an amount,
manner, and/or mode effective to improve or prevent a condition, symptom, or
parameter associated with a disorder or to prevent onset, progression, or
exacerbation
of the disorder (including secondary damage caused by the disorder), to either
a
statistically significant degree or to a degree detectable to one skilled in
the art.
Accordingly, treating can achieve therapeutic and/or prophylactic benefits. An
effective amount, manner, or mode can vary depending on the subject and may be
tailored to the subject.
[00121] The term "inadequate response", "inadequate responder" or "-IR"
refers to a patient who, as assessed by the patient or a clinician of ordinary
skill,
exhibits insufficient efficacy or intolerable or unacceptable toxicity to a
particular
treatment. Insufficient efficacy can mean a failure to meet a predetermined
level of
response to treatment. In the case of rheumatoid arthritis (RA), for example,
insufficient efficacy may be defined as failure to exhibit at least a 10%,
20%, 25%,
30%, 40%, 50% or more decrease in a clinical parameter of RA, such as tender
joint
count (TJC), swollen joint count (SJC), patient global assessment of disease
activity
[PGA visual analog scale (VAS) 0-10 cm], physician global assessment of
disease
activity (MDGA VAS 0-10 cm) and C-reactive protein (CRP in mg/dl). Intolerable
toxicity can be an adverse reaction to an agent that results in medical need
or
recommendation to discontinue use of the first agent. Examples of intolerable
or
unacceptable toxicity may include hepatic injury or dysfunction, severe
allergic
reaction, severe depression or suicidal ideation, anaphylaxis, or injection
site reaction.
[00122] As used herein, "administered in combination" means that two or more
agents (e.g., the soluble LTBR and the second agent) are administered to a
subject at
the same time or within an interval, such that there is overlap of an effect
of each
agent on the patient. Preferably the administrations of the first and second
agent are
spaced sufficiently close together such that a combinatorial effect, e.g., an
additive or
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synergistic effect, is achieved. The interval can be an interval of hours,
days or
weeks. The agents can be concurrently bioavailable, e.g., detectable, in the
subject.
In a preferred embodiment at least one administration of one of the agents,
e.g., the
first agent, is made while the other agent, e.g., the soluble LTBR, is still
present at a
therapeutic level in the subject.
[00123] The term "weekly" means not more than once within a particular 6 to 8
day period, e.g., once every 7 days.
[00124] The term "biweekly" means not more than once within a particular 12
to 16 day period, e.g., once every 14 days. The term "monthly" means once a
month,
e.g, once every 28 to 3ldays. The subject is typically a mammal, e.g., human,
non-
human primate (such as a monkey or ape), dog, cat, rabbit, or agriculture
mammal
(e.g., horse, cow, pig, and so on). For example, the subject is a human, e.g.,
a human
male or female. The subject can be at least about 18, 25, 30, 45, 50, 55, 60,
or 70
years old.
[00125] As used herein, the term "minimal average concentration" refers to the
mean minimal concentration of drug present in the circulation or in the serum
of a
subject.
[00126] Various aspects of the invention are described in further detail in
the
following subsections.
I LTBR-Ig Fusion Proteins and Compositions Thereof
[00127] The invention pertains to compositions comprising LBTR-Ig fusion
proteins that are improved for therapeutic use in that the population of LTBR-
Ig
fusions proteins has reduced molecular heterogeneity. The invention provides
compositions, including pharmaceutical compositions, comprising LTBR-Ig fusion
proteins, as well as proteins, nucleic acids, vectors, host cells, and methods
of making
the same.
[00128] A lymphotoxin-(3 receptor-immunoglobulin (LT(3R-Ig) fusion protein
can block signaling between the surface LT ligand and the receptor with
consequences on the functional state of follicular dendritic cells (Mackay and
Browning 1998). This blocking can furthermore lead to diminished autoimmune
disease in rodent models (Mackay et al, 1998, U.S. Ser. No. 08/505,606 filed
Jul. 21,
1995 and U.S. Ser. No. 60/029,060 filed Oct. 26, 1996).
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[00129] Generally, preferred soluble LTBRs of the invention are fusion
proteins. A soluble LTBR, as defined herein, is a molecule that includes an LT-
binding fragment of the extracellular domain of LTBR. For example, a soluble
LTBR
can include all or a substantial portion of the extracellular domain of LTBR
(e.g., it
can include residues 40-200, 35-200, 40-210; 35-220, 32-225, or 28-225 of SEQ
ID
NO:1). In one embodiment, a soluble LTBR includes residues 32-225 of SEQ ID
NO: 1. In some embodiments, a soluble LTBR can be modified by covalent
attachment of a moiety, e.g., a heterologous polypeptide (e.g., to make an
LTBR
fusion protein) or a non-polypeptide moiety. In some cases, such moieties can
improve a pharmacodynamic or pharmacokinetic parameter, such as solubility or
half-
life. LTBR fusion proteins can include all or part of the constant region of
an
antibody (e.g., an Fc domain), transferrin, or albumin, such as human serum
albumin
(HSA) or bovine serum albumin (BSA). The fusion protein can include a linker
region between the LTBR sequence and the non-LTBR protein domain. In some
embodiments, a soluble LTBR is modified by covalent attachment to a polymer
such
as a polyethylene glycol (PEG). While not wishing to be bound by theory or
mechanism, such soluble LTBRs can act as decoy receptors to reduce (block)
LTBR
activity. An exemplary LTBR-Fc has the amino acid sequence of SEQ ID NO:5, 6,
8,
9, or 10.
[00130] The soluble LTBR can include all or a fragment of LTBR, e.g., a
soluble fragment of LTBR, fused to one or more heterologous protein domains
(which
domain(s) may increase solubility or lifetime in the blood). An exemplary LTBR
moiety is the LTBR sequence of SEQ ID NO: 1, or a sequence which differs
therefrom
by no more than 1, 2, 3, 5, or 10 amino acid residues. The differences can be
any
difference, e.g., a substitution, deletion or insertion, but is preferably a
substitution,
e.g., a conservative substitution. Conservative substitutions are usually
exchanges of
one amino acid for another with similar polarity, steric arrangement, or of
the same
class (e.g., hydrophobic, acidic or basic). Examples of non-LTBR proteins or
domains include all or part of the constant region of an antibody, e.g., an Fc
domain,
transferrin, or albumin, such as human serum albumin (HSA) or bovine serum
albumin (BSA).
[00131] In a preferred embodiment, the polypeptide of the invention is an Fc
fusion protein containing a polypeptide such as an antibody, and preferably an
IgG
immunoglobulin, e.g., of the subtype IgG1, IgG2, IgG3, or IgG4, and
preferably, of
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the subtype IgGI or IgG4. In a preferred embodiment, the foregoing polypeptide
binds to a ligand of LTBR. Amino acid numberings herein for portions of an Fc
region of a polypeptide correspond to the Kabat numbering system as described,
e.g.,
by Kabat et al., in "Sequences of Proteins of Immunological Interest", U.S.
Dept.
Health and Human Services, 1983 and 1987. In some embodiments, sequential
amino
acid numbering, e.g., for sequences presented in the sequence listing, are
provided.
[00132] In one embodiment, a fusion protein of the invention comprises at
least
a portion of a hinge region, a CH1, a CH2, and a CH3 region of an
immunoglobulin.
[00133] Heterogeneity within a protein population may result from post-
translational modifications such as variable glycosylation patterns, N-
terminal
proteolysis, C-terminal proteolysis, and pyroglutamate formation (also
referred to
herein as pyroglu formation). The invention provides a composition comprising
a
population of lymphotoxin-(3 receptor-immunoglobulin (LT-(3-R-Ig)-fusion
proteins
having reduced heterogeneity, including, but not limited to, reduced N-
terminal
heterogeneity (e.g., with respect to variations in size of molecules or
variantion in the
form of molecules (e.g., pyroglutamic acid containing proteins) or reduced C-
terminal
heterogeneity, as well as combinations thereof. The reductions in
heterogeneity may
be attributed to deletions and/or mutations made within the sequence of the
LTBR-Ig
protein, such that heterogeneity is reduced relative to the wild-type LTBR-Ig
fusion
protein, i.e., unmutated and/or undeleted LTBR-Ig. An example of a wild-type
LTBR-Ig fusion protein is described in SEQ ID NO: 11 (mature form of protein),
also
referred to as LTBRO 1. It should be noted that the terms LT(3R-Ig and LTBR-Fc
are
used interchangeably herein.
[00134] In a preferred embodiment, an LT(3R-Ig fusion protein comprises a
variant LTBR extracellular domain and/or a variant Ig portion, e.g., Fc
portion of an
Ig. In one embodiment of the invention, the LTBR-Ig fusion protein comprises
either
a LTBR extracellular domain variant, a variant Ig portion, or a combination
thereof.
[00135] The amino acid and nucleic acid sequences of wild type LTBR are
described in the NCBI database as AAH26262 and P36941. The wild type human
amino acid sequence of LTBR is also describe as SEQ ID NO: 1. A soluble LTBR
can be an LTBR-Fc polypeptide having the sequence of SEQ ID NO: 1, or
preferably a
variant thereof. In a preferred embodiment the soluble LTBR is an LTBR-Fc
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WO 2008/112325 PCT/US2008/003548
polypeptide which differs from the sequence of SEQ ID NO:1 by no more than 1,
2, 3,
5, or 10 amino acid residues.
Human LTBR sequence (GenPept ID No. P36941)
SEQ ID NO: 1 is the immature or nonprocessed human LTBR sequence, i.e.,
contains
the signal sequence. Amino acids in italics indicate signal sequence. Amino
acids
28-225 are the extracellular region of LTBR.
1 MLLPWATSAP GLAWGPLVLG LFGLLAASQP QAVPPYASEN QTCRDQEKEY YEPQHRICCS
61 RCPPGTYVSA KCSRIRDTVC ATCAENSYNE HWNYLTICQL CRPCDPVMGL EEIAPCTSKR
121 KTQCRCQPGM FCAAWALECT HCELLSDCPP GTEAELKDEV GKGNNHCVPC KAGHFQNTSS
181 PSARCQPHTR CENQGLVEAA PGTAQSDTTC KNPLEPLPPE MSGTMLMLAV LLPLAFFLLL
241 ATVFSCIWKS HPSLCRKLGS LLKRRPQGEG PNPVAGSWEP PKAHPYFPDL VQPLLPISGD
301 VSPVSTGLPA APVLEAGVPQ QQSPLDLTRE PQLEPGEQSQ VAHGTNGIHV TGGSMTITGN
361 IYIYNGPVLG GPPGPGDLPA TPEPPYPIPE EGDPGPPGLS TPHQEDGKAW HLAETEHCGA
421 TPSNRGPRNQ FITHD (SEQ ID NO: 1)
[00136] The term "wild type LTBR-Ig" as used herein, refers to a fusion
protein comprising the extracellular domain of human wild type LTBR, e.g., the
extracellular domain of the LTBR sequence presented in SEQ ID NO: 1, and any
immunoglobulin sequence known in the art which is not modified, for example,
by
mutations, deletions, etc. An exemplary wild type Ig amino acid sequence is
provided
in SEQ ID NO: 22. An example of a wild type LTBR-Ig fusion protein having no
modifications is described in SEQ ID NO: 6.
[00137] In one aspect, the invention pertains to LTBR-Ig fusion proteins
comprising a variant LTBR extracellular domain. For example, the amino acids
"SQPQ" may be deleted from the amino terminal of the wild-type LTBR protein
(mature form), as shown in SEQ ID NO: 4(amino acid sequence of the LTBR
extracellular domain of LTBR06). As demonstrated in the Examples provided
below,
deletion of "SQPQ" from the amino terminal of LTBR improves the overall
heterogeneity within an LTBR-Ig protein population, including N-terminal
heterogeneity. As described in the Examples, expression of LTBR06 provides a
population of LTBR-Ig fusion proteins where at least 90% of the LT-(3-R-Ig-
fusion
proteins are missing no more than 5 amino acids from the N-terminus of the
mature
form of the wild type LT-(3-R extracellular domain set forth in SEQ ID NO:21.
Thus,
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WO 2008/112325 PCT/US2008/003548
expression of LTBR06 results in a population of LTBR-Ig proteins, where 90% of
the
proteins are either N-4 or N-5. In addition, LTBR06 has reduced pyroglutamic
acid
formation.
