U.S. patent application number 13/682221 was filed with the patent office on 2013-10-03 for treatment of autoimmune disorders.
This patent application is currently assigned to BIOGEN IDEC MA INC.. The applicant listed for this patent is BIOGEN IDEC MA INC.. Invention is credited to Evan BECKMAN, Jeffrey L. BROWNING, Graham K. FARRINGTON, Werner MEIER.
Application Number | 20130259861 13/682221 |
Document ID | / |
Family ID | 42131698 |
Filed Date | 2013-10-03 |
United States Patent
Application |
20130259861 |
Kind Code |
A1 |
BECKMAN; Evan ; et
al. |
October 3, 2013 |
TREATMENT OF AUTOIMMUNE DISORDERS
Abstract
This invention relates to methods of treating disease with
soluble inhibitors of the lymphotoxin pathway having improved
properties. This invention also relates to improved LTBR-Ig fusion
proteins, and pharmaceutical compositions thereof.
Inventors: |
BECKMAN; Evan; (Waban,
MA) ; FARRINGTON; Graham K.; (Acton, MA) ;
MEIER; Werner; (Burlington, MA) ; BROWNING; Jeffrey
L.; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOGEN IDEC MA INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
42131698 |
Appl. No.: |
13/682221 |
Filed: |
November 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12560257 |
Sep 15, 2009 |
8338376 |
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13682221 |
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PCT/US08/03548 |
Mar 17, 2008 |
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12560257 |
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12446041 |
Aug 31, 2009 |
8067375 |
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PCT/US08/03548 |
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60918518 |
Mar 15, 2007 |
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60862343 |
Oct 20, 2006 |
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Current U.S.
Class: |
424/134.1 ;
435/320.1; 435/328; 530/387.3; 536/23.4 |
Current CPC
Class: |
C07K 14/70578 20130101;
C07K 2319/30 20130101; A61K 38/00 20130101; A61P 37/02
20180101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 536/23.4; 435/320.1; 435/328 |
International
Class: |
C07K 14/705 20060101
C07K014/705 |
Claims
1.-83. (canceled)
84. A composition comprising an LT.beta.R-Ig fusion protein
comprising an LT.beta.R extracellular domain, wherein the LTBR
extracellular domain is non-glycosylated at one or more of its
N-linked glycosylation sites.
85. The composition of claim 84, wherein the LT.beta.R
extracellular domain contains one or more amino acid mutations as
compared to SEQ ID NO:21, which remove said one or more N-linked
glycosylation sites.
86. The composition of claim 84, wherein the LT.beta.R
extracellular domain comprises amino acids 1-194 of SEQ ID
NO:10.
87. The composition of claim 84, wherein the LT.beta.R
extracellular domain is aglycosylated.
88. The composition of claim 84, wherein the Ig portion of the
LT.beta.R-Ig fusion protein contains one or more mutations as
compared to SEQ ID NO:22, which removes its N-linked glycosylation
sites.
89. A composition comprising an LT.beta.R-Ig fusion protein
comprising one or more mutations as compared to SEQ ID NO:11,
wherein said one or more mutations is selected from the group
consisting of N13Q, N150Q and N276Q.
90. The composition of claim 89, wherein the LT.beta.R-Ig fusion
protein is missing no more than five amino acids from the
N-terminus of the mature form of the fusion protein.
91. The composition of claim 84 or 89, wherein the LT.beta.R-Ig
fusion proteins are made by expressing a nucleic acid molecule
comprising a nucleotide sequence encoding the LT.beta.R-Ig fusion
protein in a mammalian cell.
92. The composition of claim 84 or 89, wherein the Ig portion of
the LT.beta.R-Ig fusion protein comprises a variant Fc region.
93. The composition of claim 92, wherein the variant Ig portion
comprises Fc regions of an IgG1 isotype.
94. The composition of claim 92, wherein the variant Ig portion
comprises a mutation in the hinge region.
95. The composition of claim 91, wherein the Ig portion of the
LT.beta.R-Ig fusion protein is non-glycosylated.
96. The composition of claim 91, wherein the step of expressing is
done at manufacturing scale.
97. A pharmaceutical composition comprising the composition of
claim 84 or 89 and a pharmaceutically acceptable carrier.
98. A method of treating an autoimmune disorder in a patient
comprising administering an effective amount of the pharmaceutical
composition of claim 97.
99. The method of claim 98 wherein the autoimmune disorder is
multiple sclerosis or rheumatoid arthritis.
100. An isolated polypeptide comprising an LT.beta.R-Ig fusion
protein comprising one or more mutations as compared to SEQ ID
NO:11, wherein said one or more mutations is selected from the
group consisting of N13Q, N150Q and N276Q.
101. An isolated nucleic acid molecule encoding the polypeptide of
claim 100.
102. A vector comprising the nucleic acid molecule of claim
101.
103. An isolated or cultured host cell expressing the vector of
claim 102.
104. The cell of claim 103, which is a Chinese hamster Ovary (CHO)
cell.
105. An isolated polypeptide comprising an LT.beta.R-Ig fusion
protein comprising one or more mutations as compared to SEQ ID
NO:5, wherein said one or more mutations is selected from the group
consisting of N13Q, N150Q and N276Q, referencing SEQ ID NO:11.
106. An isolated nucleic acid molecule encoding the polypeptide of
claim 105.
107. A vector comprising the nucleic acid molecule of claim
106.
108. An isolated or cultured host cell expressing the vector of
claim 107.
109. The cell of claim 108, which is a Chinese hamster Ovary (CHO)
cell.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 12/560,257, filed Sep. 15, 2009 which is a Continuation of
International Application No. PCT/US2008/003548, filed on Mar. 17,
2008 which claims the benefit of U.S. Provisional Application. No.
60/918,518, filed on Mar. 15, 2007. This application is also a
Continuation-in-Part of U.S. application Ser. No. 12/446,041, filed
Aug. 31, 2009 which claims benefit of PCT Application No.
PCT/US2007/081761, filed on Oct. 18, 2007 and U.S. Provisional
Application No. 60/862,343, filed Oct. 20, 2006. Each of these
applications is incorporated in its entirety herein.
BACKGROUND
[0002] 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.
[0003] 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.
[0004] 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 LT.alpha./.beta. 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 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.
[0005] 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
[0006] In one aspect the invention pertains to a composition
comprising a population of lymphotoxin-.beta. receptor
(LT-.beta.-R)-Ig-fusion proteins which comprise a variant
LT-.beta.-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-.beta.-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-.beta.-R extracellular domain set
forth in SEQ ID NO:21 and wherein the LT-.beta.-R-Ig-fusion
proteins lack N-terminal pyroglutamic acid.
[0007] In one embodiment, the N-terminal amino acid of the variant
LT-.beta.-R-Ig fusion protein is a non-polar amino acid.
[0008] In one embodiment, the non polar amino acid is either a
valine (amino acid six of the mature form of the wild type
LT-.beta.-R extracellular domain of SEQ ID NO:21) or an alanine
(amino acid five of the mature form of the wild type LT-.beta.-R
extracellular domain of SEQ ID NO:21).
[0009] In one embodiment, the N-terminal amino acid of at least 95%
of the LT-.beta.-R-Ig-fusion proteins is either a valine (amino
acid six of the mature form of the wild type LT-.beta.-R
extracellular domain of SEQ ID NO:21) or an alanine (amino acid
five of the mature form of the wild type LT-.beta.-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.
[0012] In one embodiment, variant Ig portion comprises Fc regions
of an IgG1 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-.beta.-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-.beta.
receptor-immunoglobulin (LT-.beta.-R-Ig)-fusion proteins comprising
a variant LT-.beta.-R extracellular domain and a variant Ig
portion, wherein the variant LT-.beta.-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-.beta.
receptor-immunoglobulin (LT-.beta.-R-Ig)-fusion proteins, the
fusion proteins comprising a variant LT-.beta.-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-.beta.-R-Ig fusion proteins.
[0021] In one embodiment, at least 90% of the LT-.beta.-R-Ig-fusion
proteins comprise a variant LT-.beta.-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-.beta. receptor
(LT-.beta.-R)-Ig-fusion proteins which comprise a variant
LT-.beta.-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-.beta.-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-.beta.-R extracellular domain set
forth in SEQ ID NO:21and wherein the LT-.beta.-R-Ig-fusion proteins
lack N-terminal pyroglutamic acid and a pharmaceutically acceptable
carrier.
[0024] In one embodiment, the N-terminal amino acid of the variant
LT-.beta.-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-.beta.-R extracellular domain of SEQ ID NO:21) or an alanine
(amino acid five of the mature form of the wild type LT-.beta.-R
extracellular domain of SEQ ID NO:21).
[0026] In one embodiment, the N-terminal amino acid of at least 95%
of the LT-.beta.-R-Ig-fusion proteins is either a valine (amino
acid six of the mature form of the wild type LT-.beta.-R
extracellular domain of SEQ ID NO:21) or an alanine (amino acid
five of the mature form of the wild type LT-.beta.-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).
[0030] In one embodiment, the invention pertains to a
pharmaceutical composition comprising a population of
lymphotoxin-.beta. receptor-immunoglobulin (LT-.beta.-R-Ig)-fusion
proteins, the fusion proteins comprising a variant LT-.beta.-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-.beta.-R-Ig fusion proteins and a
pharmaceutically acceptable carrier.
[0031] In one embodiment, at least 90% of the LT-.beta.-R-Ig-fusion
proteins comprise a variant LT-.beta.-R extracellular domain as set
forth the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23.
[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-.beta.-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-.beta.-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-.beta.-R extracellular domain of SEQ ID NO:21) or an alanine
(amino acid five of the mature form of the wild type LT-.beta.-R
extracellular domain of SEQ ID NO:21).
[0042] 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.
[0045] In one embodiment, the invention 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-.beta.
receptor (LT-.beta.-R)-Ig-fusion proteins which comprise a variant
LT-.beta.-R extracellular domain and a variant Ig portion, wherein
at least 90% of the LT-.beta.-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-.beta.-R extracellular domain set forth in SEQ ID
NO:21, the process comprising, expressing a nucleic acid molecule
encoding the LT-.beta.-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-.beta.
receptor (LT-.beta.-R)-Ig-fusion proteins which comprise a variant
LT-.beta.-R extracellular domain and a variant Ig portion, wherein
at least 90% of the LT-.beta.-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-.beta.-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-.beta.-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-.beta.-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.
[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-.beta.-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-.beta.-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-.beta.-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-.beta.-R-Ig fusion
proteins comprising a variant LT-.beta.-R extracellular domain of
193 or 194 amino acids in length, wherein at least 90% of the
LT-.beta.-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-.beta.-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-.beta.-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.
[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-.beta.-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.RTM.), etanercept (Enbrel.RTM.), or infliximab
(Remicade.RTM.).
[0077] In one embodiment, LT-.beta.-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-.beta.-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-.beta.-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-.beta.-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.RTM.), etanercept (Enbrel.RTM.), or infliximab
(Remicade.RTM.).
[0084] In one embodiment, the human is an anti-TNF-inadequate
responder.