[00138] In another embodiment, the LTBR-Ig of the invention may include a
variant Ig portion having a deletion in the c-terminal amino acid of the Fc
portion, i.e.,
"K". As described in the Examples below, the deletion of the last amino acid
of an Fc
portion of an LTBR-Ig fusion protein reduces C-terminal heterogeneity. An
example
of a variant Ig portion where the last amino acid has been deleted to improve,
i.e.,
reduce, C-terminal heterogeneity is described in SEQ ID NO: 2. Examples of
LTBR-
Ig fusion proteins having variant Ig portions with a deleted last amino acid,
i.e., the
last lysine, are described in SEQ ID NOs: 5, 8, 9, and 12. The variant Ig
portion may
comprise an Fc region of an IgG isotype, including, but not limited to, an
IgGI
isotype.
[00139] The invention also includes an LTBR-Ig fusion protein having both a
variant LTBR extracellular domain and a variant Ig portion. LTBR06 is an LTBR-
Ig
fusion protein comprising a variant LTBR extracellular domain having the first
four
amino acids removed and a variant Ig portion, wherein the last amino acid (K)
is
deleted. The N-terminal and C-terminal deletions of LTBR06 reduce both N-
terminal
and C-terminal heterogeneity. SEQ ID NO: 8 below describes the amino acid
sequence of LTBR06, including the signal sequence. Amino acids in italics in
the
sequence below indicate the signal sequence; underlined amino acids indicate
sequence derived from the extracellular region of LTBR; and amino acids in
bold
indicate IgG Fc sequence.
[00140] In one embodiment, the immunoglobulin portion of an Ig fusion
protein of the invention comprises at least a portion of an immunoglobulin
hinge
region. In one embodiment, a variant Fc portion may comprise at least one
mutation
in the hinge region, e.g., a mutation of the cysteine (at amino acid position
220 (Kabat
numbering) also shown as amino acid position one of SEQ ID NO:22) of the Ig
upper
hinge to a valine. The subject valine is bolded/underlined/italicized below.
The
underlined sequence is a substantial part of the extracellular domain of LTBR
and
corresponds to amino acids 32 to 225 of SEQ ID NO:1 (above). During
proteolytic
processing, the signal sequence of the LTBR protein is cleaved. Thus, the
final LTBR
protein product for LTBR06 is described in SEQ ID NO: 5.
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WO 2008/112325 PCT/US2008/003548
M L L P W A T S A P G L A W G P L V L G L F G L L A A A V P P Y A S E
N Q T C R D Q E K E Y Y E P Q H R I C C S R C P P G T Y V S A K C S R
I R D T V C A T C A E N S Y N E H W N Y L T I C Q L C R P C D P V M G
L E E I A P C T S K R K T Q C R C Q P G M F C A A W A L E C T H C E L
L S D C P P G T E A E L K D E V G K G N N H C V P C K A G H F Q N T S
S P S A R C Q P H T R C E N Q G L V E A A P G T A Q S D T T C K N P L
E P L P P E M S G T M V D K T H T C P P C P A P E L L G G P S V F L F
P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y
V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W
L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V
Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S
N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q
Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G Stop
(SEQ ID NO: 8)
[00141] In addition to the valine mutation described in SEQ ID NO: 8, other
suitable amino acids may be used. For example, amino acids including serine,
threonine, alanine, leucine, glycine, or isoleucine may be used in place of
the valine of
LTBR06, and the other LTBR-Ig fusion proteins described herein.
[00142] In an optional embodiment, an immunoglobulin hinge be used to link
an LTBR extracellular domain to, e.g., the CH1, CH2, and CH3 domains of an
immunoglobulin molecule. For example, an IgG hinge region having the sequence
CDKTHTCPPCPAPELLGGP may be used. In one embodiment, a hinge region
having the sequence VDKTHTCPPCPAPELLGGP may be used. Other exemplary
upper and middle hinge constructs are shown in Figures 6 to 8, as well as SEQ
ID
NOs: 13 to 20. Thus, the variant Ig portion of the LTBR-Ig fusion protein may
include an upper and middle hinge region comprising at least 90% to 95%
identity to
the hinges described in SEQ ID NOs: 13 to 20. The upper and middle hinge
region
set forth in SEQ ID NO: 13 was used in the variant LTBR-Ig fusion proteins
exemplified herein. Accordingly, LTBR-Ig fusion proteins comprising variant
hinges,
e.g., those described in SEQ ID NOs: 14 to 21, would be constructed by
replacing the
sequence set forth in SEQ ID NO: 13 with the variant hinge. Other variant
hinge
molecules that can optionally be used to link an LTBR extracellular domain to,
e.g.,
the CH1, CH2, and CH3 domains of an immunoglobulin molecule are disclosed in
20050163782A1, the contents of which are incorporated by reference herein.
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[00143] When an LTBR-Ig fusion protein, such as LTBR06, is expressed, the
population of proteins which results includes a variety of overall lengths
due, at least
in part, to N-terminal and/or C-terminal proteolysis, as well as heterogeneity
of
pyroglu formation. An important aspect of the invention is the discovery that
by
deleting the first four amino acids of the LTBR-Ig fusion protein
(specifically the first
four amino acids of the LTBR extracellular domain), heterogeneity can be
reduced,
including N-terminal heterogeneity. Thus, in one embodiment, the invention
features
a composition comprising a population of LT(3R-Ig-fusion proteins which
comprise a
variant LT[3-R extracellular domain of 193 or 194 amino acids in length and a
variant
Ig portion of 227 amino acids in length, wherein at least 90% of the LT-(3-R-
Ig-fusion
proteins are missing no more than 5 amino acids from the N-terminus of the
mature
form of the wild type LT-P-R extracellular domain set forth in SEQ ID NO:21
and
wherein the LT-(3-R-Ig-fusion proteins lack pyroglutamic acid. The invention
also
features a composition comprising a population of LT-[3-R-Ig fusion proteins
comprising a variant LT-P-R extracellular domain of 193 or 194 amino acids in
length
and a variant Ig portion, wherein the population has reduced N-terminal
pyroglutamic
acid formation, and reduced C-terminal heterogeneity compared to wild-type LT-
(3-R-
Ig fusion proteins.
[001441 The amino terminal amino acid of the variant LTBR-Ig fusion protein
may be an alanine, as set forth in SEQ ID NOs: 5 and 12, or, alternatively,
may be a
non-polar amino acid. Examples of non polar amino acids which may be used as
the
first amino acid of the LTBR-Ig fusion protein include a valine (amino acid
six of the
mature form of the wild type LT-P-R portion SEQ ID NO:1) or an alanine (amino
acid five of the mature form of the wild type LT-P-R portion SEQ ID NO:1). In
addition, to non-polar amino acids, serine or threonine may be used. In one
embodiment, the composition of the invention includes a population of LTBR-Ig
fusion proteins whereby the N-terminus of at least 95% of the LT-[i-R-Ig-
fusion
proteins is either a valine (amino acid six of the mature form of wild type LT-
[3-R) or
an alanine (amino acid five of the mature form of wild type LT-(3-R).
[00145] In addition to N- and/or C-terminal heterogeneity, heterogeneity may
also result from variable glycosylation. In order to minimize heterogeneity
resulting
from glycosylation variations within a protein population of LTBR-Ig fusion
proteins,
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the LTBR and/or the Ig may be altered to reduce the amount of glycosylation.
In one
embodiment, the Ig portion of the LTBR-Ig fusion protein is non-glycosylated.
[00146] The invention also contemplates a composition comprising a
population of LT(3R-Ig fusion proteins comprising a variant LT-P-R
extracellular
domain and a variant Ig portion, wherein variant LT-P-R extracellular domain
is
aglycosylated. An example of an aglycosylated extracellular domain of LTBR is
provided in the Examples, and is also described in SEQ ID NO: 10 (see amino
acids 1
to 194 of SEQ ID NO: 10).
[00147] Thus, examples of post-translational heterogeneity that may be
decreased using the methods and compositions described herein include N-
terminal
heterogeneity, C-terminal heterogeneity, and glycosylation heterogeneity. It
is within
the scope of the invention to provide LTBR-Ig fusion proteins having reduced
heterogeneity in each of these parameters, as well as combinations thereof.
For
example, the invention includes compositions comprising a population of LT-(3-
R-Ig
fusion proteins, wherein the population has reduced N-terminal heterogeneity
and
reduced C-terminal heterogeneity, compared to a population of wild-type LT-(3-
R-Ig
fusion proteins.
[00148] Methods of treating autoimmune disorders using the above
compositions and LTBR-Ig fusion proteins, as well as pharmaceutical
compositions
comprising the same, are described in more detail in sections II and III
below.
1001491 An LTBR-Ig fusion polypeptide of the invention retains the ligand
binding activity of LTBR, and includes those polypeptides which have an amino
acid
sequence that has at least 70% homology to the LTBR-Ig polypeptides set forth
in
SEQ ID NOs: 5, 6, 8, 9, 10, 11, 12, and 23 and variants and derivatives of
each of the
foregoing. The invention also includes isolated polypeptides described in SEQ
ID
NOs: 3, 4, and 7, which provide either LTBR extracellular domains of the
invention
or Ig variants of the invention. Preferably the LTBR-Ig polypeptide (or
portion
thereof) has an amino acid sequence greater than 85% homology, more preferably
greater than 90% homology, more preferably greater than 95% homology, most
preferably greater than 99% homology, to the foregoing sequences.
[00150] In one embodiment, the invention features an isolated polypeptide
comprising an amino acid sequence which is at least 90% identical to the full
length
of the mature form of SEQ ID NO:8 or SEQ ID NO:23, wherein the amino acid
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sequence of the ligand binding domain is unchanged, the polypeptide further
comprising a truncated Fc region. In one embodiment, the polypeptide of the
invention comprises an amino acid sequence is at least 95% identical to the
full length
of the mature form of the polypeptide set forth in SEQ ID NO: 8. In one
embodiment,
the polypeptide of the invention comprises an amino acid sequence of the
mature
form of the polypeptide set forth in SEQ ID NO: 8. In one embodiment, the
polypeptide of the invention comprises the amino acid sequence set forth in
SEQ ID
NO: 5.
[00151] Variants of the polypeptides described herein are also contemplated by
the invention. Thus, variants of the polypeptide having an amino acid sequence
that
differs from the sequence presented in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO:
23, as well as portions thereof described in SEQ ID NO: 3, SEQ ID NO: 4, and
SEQ
ID NO: 7, at one or more amino acid positions, are included in the invention.
Such
variant polypeptides include the modified polypeptides described above, as
well as
conservative substitutions, splice variants, isoforms, homologues from other
species,
and polymorphisms. the
[00152] Modifications of an LTBR-Ig primary amino acid sequence may result
in proteins which have substantially equivalent activity as compared to the
unmodified counterpart polypeptide, and thus may be considered functional
analogous
of the parent proteins. Such modifications may be deliberate, e.g. as by site-
directed
mutagenesis, or they may occur spontaneous, and include splice variants,
isoforms,
homologues from other species, and polymorphisms. Such functional analogs are
also
contemplated according to the invention.
[00153] Moreover, modifications of the primary amino acid sequence may
result in proteins which do not retain the biological activity of the parent
protein,
including dominant negative forms, etc. A dominant negative protein may
interfere
with the wild-type protein by binding to, or otherwise sequestering regulating
agents,
such as upstream or downstream components, that normally interact functionally
with
the polypeptide. Such dominant negative forms are also contemplated according
to the
invention.
[00154] The LTBR-Ig proteins of the invention may be made according to
standard methods known in the art. A nucleic acid molecule encoding LTBR-Ig
may
be used to express a LTBR-Ig polypeptide, e.g., by expressing a LTBR-Ig
polypeptide
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in vivo, or by administering a nucleic acid molecule encoding LTBR-Ig to an
animal
for in vivo expression. Nucleic acid molecules encoding LTBR-Ig may be
included
within a nucleic acid vector, e.g., an expression vector or a cloning vector.
A nucleic
acid molecule encoding a LTBR-Ig may, but need not of necessity, be
maintained,
reproduced, transferred, or expressed as part of a nucleic acid vector. Thus,
another
aspect of the invention is isolated nucleic acid molecules which encode the
LTBR-Ig
proteins of the invention described herein. The invention includes a nucleic
acid
molecule comprising the nucleotide sequence set forth in SEQ ID NO:7 (mature
form
of LTBR06).