[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] FIG. 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 LTBR01, i.e., amino
acids 1-4 and the last amino acid (lysine). Three consensus sites
for N-linked glycosylation are located at Asn13, 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] FIG. 2A and FIG. 2B provide an alignment of LTBR01 (SEQ ID
NO: 11), LTBR05 (SEQ ID NO: 9), LTBR06 (SEQ ID NO: 5), and LTBR09
(SEQ ID NO: 12). The secretion sequence is omitted from the LTBR
sequences.
[0090] FIG. 3 describes LTBR06 (N-terminus residues 1-3 of SEQ ID
NO: 5 and C-terminus, residues 417-421 of SEQ ID NO: 5) in
comparison to wild type (N terminus residues 1-7 of SEQ ID NO: 11
and C-terminus, residues 421-426 of SEQ ID NO:11) and provides a
schematic of the protein.
[0091] FIG. 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] FIG. 5 describes aglycosylated hLT.beta.R hIgG1 (mature form
of protein). Asparagine to glutamine mutations in LT.beta.R
extracellular domain are shown in bold. The huLT.beta.R is residues
1-204, the huIgG1 Fc is residues 205-431 above (the Fc's
glycosylation site is intact).
[0093] FIG. 6 describes hinges (SEQ ID NOS: 13-20) of which may be
used in the LTBR IgG fusion proteins of the invention.
[0094] FIG. 7 describes a table which describes a summary of
results from a hinge expression analysis.
[0095] FIG. 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.
[0096] FIG. 9 provides a graph which depicts a decrease in Tender
Joint Counts (TJC) in patients following treatment with LTBR-Fc
(LTBR06).
[0097] FIG. 10 provides a graph which depicts a decrease in Swollen
Joint Counts n (SJC) in patients following treatment with LTBR-Fc
(LTBR06).
[0098] FIG. 11 provides a graph which depicts the median % change
in Swollen Joint Counts n (SJC) in patients following treatment
with LTBR-Fc (LTBR06).
[0099] FIG. 12 provides a graph which depicts ACR20 improvements in
patients following treatment with LTBR-Fc (LTBR06).
[0100] FIG. 13 graphically depicts serum levels of differentially
sialylated LFA3TIP and hu-LTBR-Ig (LTBR05) in mice 24 hours post
administration with 100 ug/animal.
[0101] FIG. 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
[0102] In order that the present invention may be more readily
understood, certain terms are first defined.
[0103] 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.
[0104] 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.
[0105] 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 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, IgG1,
IgG2, IgM, IgA etc. Immunoglobulin fusion proteins are referred to
herein as Ig for Fc fusion proteins.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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-terminal
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). 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 (SEQ ID NO: 24),
an N-1 protein would not comprise the initial A and would instead
begin with AGTY (SEQ ID NO:25), although both proteins begin with
A. Similarly, an N-2 protein would begin with GTY.
[0110] 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.
[0111] 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.
[0112] 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
O-linked glycosylation (where one or more sugars are linked to an
amino acid residue having a hydroxyl group (e.g., serine or
threonine).
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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).
[0118] 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.
[0119] 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. [0120] Genetically encoded
amino acids generally may be divided into four families: (1)
acidic: aspartate, glutamate; (2) basic: lysine, arginine,
histidine; (3) nonpolar: 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.
[0121] 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.
[0122] 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.
[0123] 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 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.
[0124] The term "weekly" means not more than once within a
particular 6 to 8 day period, e.g., once every 7 days.
[0125] 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 31 days. 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.
[0126] 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.
[0127] Various aspects of the invention are described in further
detail in the following subsections.
I. LTBR-Ig Fusion Proteins and Compositions Thereof
[0128] 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.
[0129] A lymphotoxin-.beta. receptor-immunoglobulin (LT.beta.R-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).
[0130] 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.
[0131] 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).
[0132] 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 the subtype
IgG1 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.
[0133] 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.
[0134] 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-.beta. receptor-immunoglobulin
(LT-.beta.-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 variation
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 LTBR01. It should be noted that the terms
LT.beta.R-Ig and LTBR-Fc are used interchangeably herein.
[0135] In a preferred embodiment, an LT.beta.R-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.
[0136] 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 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)
[0137] 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.
TABLE-US-00001 (SEQ ID NO: 1) 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
[0138] 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.
[0139] In one aspect, the invention pertains to LTBR-Ig fusion
proteins comprising a variant LTBR extracellular domain. For
example, the amino acids "SQPQ" (SEQ ID NO: 26) 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" (SEQ ID NO: 26) 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-.beta.-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-.beta.-R extracellular domain set forth in SEQ ID NO:21. Thus,
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.
[0140] 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 IgG1 isotype.
[0141] 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.
[0142] 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.
TABLE-US-00002 (SEQ ID NO: 8) 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 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 E S C S
V M H E A L H N H Y T Q K S L S L S P G Stop
[0143] 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.
[0144] In an optional embodiment, an immunoglobulin hinge [[be]] is
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 (SEQ ID NO:
27) may be used. In one embodiment, a hinge region having the
sequence VDKTHTCPPCPAPELLGGP (SEQ ID NO:28) may be used. Other
exemplary upper and middle hinge constructs are shown in FIGS. 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.
[0145] 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.beta.R-Ig-fusion proteins which comprise a variant LT.beta.-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-.beta.-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-.beta.-R extracellular domain set forth in SEQ ID NO:21 and
wherein the LT-.beta.-R-Ig-fusion proteins lack pyroglutamic acid.