[00155] A recombinant expression vector containing a LTBR-Ig polynucleotide
sequence can be introduced into and/or maintained within a cell. Cells hosting
a
LTBR-Ig vector may be prokaryotic. Alternatively, a LTBR-Ig nucleic acid can
be
introduced into a eukaryotic cell, e.g., a eukaryotic cell that contains the
appropriate
apparati for post-translational processing of a polypeptide into a mature
protein,
and/or the appropriate apparati for secreting a polypeptide into the
extracellular
environment of the cell. Also encompassed within the scope of the invention
are
vectors comprising nucleic acid molecules encoding the LTBR-Ig fusion proteins
of
the invention.
1001561 Suitable methods of making LTBR-Ig proteins of the invention are
known in the art and are described, for example, in WO 97/03687, WO 98/17313,
WO
00/21558, WO 99/38525, WO 00/36092. For example, an LTBR immunoglobulin
fusion protein can be expressed in cell culture (e.g., mammalian cell culture
(such as
monkey cos cells or Chinese hamster ovary cells) or yeast cell culture) at a
reduced
temperature, e.g, to produce an increased amount of properly folded fusion
protein.
Also included within the scope of the invention are host cells expressing LTBR-
Ig
fusions proteins of the invention, where the host cell comprises a vector
comprising a
nucleic acid encoding an LTBR-Ig fusion protein. In one embodiment, the host
cell is
a Chinese Hamster Ovary (CHO) cell. The expressed fusion protein can be
purified,
e.g., by affinity or conventional chromatography techniques. See WO 00/36092.
Expression of the LTBR-Ig fusion protein may range in scale, including
manufacturing scale.
[00157] The invention further provides a process for making a composition
comprising a population of LT[3R-Ig-fusion proteins which comprise a variant
LT-(3-
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R extracellular domain and a variant Ig portion, wherein at least 90% of the
LT[3R-Ig-
fusion proteins are missing no more than 5 amino acids from the N-terminus of
the
mature form of the wild type LT-(3-R portion set forth in SEQ ID NO: 1, the
process
comprising, expressing a nucleic acid molecule encoding the LT-(3-R- Ig fusion
protein set forth in SEQ ID NO:8 in a mammalian cell, obtaining the population
from
the culture supernatant, and, optionally, purifying the supernatant, to
thereby obtain a
composition comprising a population of LT(3R-Ig-fusion proteins which comprise
a
variant LT-(3-R extracellular domain and a variant Ig portion, wherein at
least 90% of
the LT-(3-R-Ig-fusion proteins are missing no more than 5 amino acids from the
N-
terminus of the mature form of the wild type LT-(3-R portion set forth in SEQ
ID
NO: 1. In one embodiment, the process comprises expressing a nucleic acid
molecule
set forth in SEQ ID NO:7 in a mammalian cell. In one embodiment, the process
comprises expressing a nucleic acid molecule set forth in SEQ ID NO:7 in a
mammalian cell. The invention also features expressing a nucleic acid molecule
comprising a nucleotide sequence encoding the extracellular domain of LTBR set
forth in SEQ ID NO:4, or the nucleic acid sequence set forth in SEQ ID NO: 3
in a
mammalian cell. The invention further features a composition which is made by
expressing a nucleic acid molecule encoding the LT-[i-R- Ig fusion protein set
forth
in SEQ ID NO:8, or the nucleic acid sequence set forth in SEQ ID NO: 7, in a
mammalian cell.
11. Uses of LTBR-IgG of Invention for Treating Autoimmune Disorders
[00158] Soluble LTBRs are lymphotoxin (LT) pathway inhibitors useful for
treating autoimmune disorders. Autoimmune disorders include, for example,
autoimmune arthritides (including rheumatoid arthritis (RA) and Sjogren's
syndrome)
psoriasis, multiple sclerosis, inflammatory bowel disease (IBD) (including
ulcerative
colitis and Crohn's disease), insulin-dependent diabetes, uveitis, systemic
lupus
erythematosus (SLE, or lupus), polychondritis, and transplant rejection. The
agents
and methods described herein are particulary suitable for treatment of RA.
[00159] Rheumatoid arthritis is marked by tenderness in the joints. Synovial
thickening eventually occurs in most effected joints. Stiffness lasting >30
minuts
upon arising in the morning or after prolonged inactivity is common, as is
early
afternoon fatigue and malaise. Deformities, particularly flexion contractures
may
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develop rapidly. Carpal tunnel syndrome can result from wrist synovitis. X-
rays may
reveal soft-tissue swelling in the first months of disease, and subsequently,
perarticular osteoporosis, joint space narrowing, and marginal erosions may be
present.
[00160] Various tests can be employed to assay the efficacy of a soluble LTBR
to treat autoimmune disorders. In the case of rheumatoid arthritis (RA), for
example,
insufficient efficacy may be defined as failure to exhibit at least a 10%,
20%, 25%,
30%, 40%, 50% or more decrease in a clinical parameter of RA, such as tender
joint
count (TJC), swollen joint count (SJC), patient global assessment of disease
activity
[PGA visual analogue scale (VAS) 0-10 cm, for measurement of pain], physician
global assessment of disease activity (MDGA VAS 0-10 cm), levels of C-reactive
protein (CRP in mg/dl), and erythrocyte sedimentation rate (ESR). C-reactive
protein
is produced by the liver during episodes of acute inflammation or infection.
CRP
levels in blood serum are an indicator of the severity of RA. A decrease in
CRP
levels, such as by 5%, 10%, 15%, 20%, or more, can be an indication of
effective
treatment of autoimmune disorder, such as RA, in response to treatment with a
soluble
LTBR, such as LTBR-Fc. Erythrocyte sedimentation rate (ESR) is also elevated
in
90% of RA cases. Thus a decrease in ESR following treatment with a soluble
LTBR
can also be an indication of effective treatment.
1001611 The high potency of the soluble LTBR demonstrated herein indicates
that low dosage regimes would be equally effective for treatment of other
autoimmune conditions. Autoimmune conditions suitable for treatment as
described
herein with a soluble LTBR, e.g., an LTBR-Fc, include, e.g., autoimmune
arthritides
(including rheumatoid arthritis and Sjogren's syndrome), psoriasis, multiple
sclerosis,
inflammatory bowel disease (IBD) (including ulcerative colitis and Crohn's
disease),
insulin-dependent diabetes, uveitis, systemic lupus erythematosus (SLE, or
lupus),
polychondritis, and transplant rejection.
Exemplary Dosing Regimens
[00162] The invention is based, in part, on the discovery that low dosage or
frequency regimens of a soluble form of LTBR (e.g., LTBR-Fc) can effectively
treat
symptoms of an autoimmune disorder, and rheumatoid arthritis (RA) in
particular.
Accordingly, in one aspect, the invention provides methods of treating
autoimmune
disease in a subject. The method includes administering to the subject an LTBR
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blocking agent (e.g., a soluble LTBR fusion protein, e.g., LTBR-Fc) at a low
dose
and/or frequency.
[00163] As shown in Figure 14, comparisons of LTBR-Fc and a typical
antibody, show that at identical dosing significant differences in efficacy
are observed
for prolonged time frames. This results because LTBR-Fc has high affinity for
the
target ligand, in addition to antibody like pharmacokinetics and is therefore
effective
at very low doses. Arrows A and B indicate the typical alpha and beta phases
for an
antibody or Fc-fusion protein, respectively. For an antibody the gray line
indicated by
arrow C shows typical lower limit concentration for efficacy, whereas arrow D
shows
LTBR-Fc has efficacy at significantly lower concentrations.
1001641 In one embodiment, a soluble LTBR fusion protein is administered to
achieve an average concentration of between about 0.14 ug/ml to about 3.5
ug/ml. In
one embodiment, a soluble LTBR fusion protein is administered to achieve a
minimal
average concentration of about 0.3 to about 1.0 ug/m., e.g., of about 0.6 to
0.7 ug/ml.
In another embodiment, higher doses of the fusion protein may be administered.
For
example, the fusion protein may be administered to achieve an average
concentration
of between about 5 and 15 ug/ml.
[00165] In another embodiment, a soluble LTBR fusion protein is administered
at a dose of between about 70 and about 200 mg/month to an average subject
weighing about 75 kg. In another embodiment, a soluble LTBR fusion protein is
administered at a dose of about 100, about 150, or about 175 mg/month to an
average
subject weighing about 75 kg.
[00166] Embodiments of the invention can include administration of a regimen
of LTBR blocking agent, e.g., a soluble LTBR, such as an LTBR fusion protein
(e.g.,
LTBR-Ig), for the treatment of autoimmune arthritides (including rheumatoid
arthritis
and Sjogren's syndrome), psoriasis, multiple sclerosis, inflammatory bowel
disease
(IBD) (including ulcerative colitis and Crohn's disease), insulin-dependent
diabetes,
uveitis, systemic lupus erythematosus (SLE, or lupus), polychondritis, and
transplant
rejection.
[00167] In one embodiment, the subject is treated with a soluble LTBR, e.g.,
an
LTBR immunoglobulin fusion. Exemplary LTBR-Ig fusion proteins are described
herein.
[00168] In one embodiment, the subject has one or more symptoms of RA, e.g.,
joint swelling, joint pain, joint stiffness, or difficulty moving. For
example, the
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subject has active RA disease (e.g., tender joint count (TJC) or swollen joint
count
(SJC) > 5 or synovitis). In another embodiment, the subject is at risk for
developing
RA, e.g., a family history, or an autoimmune disease.
[00169] In one embodiment, the soluble LTBR can be administered in an
amount and/or for a time sufficient to alleviate the symptoms associated with
RA.
[00170] In another embodiment, the soluble LTBR, e.g., an LTBR-Fc, is
administered in an amount sufficient to improve symptoms in one or more RA
assessment criterion, e.g., a criterion described herein. For example, the
soluble
LTBR is administered in an amount sufficient to improve symptom scores at
least
about 10%, 20%, 25%, 30%, 40%, 50% or more. Symptom scores refer, for example,
to TJC, SJC, patient global assessment of disease activity [PGA visual analog
scale
(VAS) 0-10 cm], physician global assessment of disease activity (MDGA VAS 0-10
cm) quantities of C-reactive protein (CRP in mg/dl), or Disease Activity
Score, 28-
joint version (DAS28). Symptom scores can be reported according to criteria
set forth
by the American College of Rheumatology (ACR). An ACR score is an indication
of
the percent clinical improvement in RA symptoms. For example, an ACR20 is an
indication of a 20% clinical improvement in TJC and SJC, as well as a 20%
improvement in three of the following five parameters: (i) patient's global
assessment, (ii) physician's global assessment, (iii) patient's assessment of
pain, (iv)
degree of disability, and (v) level of acute-phase reactant. ACR scores of
ACR50 and
ACR70 can also be used to indicate a 50% improvement, or a 70% improvement,
respectively.
[00171] In one embodiment, the soluble LTBR, e.g., LTBR-Fc, is administered
more than once, e.g., weekly, biweekly, or monthly. The course of treatment
can be
maintained for a period of time, such as for 1 or 2 weeks or more; 1, 2, 3, 4,
5, or 6
months or more; 1 year; or longer. In certain embodiments, the soluble LTBR is
administered at a dosage of about 0.03 to 3 mg/kg of body weight per
administration,
e.g., about 0.6 to about 1.4 mg/kg per administration. In another embodiment,
the
soluble LTBR is administered at a dosage of about 0.3 -3, e.g., 1 mg/kg of
body
weight per administration, e.g., about 0.6 to about 1.4 mg/kg per
administration. In
other embodiments, the soluble LTBR is administered at a dosage of about 2.5
to 3.5
mg/kg of body weight per administration, e.g., about 2.8 to 3 mg/kg per
administration. In yet other embodiments, the soluble LTBR is administered at
a
dosage of about 0.01 to 3 mg/kg of body weight per administration, e.g., about
0.01 to
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about 0.05 mg/kg per administration, about 0.01 to about 0.3 mg/kg per
administration, about 0.01 to 0.2 mg/kg per administration, or about 0.01 to
about 0.1
mg/kg per administration. In some embodiments, the soluble LTBR is
administered at
about 0.03 mg/kg per administration, about 0.05 mg/kg per administration,
about 0.07
mg/kg per administration, about 0.1 mg/kg per administration, or about 0.2
mg/kg per
administration. In some embodiments, the soluble LTBR is administered at a
dosage
shown in Table 1.
Table 1. Exemplary dosages of LTBR-Fc (mp,per kg of body weight of subject per
administration)
Weekly dosage Biweekly Dose Monthly Dose
0.01-0.3; 0.01-0.3; 0.3-3;
0.011-0.29; 0.011-0.29; 0.3-3.5
0.011-0.25; 0.011-0.25;
0.011-0.2; 0.011-0.2;
0.02-0.05 0.02-0.05
0.3-3.5 0.3-3.5
0.01; 0.01; 0.29;
0.011; 0.011; 0.3;
0.02; 0.02; 0.5;
0.03; 0.03; 0.6;
0.04; 0.04; 0.7;
0.05; 0.05; 0.8;
0.06; 0.06; 1;
0.1; 0.1; 1.4;
0.2; 0.2; 2;
0.29; 0.29; 2.5;
0.3; 0.3; 3;
0.4; 0.4; 3.5
0.5; 0.5;
0.6; 0.6;
1; 0.7;
1.4; 1;
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2; 1.4;
2.5; 2;
3; 2.5;
3.5 3;
3.5
[00172] Generally, the lower unit doses disclosed herein can be administered
with more frequent dosing (e.g., weekly) while higher unit doses disclosed
herein
(though still low by relative standards) can be administered with more
infrequent
dosing (e.g., monthly). In some embodiments, the soluble LTBR, e.g., LTBR-Fc,
is
administered at a dosage of about 0.3 mg/kg per administration to about 3
mg/kg per
administration, e.g., about 0.6 mg/kg per day to about 1.4 mg/kg per day. For
example, the dose is about 1 mg/kg, or about 3 mg/kg. In some embodiments, the
dose is administered once every 14-20 days, once or twice monthly, e.g., once
or
twice every 28-31 days. For example, the dose is about 1 mg/kg or about 3
mg/kg
administered every 14 to 20 days, e.g., every 12 to 16 days, or about every
two weeks.
Alternatively, the dose is about 1 mg/kg or about 3 mg/kg administered
monthly, e.g.,
about every 28 to 31 days.
[00173] In other embodiments, the soluble LTBR, e.g., LTBR-Fc, is
administered at a dosage of about 0.6 mg/kg per administration to about 1.4
mg/kg per
administration not more than twice every 20 to 40 days, e.g., not more than
twice
every 25 to 35 days, or not more than twice every 28-31 days. In other
embodiments,
the soluble LTBR, e.g., LTBR-Fc, is administered at a dosage of about 2.5
mg/kg per
administration to about 3.5 mg/kg per administration not more than twice every
20 to
40 days, e.g., not more than twice every 25 to 35 days, or not more than twice
every
28-31 days.
[00174] In yet other embodiments, the soluble LTBR, e.g., LTBR-Fc, is
administered at a dosage of about 0.01 mg/kg per administration to about 0.3
mg/kg
per administration, e.g., about 0.01 mg/kg per day to about 0.25 mg/kg per
day, e.g.,
weekly, or every 3 to 10 days, repeated at least twice. For example, the
soluble LTBR
is administered at a dosage of about 0.02, 0.03, 0.04, 0.05, 0.06, 0.1, or 0.2
mg/kg,
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administered weekly or every 3-10 days, e.g., every 4, 5, 6, 7, 8, or 9 days.
In some
embodiments, the soluble LTBR is administered at a dosage shown in Table 1.
[00175] In another embodiment, the soluble LTBR is administered at a dosage
of about 0.01 mg/kg per day to about 0.3 mg/kg per day, e.g., 0.02 mg/kg per
day to
about 0.25 mg/kg per day, e.g., biweekly, or every 5 to 20 days, repeated at
least
twice. For example, the soluble LTBR is administered at a dosage of about
0.03, 0.05,
0.08, 0.1, or 0.2 mg/kg, administered biweekly, or every 5-20 days, e.g.,
every 6, 8,
10, 12, 14, 16 or 18 days. In some embodiments, the soluble LTBR is
administered at
a dosage shown in Table 1.
[00176] In another embodiment, the soluble LTBR is administered at a dosage
of about 0.3 mg/kg per day to about 3 mg/kg per day, e.g., 1 mg/kg per day,
e.g.,
biweekly, or every 5 to 20 days, repeated at least twice. In some embodiments,
the
soluble LTBR is administered at a dosage shown in Table 1.
[00177] In another embodiment, the soluble LTBR is administered at a dosage
of about 0.3 mg/kg per day to about 3 mg/kg per day, e.g., 0.4 mg/kg per day
to about
3 mg/kg per day, e.g., monthly, or every 15 to 45 days, repeated at least
twice. For
example, the soluble LTBR is administered at a dosage of about 0.5, 0.8, 1,
1.5, or 2
mg/kg, administered monthly, or every 15-45 days, e.g., every 16, 18, 20, 25,
30, 35
or 40 days. In some embodiments, the soluble LTBR is administered at a dosage
shown in Table 1.
[00178] In certain embodiments, the dose of soluble LTBR, e.g., LTBR-Fc, is
about 0.4 mg to about 375 mg, about 0.4 mg to about 6.25 mg, about 2 mg to
about
6.25 mg, or about 4 mg to about 12.5 mg.
[00179] In certain embodiments, the invention provides for the administration
of a particularly low dose of a soluble LTBR, e.g., an LTBR-Fc, for treatment
of an
autoimmune disorder, e.g., RA.
[00180] In certain embodiments, the soluble LTBR is administered
intravenously or parenterally, e.g., subcutaneously or intramuscularly.
[00181] In another embodiment, the soluble LTBR is administered as a
monotherapy.
[00182] In one aspect, the invention provides methods of treating an
autoimmune condition, e.g., RA, in a subject, such as a human. The method
includes
administering to the human a dose of a soluble LTBR, e.g., LTBR-Fc, wherein
the
dose is between about 0.01 mg and about 3 mg LTBR-Fc per kg body weight of the
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human (mg/kg), e.g., between about 0.01 mg/kg and about 0.05 mg/kg. In one
embodiment, the human is administered a weekly dose of a soluble LTBR, e.g.,
LTBR-Fc, over the course of at least two weeks, where the dose is about 0.01
mg/kg
per day to about 0.3 mg/kg per day, e.g., about 0.01 mg/kg per day to about
0.25
mg/kg per day, administered weekly, e.g., every 3 to 10 days. For example, the
soluble LTBR is administered at a dosage of about 0.02, 0.03, 0.04, 0.05,
0.06, 0.1, or
0.2 mg/kg per day, e.g., every 4, 5, 6, 7, 8, or 9 days. In some embodiments,
the
soluble LTBR is administered at a dosage shown in Table 1.
[00183] In another embodiment, the human is administered a biweekly dose of
a soluble LTBR, e.g., LTBR-Fc, over the course of at least four weeks, where
the dose
is about 0.01 mg/kg per day to about 0.5 mg/kg per day, e.g., about 0.01 mg/kg
per
day to about 0.3 mg/kg per day repeated every 5 to 20 days. For example, the
soluble
LTBR is administered at a dosage of about 0.03, 0.05, 0.08, 0.1, 0.2, 0.3 or
0.4 mg/kg,
e.g., every 6, 8, 10, 12, 14, 16 or 18 days. In some embodiments, the soluble
LTBR is
administered at a dosage shown in Table 1.
[00184] In another embodiment, the human is administered a monthly dose of a
soluble LTBR, e.g., LTBR-Fc, over the course of at least two months, where the
dose
is about 0.1 mg to about 3 mg soluble LTBR per kg body weight of the human per
day. In one embodiment, the dose is about 0.3 mg/kg per day to about 3 mg/kg
administered monthly, e.g., every 15 to 45 days. For example, the soluble LTBR
is
administered at a dosage of about 0.5, 0.8, 1, 1.5, or 2 mg/kg per day, e.g.,
every 16,
18, 20, 25, 30, 35 or 40 days. In some embodiments, the soluble LTBR is
administered at a dosage shown in Table 1.
[00185] In some embodiments, a patient exhibiting an inadequate response to a
therapy has not shown a clinically acceptable or significant improvement in
response
to the therapy. In other embodiments, the patient initially showed an
improvement in
response to a therapy but no longer demonstrates an improvement, as assessed
by a
standard clinical measure for the specific disorder. A DMARD-IR subject is a
subject
who has had an inadequate response to a disease modifying antirheumatic drug
(DMARD), such as methotrexate, leflunomide (Arava ), anakinra (Kineret(D),
hydroxycholoquine sulfate (Plaquenil ) antimalarials, gold salts,
sulfasalazine
(Azulfidine ), minocycline (Minocin ), d-penicillamine, cyclosporin A,
cyclosporine (Neoral ), cyclophosphamide and azathioprine (Imuran ).
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[00186] In one aspect, the invention features a delivery device, e.g., a
transcutaneous delivery device, e.g., a syringe, designed for subcutaneous or
intramuscular administration, where the device is packaged with or contains at
least
one unit dose of a soluble LTBR, e.g., LTBR-Fc, such that an appropriately low
quantity of the agent will be administered to a human. In one embodiment, the
device
contains or is packaged with a unit dose of LTBR-Fc such that administration
to a
human will deliver between about 0.01 mg/kg and 3 mg/kg LTBR-Fc, e.g., about
0.01
to about 0.05 mg/kg LTBR-Fc, or about 0.6 mg/kg and 1.4 mg/kg LTBR-Fc, to the
human. In another embodiment, the device contains or is packaged with a unit
dose
of LTBR-Fc such that administration to a human will deliver between about 2.5
mg/kg and 3.5 mg/kg LTBR-Fc to the human. In certain embodiments the delivery
device will deliver about 1 mg/kg or about 3 mg/kg LTBR-Fc to the human.
Exemplary unit dose amounts appropriate for humans of various weights is
provided
in Table 2.
1001871 In one embodiment, the liquid in the second compartment is water or a
buffer. The liquid can include, e.g., a pharmaceutically acceptable carrier,
such as a
solvent, dispersion media, antibacterial or antifungal agent, or isotonic or
absorption-
delaying agent. The liquid may also include a pharmaceutically acceptable
salt.
Table 2. Exemplary dose of LTBR-Fc (mg) accordin tgo weight of human
Weight of 40-50 kg 50-60 kg 60-75 kg 75-100 kg 100-125 kg
human:
Final
concentrationa:
0.01 mg/kg 0.4-0.5 0.5-0.6 0.6-0.75 0.75-1.0 1.0-1.25
0.02 mg/kg 0.8-1.0 1.0-1.2 1.2-1.5 1.5-2.0 2.0-2.5
0.03 mg/kg 1.2-1.5 1.5-1.8 1.8-2.25 2.25-3.0 3.0-3.75
0.05 mg/kg 2-2.5 2.5-3 3-3.75 3.75-5.0 5.0-6.25
0.1 mg/kg 4-5 5-6 6-7.5 7.5-10 10-12.5
0.2 mg/kg 8-10 10-12 12-15 15-20 20-25
0.3 mg/kg 12-15 15-18 18-22.5 22.5-30 30-37.5
0.5 mg/kg 20-25 25-30 30-37.5 37.5-50 50-62.5
0.7 mg/kg 28-35 35-42 42-52.5 52.5-70 70-87.5
1.0 mg/kg 40-50 50-60 60-75 75-100 100-125
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1.5 mg/kg 60-75 75-90 90-112.5 112.5-150 150-187.5
2.0 mg/kg 80-100 100-120 120-150 150-200 200-250
2.5 mg/kg 100-125 125-150 150-187.5 187.5-250 250-312.5
3.0 mg/kg 120-150 150-180 180-225 225-300 300-375
3.5 mg/kg 140-175 175-210 210-262.5 262.5-350 350-437.5
amg LTBR-Fc per kg of body weight of human
[00188] In certain embodiments, the unit dose of the soluble LTBR, e.g.,
LTBR-Fc included in a delivery device is about 0.4 mg to about 375 mg, about
0.4 mg
to about 6.25 mg, about 2 mg to about 6.25 mg, or about 4 mg to about 12.5 mg.
In
one embodiment, the device contains lyophilized LTBR-Fc.
[00189] In one aspect, the invention features a kit including two or more unit
doses of between about 0.4 mg to about 375 mg LTBR-Fc. The unit doses are such
that an appropriately low quantity will be administered to a human, as
determined by
the weight of the human. Exemplary unit dose amounts appropriate for humans of
various weights is provided in Table 2.
[00190] In another aspect, the invention features a device, e.g., a
transcutaneous delivery device, e.g., a syringe having at least two
compartments,
where a first compartment contains a unit dose of lyophilized LTBR-Fc and a
second
compartment contains a liquid for reconstituting the LTBR-Fc prior to
administration
to a subject. For example, he unit dose is such that administration of the
LTBR-Fc
will deliver between about 0.01 to 3 mg/kg, e.g., 1.0 mg/kg LTBR-Fc to the
human.