The invention also features a composition comprising a population
of LT-.beta.-R-Ig fusion proteins comprising a variant LT-.beta.-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-.beta.-R-Ig fusion proteins.
[0146] 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-.beta.-R portion SEQ ID NO:1) or an
alanine (amino acid five of the mature form of the wild type
LT-.beta.-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-.beta.-R-Ig-fusion proteins is either a valine (amino acid six
of the mature form of wild type LT-.beta.-R) or an alanine (amino
acid five of the mature form of wild type LT-.beta.-R).
[0147] 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, 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.
[0148] The invention also contemplates a composition comprising a
population of LT.beta.R-Ig fusion proteins comprising a variant
LT-.beta.-R extracellular domain and a variant Ig portion, wherein
variant LT-.beta.-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).
[0149] 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-.beta.-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-.beta.-R-Ig fusion proteins.
[0150] 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.
[0151] 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.
[0152] 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 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.
[0153] 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
[0154] 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.
[0155] 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.
[0156] 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 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).
[0157] 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.
[0158] 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.
[0159] The invention further provides a process for making a
composition comprising a population of LT.beta.R-Ig-fusion proteins
which comprise a variant LT-.beta.-R extracellular domain and a
variant Ig portion, wherein at least 90% of the LT.beta.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-.beta.-R portion set forth
in SEQ ID NO:1, the process comprising, expressing a nucleic acid
molecule encoding the LT-.beta.-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.beta.R-Ig-fusion proteins which comprise a variant LT-.beta.-R
extracellular domain and a variant Ig portion, wherein at least 90%
of the LT-.beta.-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-.beta.-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.beta.-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.
II. Uses of LTBR-IgG of Invention for Treating Autoimmune
Disorders
[0160] 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 particularly
suitable for treatment of RA.
[0161] Rheumatoid arthritis is marked by tenderness in the joints.
Synovial thickening eventually occurs in most effected joints.
Stiffness lasting >30 minutes upon arising in the morning or
after prolonged inactivity is common, as is early afternoon fatigue
and malaise. Deformities, particularly flexion contractures may
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.
[0162] 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.
[0163] 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
[0164] 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 blocking agent (e.g., a soluble LTBR fusion
protein, e.g., LTBR-Fc) at a low dose and/or frequency.
[0165] As shown in FIG. 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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 subject has active RA disease
(e.g., tender joint count (TJC) or swollen joint count
(SJC).gtoreq.5 or synovitis). In another embodiment, the subject is
at risk for developing RA, e.g., a family history, or an autoimmune
disease.
[0171] 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.
[0172] 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.
[0173] 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 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-US-00003 TABLE 1 Exemplary dosages of LTBR-Fc (mg 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; 2;
1.4; 2.5; 2; 3; 2.5; 3.5 3; 3.5
[0174] 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.
[0175] 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.
[0176] 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, 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.
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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.
[0182] In certain embodiments, the soluble LTBR is administered
intravenously or parenterally, e.g., subcutaneously or
intramuscularly.
[0183] In another embodiment, the soluble LTBR is administered as a
monotherapy.
[0184] 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 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.
[0185] 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.
[0186] 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.
[0187] 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.RTM.), anakinra (Kineret.RTM.),
hydroxycholoquine sulfate (Plaquenil.RTM.) antimalarials, gold
salts, sulfasalazine (Azulfidine.RTM.), minocycline (Minocin.RTM.),
d-penicillamine, cyclosporin A, cyclosporine (Neoral.RTM.),
cyclophosphamide and azathioprine (Imuran.RTM.).
[0188] 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.
[0189] 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-US-00004 TABLE 2 Exemplary dose of LTBR-Fc (mg) according to
weight of human Weight of human: Final concentration.sup.a: 40-50
kg 50-60 kg 60-75 kg 75-100 kg 100-125 kg 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 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
.sup.amg LTBR-Fc per kg of body weight of human
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] In one embodiment, the patient is instructed to stop an
NSAID, corticosteroid, or DMARD therapy.
[0204] 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 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
[0205] 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.
[0206] 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.
[0207] DMARDs typically slow the progression of rheumatoid
arthritis. Suitable DMARDS include methotrexate, leflunomide
(Arava.RTM.), anakinra (Kineret.RTM.), hydroxycholoquine sulfate
(Plaquenil.RTM.) antimalarials, gold salts, sulfasalazine
(Azulfidine.RTM.), minocycline (Minocin.RTM.), d-penicillamine,
cyclosporin A, cyclosporine (Neoral.RTM.), cyclophosphamide and
azathioprine (Imuran.RTM.). 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 (Enbrel.TM.), infliximab
(Remicade.TM.), and adalimumab (Humira.TM.)
[0208] Corticosteroids typically function as anti-inflammatory
agents and can also be used in combination therapy with a soluble
LTBR.
[0209] 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.
[0210] 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).
[0211] 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).
[0212] 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 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.
[0213] 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.
[0214] 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.
[0215] In one embodiment, the subject is a DMARD inadequate
responder (DMARD-IR). For example, the subject is an anti-TNF
inadequate responder.
[0216] 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 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
[0217] 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).
[0218] 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).
[0219] 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.degree. C.
[0220] 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 intended mode of administration and therapeutic application.
Typically compositions for the agents described herein are in the
form of injectable or infusible solutions.
[0221] 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.
[0222] 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.
[0223] 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 known. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0224] 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).
[0225] 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.
[0226] 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.
[0227] 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.
[0228] In one embodiment, a pharmaceutical composition comprises a
population of lymphotoxin-.beta. receptor (LT-.beta.-R)-Ig-fusion
proteins which comprise a variant LT-.beta.-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-.beta.-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-.beta.-R extracellular domain set forth in SEQ ID NO:21and
wherein the LT-.beta.-R-Ig-fusion proteins lack N-terminal
pyroglutamic acid and a pharmaceutically acceptable carrier.
[0229] In another embodiment, a pharmaceutical composition
comprises a population of lymphotoxin-.beta.
receptor-immunoglobulin (LT-.beta.-R-Ig)-fusion proteins, the
fusion proteins comprising a variant LT-.beta.-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-.beta.-R-Ig fusion proteins and a pharmaceutically
acceptable carrier.
[0230] In yet another embodiment a pharmaceutical composition of
the invention comprises the amino acid sequence set forth in SEQ ID
NO:5.
Administration.
[0231] 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.
[0232] The dose can also be chosen to reduce or avoid production of
antibodies against the agent.
[0233] 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.
[0234] 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.
[0235] 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 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.
[0236] 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
[0237] 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.
[0238] 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.
[0239] In other examples, the pharmaceutical composition can be
administered with a needleless hypodermic injection device, such as
the devices disclosed in U.S. Pat. Nos. 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: U.S. Pat. No.
4,487,603, which discloses an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system.
Many other devices, implants, delivery systems, and modules are
also known.
[0240] 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.
[0241] 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.
[0242] 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 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.
[0243] 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.
[0244] 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
[0245] 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.
N-Terminal Heterogeneity
[0246] Predictions of the N-termini encoded by the full length mRNA
of LTBR (NP.sub.--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.
[0247] LTBR01 (LTBR-IgG fusion protein version 01) was the initial
molecule that was cloned. The N-terminus of LTBR01 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 IgG1 hinge
to the structurally similar amino acid valine (V199 in LTBR01) was
included LTBRIgG. This mutation eliminated the problematic and
unwanted formation of a free unpaired thiol in the LTBRIgG, in the
IgG1 C220 of the heavy chain is normally disulfide bonded with the
C107 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.
[0248] 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 FIG. 1, including the signal sequence) and
was missing the C-terminal K426 from the Fc domain.
[0249] 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.
[0250] 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 elimination of the C-terminal
lysine, but comparable N-terminal variability to LTBR01.
[0251] 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 FIGS. 1 and 3.
[0252] 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 LTBR01
(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 FIG. 2.
TABLE-US-00005 TABLE 3 Comparison of LTBR05 and LTBR06 vs. LTBR01
Construct name Features Purpose Results LTBR01 Wt sequence 1.
Characterization of 1. N-terminus N-1, N-3, N-4 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, C- terminal splice variant terminus
homogeneous of pMDR plasmid LTBR06 N-4/C-1 1. Reduce N-terminal 1.
N-terminus N-4 and N-5, no N and C-terminal terminal pryoGlu
heterogeneity 2. Fully active ligand binding 2. Maintain
activity
[0253] 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-US-00006 TABLE 4 Summary of the biochemical characteristics
of LTBR05 Attribute Test Result Concentration UV absorbance 5.0
A280/ml (5.0 mg/ml) 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.
[0254] 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-US-00007 TABLE 5 Summary of the biochemical characteristics
of LTBR06 Attribute Test Result Concentration UV absorbance 25.0
A280/ml (25.0 mg/ml) Purity SDS-PAGE Red >90% SDS-PAGE Nonred.
>90% Aggregation SEC assay <1% % Inactive HIC assay <1%
Endotoxin Chromogenic assay 0.052 EU/mg or 1.3 EU/ml N-terminal
Edman degradation 56% N-5, 40% N-4, ~4% N-11 Sequence Peptide map
LC chromatography of Corresponds to characterized the Endo Lys-C
digest map of LTBR05 LC/MS Endo Lys-C digest 31 peptides identified
account for 95% of the predicted amino acid sequence.
[0255] 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
[0256] 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 FIG. 2).
[0257] Table 6 describes the glycosylation occupancy for each of
three LTBR constructs at Asn13 and Asn150.
TABLE-US-00008 TABLE 6 Glycosylation occupancy Construct % site
occupancy Asn13 % occupancy Asn150 LTBR05 28* 86 LTBR06 26** 85
LTBR09 22** 84 *Calculated as an average of intact N-terminus, N-1
<Q and N-3 <Q **Calculated as an average of N-4 and N-5
[0258] As shown in Table 6, glycosylation occupancy was relatively
invariant within the different constructs for Asn13 and Asn150.
[0259] 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 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-US-00009 TABLE 7 Additional glycosylation mutants Construct
name Features Purpose Results LTBR02 N13Q 1. Expression level of 1
1. Expression slightly glyc. mutation in animal reduced cells 2.
Active in ligand 2. Activity assessment binding LTBR03 N13Q, 1.
Expression level of 2 1. Expression reduced N150Q glyc. mutations
in significantly: about 10% animal cells of wt in CHO stable 2.
Activity assessment 2. Wildtype ligand binding. 3. No effect on
folding. LTBR04 N13Q, 1. Expression level of 3 1. Expression
reduced. N150Q, glyc. mutations in a lot N275Q animal cells 2.
Active in ligand 2. Activity assessment binding 3. Microbial 3.
Expression from yeast possible.
[0260] LTBR03 is shown in FIG. 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.
[0261] 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.
Example 3
Sialylation Effect on LTBRIgG Pharmacokinetics
[0262] In order to study the effects of sialylation on LTBRIgG
pharmacokinetics, sialylation of LTBRIgG was forced using
.alpha.-2,6-Sialyltransferase from rat liver (Boehringer Mannheim).