For example, in some embodiments, the unit dose of LTBR-Fc is between about
0.4
mg and about 375 mg LTBR-Fc, e.g., between about 0.4 mg and about 6.25 mg
LTBR-Fc. The unit doses are such that an appropriately low quantity will be
administered to a human, as determined by the weight of the human. Exemplary
unit
dose amounts appropriate for humans of various weights is provided in Table 2.
1001911 In another aspect, the invention features a method of instructing a
patient having rheumatoid arthritis to treat the RA by (i).providing the
patient with at
least two unit doses of LTBR-Fc; and (ii) instructing the patient to self-
administer the
unit doses, e.g., subcutaneously, one dose at a time. In certain embodiments,
the dose
delivered will be between about 0.01 mg and 3 mg LTBR-Fc per kg of body weight
of
the patient (mg/kg), e.g., between about 0.6 mg/kg and 1.4 mg/kg, or about
0.01
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mg/kg and 0.05 mg/kg. In one embodiment, the dose delivered will be between
about
2.5 to 3.5 mg/kg LTBR-Fc per kg of body weight of the patient (mg/kg). In
certain
embodiments, the patient is instructed to self-administer the doses weekly,
over the
course of at least 2 weeks; biweekly, over the course of at least 4 weeks; or
monthly,
over the course of at least 2 months. In certain embodiments, the unit dose is
about
0.01 to about 3.0 mg/kg LTBR-Fc per administration. In certain embodiments,
the
unit dose is about 0.01 to about 2.5 mg/kg per administration, about 0.02 to
about 0.5
mg/kg per administration, about 0.01 to about 0.3 mg/kg per administration,
about
0.01 to about 0.2 mg/kg per administration, about 0.01 to about 0.1 mg/kg per
administration, or about 0.01 to about 0.05 mg/kg per administration. In
certain
embodiments, the unit dose is about 0.03 mg/kg, about 0.05 mg/kg, about 0.07
mg/kg,
about 0.1 mg/kg, or about 0.2 mg/kg per administration.
[00192] In one embodiment, the patient is instructed to self-administer the
doses weekly, over the course of at least 2 weeks, where the unit dose is
about 0.01
mg/kg to about 3.0 mg/kg LTBR-Fc per administration, e.g., about 0.01 mg/kg to
about 0.25 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg LTBR-Fc per
administration. For example, the unit dose is about 0.02, 0.03, 0.04, 0.05,
0.06, 0.1,
or 0.2 mg/kg per administration.
1001931 In one embodiment, the patient is instructed to self-administer the
doses biweekly, over the course of at least 4 weeks, where the unit dose is
between
about 0.01 and1.5 mg/kg, e.g., about 0.01 to about 0.3, or about 0.02 mg/kg to
about
0.3 mg/kg or about 0.5-1.25 mg/kg LTBR-Fc. For example, the unit dose is about
0.03, 0.05, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, or 1.0 mg/kg per
administration.
[00194] In one embodiment, the patient is instructed to self-administer the
doses monthly, over the course of at least 2 months, where the unit dose is
between
about 0.03 to about 3 mg LTBR-Fc per kg of body weight of the patient (mg/kg).
For
example, the unit dose is about 0.2, 0.5, 0.8, 1, 1.5, or 2 mg/kg.
[00195] In another aspect, the invention features a method of managing RA in a
patient including (i) instructing a patient to stop taking a therapy to treat
RA, and (ii)
administering to the patient a unit dose of LTBR-Fc. In one embodiment, the
unit
dose is between about 0.03 to about 3 mg/kg LTBR-Fc, e.g., about 0.6 to about
1.4
mg/kg LTBR-Fc. In another embodiment, the unit dose is about 2.5 to about 3.5
mg/kg LTBR-Fc. In other embodiments, the unit dose is between about 0.01 to
about
0.05 mg/kg LTBR-Fc.
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[00196] In another embodiment, the unit dose is between about 0.3 mg/kg and
3 mg/kg LTBR-Fc, and the unit dose is administered not more than twice every
20-40
days, e.g., every 25-35 days, or 28-31 days. In another embodiment, the unit
dose is
between about 0.6 mg/kg and about 1.4 mg/kg LTBR-Fc, administered not more
than
twice every 20-40 days, e.g., every 25-25 days, or every 28-31 days.
[00197] In another embodiment, the unit dose is between about 0.01 mg/kg and
0.3 mg/kg LTBR-Fc, and the unit dose is administered not more than once every
3-10
days, e.g., weekly. In another embodiment, the unit dose is between about 0.01
mg/kg
and about 0.3 mg/kg LTBR-Fc administered not more than once every 5-20 days,
e.g.,
biweekly. In another embodiment, the unit dose is between about 0.3 mg/kg and
about 3 mg/kg LTBR-Fc administered not more than once every 28-31 days, e.g.,
monthly.
[00198] In one embodiment, the unit dose is about 0.01 mg/kg to about 0.25
mg/kg LTBR-Fc, and the unit dose is administered weekly over the course of at
least
2 weeks. For example, the unit dose is about 0.02, 0.03, 0.04, 0.05, 0.06,
0.1, or 0.2
mg/kg per administration.
[00199] In another embodiment, the unit dose is about 0.02 mg/kg to about 0.5
mg/kg LTBR-Fc, and the unit dose is administered biweekly over the course of
at
least 4 weeks. For example, the unit dose is about 0.03, 0.05, 0.08, 0.1, 0.2,
0.3 or 0.4
mg/kg per administration.
[00200] In another embodiment, the unit dose is about 0.03 to about 3 mg
LTBR-Fc, and the unit dose is administered monthly over the course of at least
2
months. For example, the unit dose is about 0.2, 0.5, 0.8, 1, 1.5, or 2 mg/kg
per
administration.
[00201] In one embodiment, the patient is instructed to stop an NSAID,
corticosteroid, or DMARD therapy.
[00202] In one aspect, the invention features a method of managing RA in a
patient that includes administering to the patient a unit dose of a soluble
LTBR, e.g.,
LTBR-Fc, where (i) the unit dose is between about 0.01 mg and about 0.05 mg of
the
soluble LTBR per kg of body weight of the patient (mg/kg); (ii) the unit dose
is
between about 0.01 mg/kg and 0.3 mg/kg LTBR-Fc administered not more than once
every 3-10 days, e.g., weekly; (iii) the unit dose is between about 0.01 mg/kg
and 1
mg/kg LTBR-Fc administered not more than once every 5-20 days, e.g., biweekly;
(iv) the unit dose is between about 0.3 mg/kg and 3 mg/kg LTBR-Fc (e.g.,
between
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about 0.6 and 1.4 mg/kg LTBR-Fc) administered not more than twice every 20-40
days, e.g., every 25-35 days, or every 28-31 days, or (v) the unit dose is
between
about 2.5 mg/kg and 3.5 mg/kg LTBR-Fc administered not more than once every 20-
40 days, e.g., every 25-35 days, or every 28-31 days. By this method, the
patient
receives a first therapy, e.g., an NSAID, corticosteroid, or DMARD therapy, to
treat
RA prior to the administration of the soluble LTBR, and the levels of the
first therapy
are maintained in the patient at least when the first unit dose of the soluble
LTBR is
administered to the patient. In one embodiment, the levels of the first
therapy are not
maintained in the patient after the first or second unit dose of the soluble
LTBR. For
example, the levels of the first therapeutic agent are diminished, e.g., by
administration of lower doses of the first therapeutic agent, or by ceasing
administration and allowing the levels of the first therapeutic agent to be
cleared from
the body. In another embodiment, the patient continues to receive the first
therapy
during the administration of the soluble LTBR.
Combination Therapies
[00203] The methods and compositions described herein can be used in
combination with other therapies, such as non-steroidal anti-inflammatory
agents
(NSAIDs), corticosteroids, and DMARDs.
[00204] NSAIDs typically relieve pain by reducing inflammation. Suitable
NSAIDs include, for example, aspirin, ibuprofen, naproxen, and ketoprofen. COX-
2
inhibitors are a similar class of drugs that can be used in combination with a
soluble
LTBR for treatment of an autoimmune disorder.
[002051 DMARDs typically slow the progression of rheumatoid arthritis.
Suitable DMARDS include methotrexate, leflunomide (Arava ), anakinra
(Kineret ), hydroxycholoquine sulfate (Plaquenil ) antimalarials, gold salts,
sulfasalazine (Azulfidine ), minocycline (Minocin ), d-penicillamine,
cyclosporin
A, cyclosporine (Neoral ), cyclophosphamide and azathioprine (Imuran ).
DMARDs also include TNF inhibitors. Tumor necrosis factor alpha (TNF-a) is a
pro-
inflammatory cytokine produced by macrophages and lymphocytes. TNF inhibitors
help to relieve the proinflammatory effects of this molecule. TNF inhibitors
include
etanercept (EnbrelTM), infliximab (RemicadeTM), and adalimumab (HumiraTM).
[00206] Corticosteroids typically function as anti-inflammatory agents and can
also be used in combination therapy with a soluble LTBR.
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[00207] In certain embodiments, a soluble LTBR is use as a second line
therapy. For example, a patient who is determined to be a DMARD-IR will stop
receiving treatment with DMARD and will begin treatment with a soluble LTBR,
e.g.,
LTBR-Fc. In some embodiments, a patient receiving a combination therapy of
NSAID and DMARD, will stop receiving the DMARD and will begin receiving a
soluble LTBR in combination with the NSAID. The soluble LTBR may be
administered in combination with NSAID, or separately. For example, a patient
receiving a daily dose of NSAID, may only receive a weekly, or biweekly, or
monthly
dose of a soluble LTBR. Alternatively, the patient will continue to receive
the
DMARD and/or NSAID therapies while receiving treatment with the soluble LTBR.
[00208] In another embodiment, the soluble LTBR is administered in
combination with a second treatment for RA. For example, the combination
therapy
includes administering a second agent that provides a therapeutic benefit to a
patient
who has or is at risk for RA. Exemplary second agents include, e.g., non-
steroidal
anti-inflammatory agents (NSAIDs), corticosteroids, or disease modifying
antirheumatic drugs (DMARDs).
[00209] In one embodiment, the subject is treated with an RA drug after being
diagnosed with RA and prior to administration of a soluble LTBR. The RA drug
can
be, for example, an NSAID, corticosteroid, or DMARD. In another embodiment,
the
subject is evaluated to determine if the response to the RA drug is inadequate
prior to
administration of the soluble LTBR. In certain embodiments, if the subject is
determined to have an inadequate response to the RA drug, then the subject is
administered a soluble LTBR. In one embodiment, the subject is determined to
be
asymptomatic, or an adequate responder, for a first manifestation of RA, such
as TJC
or SJC. In another embodiment, the subject is determined to be asymptomatic,
or an
adequate responder, for a first manifestation of RA, such as TJC or SJC, and
symptomatic, or an inadequate responder for a second manifestation of
rheumatoid
arthritis, such as synovitis. Synovitis can be detected by any method known in
the art,
including, for example, by magnetic resonance imaging (MRI).
[00210] In one embodiment, the soluble LTBR and the second agent are
administered at the same time. In another embodiment, the soluble LTBR is
administered first in time and the second agent is administered second in
time. In
another embodiment, the second agent is administered first in time and the
soluble
LTBR is administered second in time. The soluble LTBR can replace or augment a
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previously or currently administered therapy. For example, upon treating with
LTBR,
administration of the second agent can cease or diminish, e.g., be
administered at
lower levels. In other embodiments, administration of the previous therapy is
maintained. In some embodiments, a previous therapy will be maintained until
the
level of LTBR reaches a level sufficient to provide a therapeutic effect. The
two drugs
can be administered in combination.
[00211] In one embodiment, a human receiving a first therapy for RA, e.g., an
NSAID, corticosteroid, or DMARD, can also be administered a soluble LTBR,
e.g.,
LTBR-Fc. In one embodiment, when the human is administered the soluble LTBR,
the first therapy is halted. In another embodiment, the human is monitored for
a first
preselected result, e.g., an improvement in RA symptoms, e.g., a decrease in
TJC or
SJC by 10%, 20%, 30%, or more. In one embodiment, when the first preselected
result is observed, treatment with the soluble LTBR is decreased or halted. In
one
embodiment, the human is then monitored for a second preselected result after
treatment with the soluble LTBR is halted, e.g., a worsening of an RA symptom,
e.g.,
an increase in TJC or SJC by 10%, 20%, 30% or more. When the second
preselected
result is observed, administration of the soluble LTBR to the human is
reinstated or
increased, or administration of the first therapy is reinstated, or the human
is
administered both a soluble LTBR, or an increased amount of soluble LTBR, and
the
first therapeutic regimen.