.alpha.-2,6-Sialytransferase is specific for
Ga.beta.1.fwdarw.4GlcNAc, 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-US-00010 TABLE 8 Sialylation (SA) of LTBR-Ig PK Data
(concentrations in ug/ml) time time time time time time (hr) (hr)
(hr) (hr) (hr) (hr) sialylation mouse # 0 0.25 7 24 72 168 low 1
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-US-00011 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
FIG. 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 FIG. 13, LTBRIgG had increased
serum levels regardless of sialylation in comparison to
LFA3TIP.
Example 4
Effects of Domain Truncation of LTBRIgG
[0263] 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
LTa1b2 (see Table 2 above).
[0264] Binding affinities of the various truncations (including D
3-1, D 2-1 (wt=D1-4)) were determined. None of the purified
truncated LTBRIgG molecules retained high affinity for the LTa1b2
showing modeling contacts between LTBR/LTa1b2. Thus, TNFR/TNFa was
poor predictor of contact residues between LTBR and LTa1b2.
Example 5
Design of Hinges for LTBRIgG
[0265] 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 FIG. 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.
TABLE-US-00012 LTBR06 hinge: (SEQ ID NO: 13) VDKTHTCPPCPAP Hinge
2211 (D169N): (SEQ ID NO: 14) VNKTHTCPPCPAP Hinge 2212 (T198N):
(SEQ ID NO: 15) VDKNHTCPPCPAP Hinge 2221 (valine deletion): (SEQ ID
NO: 16) DKTHTCPPCPAP Hinge 2217 (full length): (SEQ ID NO: 17)
EPKSCDKTHTCPPCPAP Hinge 2219 (G4-hinge-G4Fc): (SEQ ID NO: 18)
ESCDKYGPPCPPCPAP Hinge 2218 (G2-hinge-G2Fc): (SEQ ID NO: 19)
CCVECPPCPAPPVAGP Hinge 2220 (short hinge-G1Fc): (SEQ ID NO: 20)
CPPCPAP
[0266] Analysis of the expression for each of the hinges in LTBRIgG
in comparison to LTBR06 is described in FIG. 7.
[0267] 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
[0268] 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.
[0269] Exemplary results are shown in FIG. 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.
[0270] 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 (0.1 mg/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.
Example 7
Clinical Study of LTBR-Fc (LTBR06) for Treatment of RA (Phase IIA
Data)
[0271] The following example describes results from a phase IIa
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).
[0272] 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 3.0
mg/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.
[0273] 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 FIGS. 10
to 12). As shown in FIGS. 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 FIG. 12 and Table 10.
TABLE-US-00013 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 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
[0274] 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).
[0275] 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.
[0276] 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
[0277] 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 parameters will be
determined: American College of Rheumatology (ACR) Core Set of
measurements, 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
[0278] All patents, patent applications, and references are hereby
incorporated by reference in their entirety. In the case of
conflict, the present application controls.
[0279] 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.
[0280] Forming part of the present disclosure is the appended
Sequence Listing, the contents of which are summarized in the table
below:
TABLE-US-00014 SEQ ID NO: DESCRIPTION 1 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) 5 LTBR06, mature
form (amino acid) 6 LTBR01 (amino acid) 7 LTBR06 (nucleic acid) 8
LTBR06 (amino acid) 9 LTBR05, mature form (amino acid) 10
non-glycosylated LTBR-IgG (amino acid) (pEAG 1980) 11 LTBR01,
mature form (amino acid) 12 LTBR09, mature form (amino acid) 13
Hinge (amino acid) 14 2211 hinge (amino acid) 15 2212 hinge (amino
acid) 16 2221 hinge (amino acid) 17 2217 hinge (amino acid) 18 2219
hinge (amino acid) 19 2218 hinge (amino acid) 20 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)
Sequence CWU 1
1
281435PRTHomo sapiensmisc_featureFull length human wild type LTBR
amino acid sequence (immature form) corresponding to GenPept ID No.
P36941 1Met Leu Leu Pro Trp Ala Thr Ser Ala Pro Gly Leu Ala Trp Gly
Pro 1 5 10 15 Leu Val Leu Gly Leu Phe Gly Leu Leu Ala Ala Ser Gln
Pro Gln Ala 20 25 30 Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr Cys
Arg Asp Gln Glu Lys 35 40 45 Glu Tyr Tyr Glu Pro Gln His Arg Ile
Cys Cys Ser Arg Cys Pro Pro 50 55 60 Gly Thr Tyr Val Ser Ala Lys
Cys Ser Arg Ile Arg Asp Thr Val Cys 65 70 75 80 Ala Thr Cys Ala Glu
Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu Thr 85 90 95 Ile Cys Gln
Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu Glu 100 105 110 Ile
Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys Gln Pro 115 120
125 Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys Glu Leu
130 135 140 Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp
Glu Val 145 150 155 160 Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys
Ala Gly His Phe Gln 165 170 175 Asn Thr Ser Ser Pro Ser Ala Arg Cys
Gln Pro His Thr Arg Cys Glu 180 185 190 Asn Gln Gly Leu Val Glu Ala
Ala Pro Gly Thr Ala Gln Ser Asp Thr 195 200 205 Thr Cys Lys Asn Pro
Leu Glu Pro Leu Pro Pro Glu Met Ser Gly Thr 210 215 220 Met Leu Met
Leu Ala Val Leu Leu Pro Leu Ala Phe Phe Leu Leu Leu 225 230 235 240
Ala Thr Val Phe Ser Cys Ile Trp Lys Ser His Pro Ser Leu Cys Arg 245
250 255 Lys Leu Gly Ser Leu Leu Lys Arg Arg Pro Gln Gly Glu Gly Pro
Asn 260 265 270 Pro Val Ala Gly Ser Trp Glu Pro Pro Lys Ala His Pro
Tyr Phe Pro 275 280 285 Asp Leu Val Gln Pro Leu Leu Pro Ile Ser Gly
Asp Val Ser Pro Val 290 295 300 Ser Thr Gly Leu Pro Ala Ala Pro Val
Leu Glu Ala Gly Val Pro Gln 305 310 315 320 Gln Gln Ser Pro Leu Asp
Leu Thr Arg Glu Pro Gln Leu Glu Pro Gly 325 330 335 Glu Gln Ser Gln
Val Ala His Gly Thr Asn Gly Ile His Val Thr Gly 340 345 350 Gly Ser
Met Thr Ile Thr Gly Asn Ile Tyr Ile Tyr Asn Gly Pro Val 355 360 365
Leu Gly Gly Pro Pro Gly Pro Gly Asp Leu Pro Ala Thr Pro Glu Pro 370
375 380 Pro Tyr Pro Ile Pro Glu Glu Gly Asp Pro Gly Pro Pro Gly Leu
Ser 385 390 395 400 Thr Pro His Gln Glu Asp Gly Lys Ala Trp His Leu
Ala Glu Thr Glu 405 410 415 His Cys Gly Ala Thr Pro Ser Asn Arg Gly
Pro Arg Asn Gln Phe Ile 420 425 430 Thr His Asp 435
2227PRTArtificial SequenceChemically synthesized Fc portion of
LTBR06, including valine mutation 2Val Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65
70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185
190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 210 215 220 Ser Pro Gly 225 3663DNAArtificial
SequenceChemically synthesized extracellular domain of LTBR06
3atgctcctgc cttgggccac ctctgccccc ggcctggcct gggggcctct ggtgctgggc
60ctcttcgggc tcctggcagc agcggtgcct ccatatgcgt cggagaacca gacctgcagg
120gaccaggaaa aggaatacta tgagccccag caccgcatct gctgctcccg
ctgcccgcca 180ggcacctatg tctcagctaa atgtagccgc atccgggaca
cagtttgtgc cacatgtgcc 240gagaattcct acaacgagca ctggaactac
ctgaccatct gccagctgtg ccgcccctgt 300gacccagtga tgggcctcga
ggagattgcc ccctgcacaa gcaaacggaa gacccagtgc 360cgctgccagc
cgggaatgtt ctgtgctgcc tgggccctcg agtgtacaca ctgcgagcta
420ctttctgact gcccgcctgg cactgaagcc gagctcaaag atgaagttgg
gaagggtaac 480aaccactgcg tcccctgcaa ggcagggcac ttccagaata
cctcctcccc cagcgcccgc 540tgccagcccc acaccaggtg tgagaaccaa
ggtctggtgg aggcagctcc aggcactgcc 600cagtccgaca caacctgcaa
aaatccatta gagccactgc ccccagagat gtcaggaacc 660atg
6634194PRTArtificial SequenceChemically synthesized extracellular
domain of LTBR06 4Ala Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr Cys
Arg Asp Gln Glu 1 5 10 15 Lys Glu Tyr Tyr Glu Pro Gln His Arg Ile
Cys Cys Ser Arg Cys Pro 20 25 30 Pro Gly Thr Tyr Val Ser Ala Lys
Cys Ser Arg Ile Arg Asp Thr Val 35 40 45 Cys Ala Thr Cys Ala Glu
Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu 50 55 60 Thr Ile Cys Gln
Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu 65 70 75 80 Glu Ile
Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys Gln 85 90 95
Pro Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys Glu 100
105 110 Leu Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp
Glu 115 120 125 Val Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys Ala
Gly His Phe 130 135 140 Gln Asn Thr Ser Ser Pro Ser Ala Arg Cys Gln
Pro His Thr Arg Cys 145 150 155 160 Glu Asn Gln Gly Leu Val Glu Ala
Ala Pro Gly Thr Ala Gln Ser Asp 165 170 175 Thr Thr Cys Lys Asn Pro
Leu Glu Pro Leu Pro Pro Glu Met Ser Gly 180 185 190 Thr Met
5421PRTArtificial SequenceChemically synthesized amino acid
sequence of the mature form of LTBR06 5Ala Val Pro Pro Tyr Ala Ser
Glu Asn Gln Thr Cys Arg Asp Gln Glu 1 5 10 15 Lys Glu Tyr Tyr Glu
Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro 20 25 30 Pro Gly Thr
Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val 35 40 45 Cys
Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu 50 55
60 Thr Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu
65 70 75 80 Glu Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg
Cys Gln 85 90 95 Pro Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys
Thr His Cys Glu 100 105 110 Leu Leu Ser Asp Cys Pro Pro Gly Thr Glu
Ala Glu Leu Lys Asp Glu 115 120 125 Val Gly Lys Gly Asn Asn His Cys
Val Pro Cys Lys Ala Gly His Phe 130 135 140 Gln Asn Thr Ser Ser Pro
Ser Ala Arg Cys Gln Pro His Thr Arg Cys 145 150 155 160 Glu Asn Gln
Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp 165 170 175 Thr
Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser Gly 180 185
190 Thr Met Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
195 200 205 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 210 215 220 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 225 230 235 240 Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly 245 250 255 Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn 260 265 270 Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp 275 280 285 Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 290 295 300 Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 305 310