1002121 In one embodiment, a human receiving a first therapy for RA, who is
then treated with a soluble LTBR, e.g., an LTBR-Fc, continues to receive the
first
therapy at the same or a reduced amount. In another embodiment, treatment with
the
first therapy overlaps for a time with treatment with the soluble LTBR, but
treatment
with the first therapy is subsequently halted.
[00213] In one embodiment, the subject is a DMARD inadequate responder
(DMARD-IR). For example, the subject is an anti-TNF inadequate responder.
[00214] In one embodiment, the method includes evaluating the subject for an
improvement in RA symptoms. In some embodiments, the evaluation is performed
at
least 1 hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1
day, 2 days, 4
days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks,
10
weeks, 13 weeks, 20 weeks or more, after the administration of the soluble
LTBR.
The subject can be evaluated in one or more of the following periods: prior to
beginning of treatment; during treatment; or after one or more elements of the
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treatment have been administered. Evaluating can include evaluating the need
for
further treatment with the same soluble LTBR or for additional treatment with
additional agents. In a preferred embodiment, if a preselected outcome of the
evaluation is obtained, an additional step is taken, e.g., the subject is
administered
another treatment or another evaluation or test is performed.
III. Pharmaceutical Compositions and Pharmaceutical Administration of
Invention
[00215] A soluble LTBR, e.g., LTBR-Fc, can be formulated as a pharmaceutical
composition, e.g., for administration to a subject to treat an autoimmune
disorder,
such as RA. Typically, a pharmaceutical composition includes a
pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable carrier"
includes any
and all solvents, dispersion media, coatings, antibacterial and antifungal
agents,
isotonic and absorption delaying agents, and the like that are physiologically
compatible. The composition can include a pharmaceutically acceptable salt,
e.g., an
acid addition salt or a base addition salt (see e.g., Berge et al., J. Pharm.
Sci. 66:1-19,
1977).
[00216] The soluble LTBR can be formulated according to standard methods.
Pharmaceutical formulation is a well-established art, and is further
described, e.g., in
Gennaro (ed.), Remington: The Science and Practice,:of Pharmacy, 20th ed.,
Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,
:.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott
Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.),
Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd
ed. (2000) (ISBN: 091733096X).
[00217] In one embodiment, a soluble LTBR (e.g., LTBR-Fc) can be
formulated with excipient materials, such as sodium chloride, sodium dibasic
phosphate heptahydrate, sodium monobasic phosphate, and a stabilizer. It can
be
provided, for example, in a buffered solution at a suitable concentration and
can be
stored at 2-8 C.
1002181 The pharmaceutical compositions may be in a variety of forms. These
include, for example, liquid, semi-solid and solid dosage forms, such as
liquid
solutions (e.g., injectable and infusible solutions), dispersions or
suspensions, tablets,
pills, powders, liposomes and suppositories. The preferred form can depend on
the
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intended mode of administration and therapeutic application. Typically
compositions
for the agents described herein are in the form of injectable or infusible
solutions.
[00219] Such compositions can be administered by a parenteral mode (e.g.,
intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The
phrases
"parenteral administration" and "administered parenterally" as used herein
mean
modes of administration other than enteral and topical administration, usually
by
injection, and include, without limitation, intravenous, intramuscular,
intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid,
intraspinal, epidural, intracerebral, intracranial, intracarotid and
intrasternal injection
and infusion.
[00220] The composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable for stable storage
at high
concentration. Sterile injectable solutions can be prepared by incorporating
an agent
described herein in the required amount in an appropriate solvent with one or
a
combination of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating an agent
described
herein into a sterile vehicle that contains a basic dispersion medium and the
required
other ingredients from those enumerated above. In the case of sterile powders
for the
preparation of sterile injectable solutions, the preferred methods of
preparation are
vacuum drying and freeze-drying that yields a powder of an agent described
herein
plus any additional desired ingredient from a previously sterile-filtered
solution
thereof. The proper fluidity of a solution can be maintained, for example, by
the use
of a coating such as lecithin, by the maintenance of the required particle
size in the
case of dispersion and by the use of surfactants. Prolonged absorption of
injectable
compositions can be brought about by including in the composition an agent
that
delays absorption, for example, monostearate salts and gelatin.
[00221] In certain embodiments, the soluble LTBR may be prepared with a
carrier that will protect the compound against rapid release, such as a
controlled
release formulation, including implants, and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Many methods for the preparation of such formulations are patented or
generally
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known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[00222] A soluble LTBR (e.g., LTBR-Fc) can be modified, e.g., with a moiety
that improves its stabilization and/or retention in circulation, e.g., in
blood, serum, or
other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. The modified agent
can be
evaluated to assess whether it can reach sites of inflammation such as may
occur in an
autoimmune disorder, such as RA (e.g., by using a labeled form of the agent).
[00223] For example, the soluble LTBR can be associated with a polymer, e.g.,
a substantially non-antigenic polymer, such as a polyalkylene oxide or a
polyethylene
oxide. Suitable polymers will vary substantially by weight. Polymers having
molecular number average weights ranging from about 200 to about 35,000
Daltons
(or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
[00224] For example, a soluble LTBR can be conjugated to a water soluble
polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or
polyvinylpyrrolidone. A non-limiting list of such polymers include
polyalkylene
oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers thereof,
provided that the water solubility of the block copolymers is maintained.
Additional
useful polymers include polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene
(Pluronics); polymethacrylates; carbomers; and branched or unbranched
polysaccharides.
[00225] When the soluble LTBR (e.g., LTBR-Fc) is used in combination with a
second agent (e.g., a DMARD), the two agents can be formulated separately or
together. For example, the respective pharmaceutical compositions can be
mixed,
e.g., just prior to administration, and administered together or can be
administered
separately, e.g., at the same or different times.
[00226] In one embodiment, a pharmaceutical composition comprises a
population of lymphotoxin-[3 receptor (LT-0-R)-Ig-fusion proteins which
comprise a
variant LT-(3-R extracellular domain of 193 or 194 amino acids in length and a
variant
Ig portion of 227 amino acids in length, wherein at least 90% of the LT-0-R-Ig-
fusion
proteins are missing no more than 5 amino acids from the N-terminus of the
mature
form of the wild type LT-(3-R extracellular domain set forth in SEQ ID NO:21
and
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wherein the LT-[3-R-Ig-fusion proteins lack N-terminal pyroglutamic acid and a
pharmaceutically acceptable carrier.
[00227] In another embodiment, a pharmaceutical composition comprises a
population of lymphotoxin-(3 receptor-immunoglobulin (LT-[i-R-Ig)-fusion
proteins,
the fusion proteins comprising a variant LT-0-R extracellular domain of 193 or
194
amino acids in length and a variant Ig portion, wherein the population has
reduced N-
terminal pyroglutamic acid formation and reduced C-terminal heterogeneity
compared
to wild-type LT-[i-R-Ig fusion proteins and a pharmaceutically acceptable
carrier.
[00228] In yet another embodiment a pharmaceutical composition of the
invention comprises the amino acid sequence set forth in SEQ ID NO:5.
Administration.
[00229] A soluble LTBR (e.g., LTBR-Fc) can be administered to a subject, e.g.,
a human subject, by a variety of methods. For many applications, the route of
administration is one of: intravenous injection or infusion (IV), subcutaneous
injection (SC), intraperitoneally (IP), or intramuscular injection. In some
cases,
administration may be directly into the CNS, e.g., intrathecal,
intracerebroventricular
(ICV), intracerebral or intracranial. The agent can be administered as a fixed
dose, or
in a mg/kg dose.
[00230] The dose can also be chosen to reduce or avoid production of
antibodies against the agent.
[00231] The route and/or mode of administration of the soluble LTBR can also
be tailored for the individual case, e.g., by monitoring the subject, e.g.,
using TJC,
SJC, CRP levels and standard parameters associated with RA or other autoimmune
diseases, e.g., the assessment criteria described herein.
[00232] Dosage regimens are adjusted to provide the desired response, e.g., a
therapeutic response or a combinatorial therapeutic effect. Generally, a low
dose of a
soluble LTBR (e.g., LTBR-Fc) optionally formulated separately or together with
an
appropriate dose of a second therapeutic agent can be used to provide a
subject with
the soluble LTBR. Exemplary doses of the soluble LTBR are described herein.
[00233] Dosage unit form or "fixed dose" as used herein refers to physically
discrete units suited as unitary dosages for the subjects to be treated; each
unit
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contains a predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required pharmaceutical
carrier and
optionally in association with the other agent. Suitable administration
frequencies are
described elsewhere herein.
[00234] A pharmaceutical composition may include a therapeutically effective
amount of a soluble LTBR described herein. Such effective amounts can be
determined based on the effect of the administered agent, or the combinatorial
effect
of an agent and secondary agent if more than one agent is used. A
therapeutically
effective amount of an agent may also vary according to factors such as the
disease
state, age, sex, and weight of the individual, and the ability of the compound
to elicit a
desired response in the individual, e.g., amelioration of at least one
disorder
parameter, e.g., RA parameter, or amelioration of at least one symptom of the
disorder, e.g., RA. A therapeutically effective amount is also one in which
any toxic
or detrimental effects of the composition is outweighed by the therapeutically
beneficial effects. Typically, a therapeutically effective amount is a low
dose as
described elsewhere herein.
Devices and Kits
[00235] Pharmaceutical compositions that include a soluble LTBR (e.g., an
LTBR-Fc) can be administered with a medical device. The device can be designed
with features such as portability, room temperature storage, and ease of use
so that it
can be used in emergency situations, e.g., by an untrained subject or by
emergency
personnel in the field, removed to medical facilities and other medical
equipment.
The device can include, e.g., one or more housings for storing pharmaceutical
preparations that include a soluble LTBR, and can be configured to deliver one
or
more unit doses of the agent.
[00236] For example, the pharmaceutical composition can be administered with
a transcutaneous delivery device, such as a syringe, including a hypodermic or
multichamber syringe. Other suitable deliver devices include stents,
catheters,
transcutaneous patches, microneedles, and implantable controlled release
devices.
[00237] In other examples, the pharmaceutical composition can be
administered with a needleless hypodermic injection device, such as the
devices
disclosed in US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880;
4,790,824; or
4,596,556. Examples of well-known implants and modules include: US 4,487,603,
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which discloses an implantable micro-infusion pump for dispensing medication
at a
controlled rate; US 4,486,194, which discloses a therapeutic device for
administering
medicants through the skin; US 4,447,233, which discloses a medication
infusion
pump for delivering medication at a precise infusion rate; US 4,447,224, which
discloses a variable flow implantable infusion apparatus for continuous drug
delivery;
US 4,439,196, which discloses an osmotic drug delivery system having multi-
chamber compartments; and US 4,475,196, which discloses an osmotic drug
delivery
system. Many other devices, implants, delivery systems, and modules are also
known.
[00238] A soluble LTBR (e.g., an LTBR-Fc) can be provided in a kit. In one
embodiment, the kit includes (a) a container that contains a composition that
includes
a soluble LTBR, and optionally (b) informational material. The informational
material can be descriptive, instructional, marketing or other material that
relates to
the methods described herein and/or the use of the agents for therapeutic
benefit. In
one embodiment, the kit also includes a second agent for treating an
autoimmune
disorder, such as a DMARD for treatment of RA. For example, the kit includes a
first
container that contains a composition that includes the soluble LTBR, and a
second
container that includes the second agent.
[00239] The informational material of the kits is not limited in its form. In
one
embodiment, the informational material can include information about
production of
the compound, molecular weight of the compound, concentration, date of
expiration,
batch or production site information, and so forth. In one embodiment, the
informational material relates to methods of administering the soluble LTBR
(e.g.,
LTBR-Fc), e.g., in a suitable dose, dosage form, or mode of administration
(e.g., a
dose, dosage form, or mode of administration described herein), to treat a
subject who
has an autoimmune disorder, or who is at risk for experiencing an episode
associated
with an autoimmune disorder. The information can be provided in a variety of
formats, including printed text, computer readable material, video recording,
or audio
recording, or information that provides a link or address to substantive
material.
[00240] In addition to the agent, the composition in the kit can include other
ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The
agent can
be provided in any form, e.g., liquid, dried or lyophilized form, preferably
substantially pure and/or sterile. When the agents are provided in a liquid
solution,
the liquid solution preferably is an aqueous solution. When the agents are
provided as
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a dried form, reconstitution generally is by the addition of a suitable
solvent. The
solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
1002411 The kit can include one or more containers for the composition or
compositions containing the agents. In some embodiments, the kit contains
separate
containers, dividers or compartments for the composition and informational
material.
For example, the composition can be contained in a bottle, vial, or syringe,
and the
informational material can be contained in a plastic sleeve or packet. In
other
embodiments, the separate elements of the kit are contained within a single,
undivided
container. For example, the composition is contained in a bottle, vial or
syringe that
has attached thereto the informational material in the form of a label. In
some
embodiments, the kit includes a plurality (e.g., a pack) of individual
containers, each
containing one or more unit dosage forms (e.g., a dosage form described
herein) of
the agents. The containers can include a combination unit dosage, e.g., a unit
that
includes both the soluble LTBR and the second agent, e.g., in a desired ratio.
For
example, the kit includes a plurality of syringes, ampules, foil packets,
blister packs,
or medical devices, e.g., each containing a single combination unit dose. The
containers of the kits can be air tight, waterproof (e.g., impermeable to
changes in
moisture or evaporation), and/or light-tight.
[00242] The kit optionally includes a device suitable for administration of
the
composition, e.g., a syringe or other suitable delivery device. The device can
be
provided pre-loaded with one or both of the agents or can be empty, but
suitable for
loading.
EXAMPLES
Example 1: Construction and characterization of LTBR-IgG constructs to
reduce heterogeneity
[00243] The following example describes the characterization of a number of
different LTBR immunoglobulin fusion proteins which were created to solve
molecular heterogeneities found in the expression of LTBRIgG, including N-
terminal
heterogeneity, C-terminal heterogeneity, pyroglutamic formation, and
sialylation.
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N-terminal heterogeneity
[00244] Predictions of the N-termini encoded by the full length mRNA of
LTBR (NP_002333) were done by programs such as SignalP using either neural
networks (NN) predictions or with hidden Markov models (HMM) trained on
eukaryotes. These two NN and HMM prediction models gave different results for
the
suggested N-termini of the protein, suggesting S28 or P30 and Q31,
respectively
(amino acid numbers refer to numbering including the signal sequence). Thus,
numerous N-terminal sequence predictions were possible and only by expressing
the
protein and evaluating the posttranslational modifications could the optimal N-
termini
with limited variability be determined.
[00245] LTBRO l(LTBR-IgG fusion protein version 01) was the initial
molecule that was cloned. The N-terminus of LTBROI started at S28 based on
intact
LTBR numbering, i.e., including signal sequence. To the LTBR C-termini at M225
was fused the hinge Fc of an IgG1 starting at C220 (Kabat numbering) of an IgG
heavy chain. A mutation of the antibody derived cysteine (C220) of the IgG 1
hinge to
the structurally similar amino acid valine (V 199 in LTBRO 1) was included
LTBRIgG.
This mutation eliminated the problematic and unwanted formation of a free
unpaired
thiol in the LTBRIgG, in the IgGl C220 of the heavy chain is normally
disulfide
bonded with the C 107 of the light chain, lacking the light chain the cysteine
(C220)
would initially remain unpaired in LTBRIgG it would cause either unwanted
scrambling of the CRDs within LTBR or aggregation of the LTBRIgG.
[002461 For each LTBRIgG molecule described the genes were constructed,
expressed, purified and evaluated for variability in the N-terminal sequence
and C-
terminal sequence. Results showed that LTBR01 with the S28 N-termini of LTBR
was heterogeneous with multiple clipping sites at N-1, N-3 and N-4 as well as
a
pyroglutamic generated at Q31(numbering based on full length LTBR sequence
described in Figure 1, including the signal sequence) and was missing the C-
terminal
K426 from the Fc domain.
1002471 Based on these findings, two different constructs, named LTBR05 and
LTBR06, were created to improve heterogeneity among the expressed LTBRIgG
fusion proteins, particularly with respect to the N and C terminal
proteolysis.
[00248] LTBR05 was constructed with a C-terminal deletion mutant to avoid
having a C-terminal lysine that might undergo proteolysis. Analysis of LTBR05
by
mass spectrophotometry (MS) confirmed no further C-terminal truncations upon
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elimination of the C-terminal lysine, but comparable N-terminal variability to
LTBR01.
[00249] Therefore, to improve N-terminal heterogeneity, LTBR06 was
constructed. LTBR06 was designed to have four amino acids deleted from the N-
terminus. This deletion resulted in dramatically decreased N-terminal sequence
variability, as described in Table 1. LTBR06 had more than 90% of the desired
N-
termini (N-4 and N-5 relative to wild type) and N-1 N-termini. The deletions
of
LTBR06 are also described in Figures 1 and 3.
[00250] Table 3 provides an overview of the different constructs which were
created by deleting portions of the N and C termini of LTBR, as well as the
results
from such deletions. LTBR05 had a single amino acid removed from the carboxy
terminus of LTBROI (C-1), and LTBR06 had the single C-1 amino acid deletion as
well as 4 amino acids deleted from the amino terminus (following the signal
sequence
- termed N-4). Thus, LTBR06 was engineered to reduce both N- and C-terminal
heterogeneity. A comparison of the mature forms of the different constructs is
set
forth in Figure 2.
Table 3: Comparison of LTBR05 and LTBR06 vs. LTBRO 1
Construct name Features Purpose Results
LTBR01 Wt sequence 1. Characterization 1. N-terminus N-1, N-3, N-4
of protein sequence 2. pyroGlu in N-1 and N-3 forms
2. Activity 3. C-terminus w/splice variant
4. Live and dead material
LTBR05 C-1 1. Remove C 1. No C-terminal splice variant,
terminal splice C-terminus homogeneous
variant of pMDR
plasmid
LTBR06 N-4 / C-1 1. Reduce N- 1. N-terminus N-4 and N-5, no N
terminal and C- terminal pryoGlu
terminal 2. Fully active ligand binding
heterogeneity
2. Maintain activity
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1002511 LTBR05 was biochemically characterized, including the N-terminal
heterogeneity (results summarized in Table 4). The amino-terminus of LTBR05
was
found to exist as N, N-1, and N-3 with the N-1 and N-3 forms converted
predominantly to pyroglutamate (<Q).
Table 4: Summary of the biochemical characteristics of LTBR05
tAttribute Test - Result
Concentration UV absorbance 15.0 A280/ml (5.0 mg/mi)
Purity SDS-PAGE Red ~90 %
SDS-PAGE Nonred. > 90 %
Aggregation SEC assay < 1 %
..... ........ ._.... ..._..... .-.-....................................... -
.......... ;% Inactive ~HIC assay (~ 1 %
Endotoxin Chromogenic assay 0.3 EU/mg or 1.5 EU/ml
N-terminal Edman degradation 33% Sequencing yield (due to <Q)
Sequence ~ 71%N, 29%N-1
Intact MS After deglycosylation, reduction Theor. Observed
and alkylation.
N/C-1 50271.55 50269
N-1<Q/C-1 50166.47 50166
N-3<Q/C-1 49941.22 49939
Peptide map LC chromatography of the Endo Corresponds to characterized map of
Lys-C digest LTBR C-1
;LC/MS Endo Lys-C digest 23 peptides identified that account for
95% of the predicted sequence.
[002521 LTBR06 was biochemically characterized to determine whether
heterogeneity was improved, including whether the N- and C- terminal
heterogeneity
was improved based on the amino acid substitutions (results summarized in
Table 5).
Table 5: Summary of the biochemical characteristics of LTBR06
Attribute Test Result
Concentration UV absorbance 25.0 A280/ml (25.0 m ml
Purity SDS-PAGE Red SDS-PAGE > 90 %
Nonred. > 90 %
A re ation SEC assay < 1%
% Inactive HIC assay < 1 %
Endotoxin Chromogenic assay 0.052 EU/mg or 1.3EU/ml
N-terminal Edman degradation 56% N-5, 40% N-4, -4% N-11
Sequence
Peptide map LC chromatography of the Corresponds to characterized map of
Endo Lys-C digest LTBR05
LC/MS Endo Lys-C digest 31 peptides identified account for 95% of
the predicted amino acid sequence.
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[00253] Edman degradation was performed on LTBR06 from LTBR06
expressing cells. As described in Table 5, 56% of the population of LTBR06
proteins
were found to be N-5, 40% were N-4, and about 4% were N-11. The sequence of
the
N-5 species of LTBRIg is described in SEQ ID NO: 23. Using the N-4 and C-1
deletions, the heterogeneity of the protein population was decreased, in
comparison to
LTBR05 (which represents unmodified extracellular domain of the LTBR fused to
a
variant).
Example 2: Glycosylation studies of LTBRIgG constructs
[00254] Glycosylation can also have an impact on protein heterogeneity, as
well as activity. To reduce heterogeneity further, therefore, glycosylation
mutants
were explored to determine whether variable LTBR domain glycosylation impacted
binding affinity, expression levels, and folding. LTBR09 was a construct based
on
LTBR06, with an additional N275Q mutation (amino acid position refers to
mature
protein - for comparison of sequence and changes between various constructs
see
Figure 2).
1002551 Table 6 describes the glycosylation occupancy for each of three LTBR
constructs at Asn13 and Asn150.
Table 6: Glycosylation occupancy
Construct % site occupancy Asn13 % occupancy Asn150
LTBRO5 28* 86
LTBR06 26** 85
LTBR09 22 * * 84
* Calculated as an average of intact N-terminus, N-I <Q and N-3 <Q
**Calculated as an average of N-4 and N-5
[00256] As shown in Table 6, glycosylation occupancy was relatively invariant
within the different constructs for Asn 13 and Asn150.
1002571 To reduce heterogeneity further, glycosylation mutants were explored
in which the N-terminal glycosylation of LTBRIgG was eliminated in either the
LTBR extracellular domain or Fc portion of the protein. Elimination of
glycosylation
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sites in the LTBR domain successfully reduced glycosylation and also decreased
expression of this protein about ten fold. Table 7 provides an overview of the
various
mutants and the results from studies which characterized the effect(s) of the
deletion
of glycosylation sites.
Table 7: Additional glycosylation mutants
Construct Features Purpose Results
name
LTBR02 N13Q 1. Expression level of 1 1. Expression slightly reduced
glyc. mutation in animal 2. Active in ligand binding
cells
2. Activity assessment
LTBR03 N13Q, N150Q 1. Expression level of 2 1. Expression reduced
glyc. mutations in significantly: about 10% of wt
animal cells in CHO stable
2. Activity assessment 2. Wildtype ligand binding.
3. No effect on folding.
LTBR04 N13Q, N150Q, 1. Expression level of 3 1.Expression reduced a lot.
N275Q glyc. mutations in 2.Active in ligand binding
animal cells 3. Expression from yeast
2. Activity assessment possible.
3. Microbial
[00258] LTBR03 is shown in Figure 5 and SEQ ID NO: 10. Aglycosylated
LTBR03 showed binding equivalency to wildtype LTBRIgG (unmodified LTBR
extracellular domain and unmodified Fc region) in a competitive FACS assay.
[002591 Soluble aglycosylated LTBRIgG was also generated by first capturing
the protein using Protein A (primary capture). The captured protein was then
run over
a phenyl column to remove misfolded and aggregated proteins. Following phenyl
chromatography, agly LTBRIgG was purified into a homogenous population using
sephacryl S-200. Soluble LTBR was then generated using Asp N digestion, where
the
digest was subsequently re-exposed to Protein A to remove the Fc portion of
the
fusion protein. The sLTBR was then purified using DEAE (which removed the Asp
N) and gel filtration / characterization, the latter of which is an optional
polishing
step. Finally, a primary crystal screen was performed.
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Example 3: Sialylation effect on LTBRIgG pharmacokinetics
1002601 In order to study the effects of sialylation on LTBRIgG
pharmacokinetics, sialyation of LTBRIgG was forced using a-2,6-
Sialyltransferase
from rat liver (Boehringer Mannheim). a-2,6-Sialytransferase is specific for
Ga(31 --> 4G1cNAc, and will sialylate 75-100% of terminal N-linked galactose
residues. Sialylation of LTBRIgG achieved various levels of sialylated
protein, which
were then evaluated for pharmacokinetics. Pharmacokinetic data of low, medium,
and
high sialylated LTBRIg (version LTBR05) was analyzed in mouse sera. The
results
are described below in Tables 8 and 9.
Table 8: Sialylation (SA) of LTBR-Ig PK Data (concentrations in uv/ml)
time time time time time time (hr)
(hr) (hr) (hr) (hr) (hr)
sialylation mouse # 0 0.25 7 24 72 168
low I blq 75 23 17 1.4 0.11
low 2 blq 130* 30 22 2.5 0.62
low 3 blq 62 23 19 1.6 < 0.02
middle 4 blq 92 42 29 3.2 0.94
middle 5 blq 89 38 26 3.8 0.050
middle 6 blq 73 41 29 2.9 0.087
high 7 blq 110 47 32 3.9 0.3
high 8 blq 110 50 34 3.9 0.49
high 9 blq 110 42 34 4.3 0.64
blq=below limit of quantitation
* not included in average described in Table 8
Table 9: Average SA from Table 8
time (hr) time (hr) time (hr) time (hr) time (hr) time (hr)
0 0.25 7 24 72 168
low 0 69 25 19 2 0
middle 0 85 40 28 3 0
high 0 110 40 33 4 0
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Figure 13 describes the difference in pharmacokinetics between huLTBRIgG and
LFA3TIP (dimeric fusion protein that consists of the extracellular CD2-binding
portion of the human leukocyte function antigen-3 (LFA-3) linked to the Fc
(hinge,
CH2 and CH3 domains) portion of human IgG1). As shown in Figure 13, LTBRIgG
had increased serum levels regardless of sialylation in comparison to LFA3TIP.
Example 4: Effects of domain truncation of LTBRIgG
[00261] The TNFRIgG product entanercept has significant misfolding
heterogenities within the protein, a situation similar to LTBRIgG. To help
decrease
the extent of misfolding within the TNFR portion a based on the contact areas
shown
in the cocrystal structure of TNFR/TNF, a truncated version of TNFRFc was
created
using 2.6 domains of TNFR. The truncated TNF receptor molecule retained full
binding activity to the TNFa ligand. Based on the CRD structural alignment of
LTBR
and TNFR domains 4 and the C-terminal half of domain 3, as well as several
other
variations, truncated versions of LTBRIgG were expressed and evaluated for
their
affinity to LTalb2 (see Table 2 above).
[00262] Binding affinities of the various truncations (including D 3-1, D 2-1
(wt = D 1-4)) were determined. None of the purified truncated LTBRIgG
molecules
retained high affinity for the LTalb2 showing modeling contacts between
LTBR/LTalb2. Thus, TNFR/TNFa was poor predictor of contact residues between
LTBR and LTalb2.
Example 5: Design of Hinges for LTBRIgG
[002631 A number of different hinge constructs were created which could be
used to connect LTBR and IgG. A description of the different hinges is
described in
Figure 6, including the following hinge sequences. If used, the sequences
below
would be placed following the C-terminal amino acid of the LTBR extracellular
domain, i.e., M.
LTBR06 hinge: VDKTHTCPPCPAP (SEQ ID NO: 13)
Hinge 2211 (D 169N): VNKTHTCPPCPAP (SEQ ID NO: 14)
Hinge 2212 (T198N): VDKNHTCPPCPAP (SEQ ID NO: 15)
Hinge 2221 (valine deletion): DKTHTCPPCPAP (SEQ ID NO: 16)
Hinge 2217 (full length): EPKSCDKTHTCPPCPAP (SEQ ID NO: 17)
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Hinge 2219 (G4-hinge-G4Fc): ESCDKYGPPCPPCPAP (SEQ ID NO: 18)
Hinge 2218 (G2-hinge-G2Fc): CCVECPPCPAPPVAGP (SEQ ID NO: 19)
Hinge 2220 (short hinge - G1Fc): CPPCPAP (SEQ ID NO: 20)
[00264] Analysis of the expression for each of the hinges in LTBRIgG in
comparison to LTBR06 is described in Figure 7.
[00265] Examples 6 to 8 describe clinical studies showing the efficacy of
LTBRIgG (more specifically LTBR06) for the treatment of an autoimmune
disorder.
Example 6. Low doses of LTBR-Fc are effective to treat rheumatoid arthritis.
1002661 A dose of 0.05 mg/kg LTBR-Fc (version LTBR06) was chosen as the
lowest of five dose cohorts in randomized, blinded, placebo-controlled, drug
escalating clinical study to evaluate the safety, tolerability and
pharmacokinetics of
multiple doses of LTBR-Fc in RA patients. 0.05 mg/kg was chosen as the lowest
dose
cohort because it was expected to be a no-effect dose with regard to safety
and
pharmacokinetic findings. Indeed this was a no-effect dose with regard to
acute phase
response in earlier studies. Assessing efficacy was not a primary or secondary
objective of the study. Dosing was performed once weekly for 4 weeks. All
members
of the cohort were DMARD-IR. The patients received methotrexate therapy while
receiving the LTBR-Fc of the study.
[00267] Exemplary results are shown in Figure 8. This figure illustrates data
for a
placebo cohort (n=4); a cohort receiving 0.05 mg/kg LTBR-Fc (n=6); and a
cohort
receiving 0.1 mg/kg LTBR-Fc (n=3). Tender joint counts (TJC) and swollen joint
counts (SJC) were evaluated for each subject.
[00268] Remarkably, the 0.05 mg/kg cohort showed a dramatic (50-70%)
improvement in TJC and SJC compared to placebo. Similar improvement was also
seen with the next higher dose cohort (0.lmg/kg). This surprising observation
resulted in a complete redesign of the study, necessitating the addition of an
additional
dose cohort lower than 0.05 mg/kg (so that a lower dosage range could be
tested, and
a linear dose response could be seen and a lower dose response determined),
and in
the cancellation of the highest dose cohort (3 mg/kg) from the study.
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Example 7. Clinical Study of LTBR-Fc (LTBR06) for Treatment of RA (Phase
IIA data)
[002691 The following example describes results from a phase Ila clinical
study
which examined the efficacy of a range of doses of LTBR-Fc (version LTBR06)
administered to patients for the treatment of rheumatoid arthritis (RA).
[00270] The study was a blinded, randomized, placebo-controlled, dose-ranging
study in 47 DMARD-IR RA patients. Screening for the study included identifying
patients with active RA who were methotrexate (mtx)-IR. Cohort doses included
the
following groups: 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg or
3.0mg/kg. LTBR-Fc was administered in combination with mtx. The randomized to
LTBR-Fc or placebo ratio was 3:1. Subcutaneous injection once weekly for 4
weeks
followed by 8 weeks of observation. Baseline demographics and disease
characteristics were similar throughout the placebo and experimental groups
(11
placebo, 36 LTBR06 patients). The average duration of RA including all of the
patients was 3.1 years. 39 of the patients (83%) had had prior DMARD
treatment,
and 3 of 6% had had prior anti-TNF treatment. The median dose of methotrexate
(mtx) at baseline was 15 mg/week. For the total number of patients, the median
of
ACR core set of measurements was 16 SJC, 21 TJC, and 1.33 HAQ.
[00271] LTBR-Fc proved effective at treating RA in patients. The results
showed
substantial improvement in the majority of ACR core set measurements in a dose
dependent manner. At Day 35 (2 weeks after the last dose), mean improvement
was
SJC 60% vs 4.6 % (LTBR-Fc vs placebo) and TJC 47% vs 6.7% (LTBR-Fc vs
placebo) (see Figures 10 to 12). As shown in Figures 9-11, improvements in SJC
and
TJC were durable, persisting 8 weeks after the final LTBR-Fc dose through Day
77.
The greatest improvements were reported in the LTBR-Fc 1.0 mg/kg and 3.0 mg/kg
groups, as the ACR20 response rates for these two dosing groups was 65% and
85%
at Day 77, respectively. Improvement in the ACR20 response was also greater
than
placebo for all dose groups, as shown in Figure 12 and Table 10.
Table 10: ACR20/50/70 response rates (% of patients) at Day 77
Doses ACR20 ACR50 ACR70
Placebo (n=10) 30 0 0
0.01 mg/kg (n=6) 0 0 0
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0.05 mg/kg (n=6) 0 0 0
0.1 mg/kg (n=4) 50 25 25
0.3 mg/kg (n=5) 40 40 20
1.0 mg/kg (n=6) 50 33 17
3.0 mg/kg (n=6) 67 50 33
[00272] ACR20 (ACR50, ACR70) response is defined as a 20% (50%, 70%)
improvement in SJC and TJC, with a 20% (50%, 70%) improvement in at least 3 of
the following indices: IGA, PGA, pain-VAS, HAQ, CRP (ESR if CRP is missing).
1002731 Overall, LTBR-Fc proved safe in patients who received treatment. 55%
of
patients (6/11) receiving placebo and 67% of patients (24/36) receiving LTBR-
Fc
(LTBR06) experienced adverse events (AEs) (e.g., headaches, chills, fatigue)
although none were severe. The most common AE in patients receiving LTBR-Fc
(LTBR06) was headache, reported in 19% of patients vs 9% of patient receiving
placebo. No drug-related serious infections or drug-related serious AEs were
reported
during the study period. One serious infection was reported in the 3.0 mg/kg
group
on Day 91 (acute bronchitis, unlikely related to treatment). Transient mild-to-
moderate flu-like symptoms were observed in 25% of patients within 24 h after
the
first dose of LTBR-Fc. These symptoms responded well to acetaminophen and did
not usually recur after subsequent doses (decreased on subsequent doses -
reported in
8%, 9% and 6% of patients after the second, third, and fourth doses,
respectively).
Decreased immunogenicity was observed with increased dose, there were no
effects
on PK, AE's or efficacy.
[00274] Overall, the results showed that LTBR-Fc was effective at treating RA
in
doses ranging from 0.01 mg/kg to 3 mg/kg.
Example 8. Clinical Study of LTBR-Fc (LTBR06) for Treatment of RA
1002751 A long term extension study is used to further evaluate the safety,
tolerability, and efficacy of LTBR-Fc (LTBR06) in subjects with RA. Patients
from
previous studies, such as the study described in Example 7 may be included in
the
study. The study design includes administering LTBR-Fc (LTBR06) to RA patients
in
one of the following doses subcutaneously: 70 mg every other week; 200 mg
every
other week; 70 mg monthly; 200 mg monthly; or a placebo dose. Patients are
evaluated for improvements in the responses / scores of the following efficacy
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parameters will be determined: American College of Rheumatology (ACR) Core Set
of maeasurements,, DAS28, Short Form (SF-36), Functional Assessment of Chronic
Illness Therapy (FACIT) questionnaires, Sjogren's Assessment. Statistically
significant improvements in such parameters in comparison to the placebo
control
group will indicate efficacy.
EQUIVALENTS
[00276] All patents, patent applications, and references are hereby
incorporated
by reference in their entirety. In the case of conflict, the present
application controls.
[00277] Each of the limitations of the invention can encompass various
embodiments of the invention. It is, therefore anticipated that each of the
limitations
of the invention involving any one element or combinations of elements can be
included in each aspect of the invention.
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Forming part of the present disclosure is the appended Sequence Listing, the
contents of which are summarized in the table below:
SEQ ID DESCRIPTION
NO:
I Wild type human LTBR sequence (amino acid)
2 LTBR06 Ig (Fc) domain (amino acid)
3 LTBR06, variant LTBR extracellular domain (nucleic acid)
4 LTBR06, variant LTBR extracellular domain (amino acid)
LTBR06, mature form (amino acid)
6 LTBR01 (amino acid)
7 LTBR06 (nucleic acid)
8 LTBR06 (amino acid)
9 LTBR05, mature form (amino acid)
non-glycosylated LTBR-IgG (amino acid) (pEAG 1980)
11 LTBROI, mature form (amino acid)
12 LTBR09, mature form (amino acid)
13 Hinge (amino acid)
14 2211 hinge (amino acid)
2212 hinge (amino acid)
16 2221 hinge (amino acid)
17 2217 hinge (amino acid)
18 2219 hinge (amino acid)
19 2218 hinge (amino acid)
2220 hinge (amino acid)
21 Wild type human LTBR, extracellular domain (amino acid)
22 Ig (Fc) domain unmodified (amino acid)
23 N-5 variant LTBR extracellular domain (amino acid)
5
58