315 320 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn 325 330 335 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile 340 345 350 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr 355 360 365 Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 370 375 380 Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 385 390 395 400 Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 405 410 415 Ser Leu
Ser Pro Gly 420 6453PRTArtificial SequenceChemically synthesized
wild type LTBR-Ig fusion protein 6Met Leu Leu Pro Trp Ala Thr Ser
Ala Pro Gly Leu Ala Trp Gly Pro 1 5 10 15 Leu Val Leu Gly Leu Phe
Gly Leu Leu Ala Ala Ser Gln Pro Gln Ala 20 25 30 Val Pro Pro Tyr
Ala Ser Glu Asn Gln Thr Cys Arg Asp Gln Glu Lys 35 40 45 Glu Tyr
Tyr Glu Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro Pro 50 55 60
Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val Cys 65
70 75 80 Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr
Leu Thr 85 90 95 Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met
Gly Leu Glu Glu 100 105 110 Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr
Gln Cys Arg Cys Gln Pro 115 120 125 Gly Met Phe Cys Ala Ala Trp Ala
Leu Glu Cys Thr His Cys Glu Leu 130 135 140 Leu Ser Asp Cys Pro Pro
Gly Thr Glu Ala Glu Leu Lys Asp Glu Val 145 150 155 160 Gly Lys Gly
Asn Asn His Cys Val Pro Cys Lys Ala Gly His Phe Gln 165 170 175 Asn
Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys Glu 180 185
190 Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp Thr
195 200 205 Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser
Gly Thr 210 215 220 Met Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310
315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435
440 445 Leu Ser Pro Gly Lys 450 71344DNAArtificial
SequenceChemically synthesized LTBR06 7atgctcctgc cttgggccac
ctctgccccc ggcctggcct gggggcctct ggtgctgggc 60ctcttcgggc tcctggcagc
agcggtgcct ccatatgcgt cggagaacca gacctgcagg 120gaccaggaaa
aggaatacta tgagccccag caccgcatct gctgctcccg ctgcccgcca
180ggcacctatg tctcagctaa atgtagccgc atccgggaca cagtttgtgc
cacatgtgcc 240gagaattcct acaacgagca ctggaactac ctgaccatct
gccagctgtg ccgcccctgt 300gacccagtga tgggcctcga ggagattgcc
ccctgcacaa gcaaacggaa gacccagtgc 360cgctgccagc cgggaatgtt
ctgtgctgcc tgggccctcg agtgtacaca ctgcgagcta 420ctttctgact
gcccgcctgg cactgaagcc gagctcaaag atgaagttgg gaagggtaac
480aaccactgcg tcccctgcaa ggcagggcac ttccagaata cctcctcccc
cagcgcccgc 540tgccagcccc acaccaggtg tgagaaccaa ggtctggtgg
aggcagctcc aggcactgcc 600cagtccgaca caacctgcaa aaatccatta
gagccactgc ccccagagat gtcaggaacc 660atggtcgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag
780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt
caactggtac 840gtggacggcg tggaggtgca taatgccaag acaaagccgc
gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt cctcaccgtc
ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca aggtctccaa
caaagccctc ccagccccca tcgagaaaac catctccaaa 1020gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg
1080accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag
cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag aacaactaca
agaccacgcc tcccgtgttg 1200gactccgacg gctccttctt cctctacagc
aagctcaccg tggacaagag caggtggcag 1260caggggaacg tcttctcatg
ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320aagagcctct
ccctgtctcc gggt 13448448PRTArtificial SequenceChemically
synthesized LTBR06 amino acid sequence, including signal sequence
8Met Leu Leu Pro Trp Ala Thr Ser Ala Pro Gly Leu Ala Trp Gly Pro 1
5 10 15 Leu Val Leu Gly Leu Phe Gly Leu Leu Ala Ala Ala Val Pro Pro
Tyr 20 25 30 Ala Ser Glu Asn Gln Thr Cys Arg Asp Gln Glu Lys Glu
Tyr Tyr Glu 35 40 45 Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro
Pro Gly Thr Tyr Val 50 55 60 Ser Ala Lys Cys Ser Arg Ile Arg Asp
Thr Val Cys Ala Thr Cys Ala 65 70 75 80 Glu Asn Ser Tyr Asn Glu His
Trp Asn Tyr Leu Thr Ile Cys Gln Leu 85 90 95 Cys Arg Pro Cys Asp
Pro Val Met Gly Leu Glu Glu Ile Ala Pro Cys 100 105 110 Thr Ser Lys
Arg Lys Thr Gln Cys Arg Cys Gln Pro Gly Met Phe Cys 115 120 125 Ala
Ala Trp Ala Leu Glu Cys Thr His Cys Glu Leu Leu Ser Asp Cys 130 135
140 Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp Glu Val Gly Lys Gly Asn
145 150
155 160 Asn His Cys Val Pro Cys Lys Ala Gly His Phe Gln Asn Thr Ser
Ser 165 170 175 Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys Glu Asn
Gln Gly Leu 180 185 190 Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp
Thr Thr Cys Lys Asn 195 200 205 Pro Leu Glu Pro Leu Pro Pro Glu Met
Ser Gly Thr Met Val Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275
280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395
400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445 9425PRTArtificial SequenceChemically
synthesized LTBR05, mature form amino acid sequence 9Ser Gln Pro
Gln Ala Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr Cys 1 5 10 15 Arg
Asp Gln Glu Lys Glu Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys 20 25
30 Ser Arg Cys Pro Pro Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile
35 40 45 Arg Asp Thr Val Cys Ala Thr Cys Ala Glu Asn Ser Tyr Asn
Glu His 50 55 60 Trp Asn Tyr Leu Thr Ile Cys Gln Leu Cys Arg Pro
Cys Asp Pro Val 65 70 75 80 Met Gly Leu Glu Glu Ile Ala Pro Cys Thr
Ser Lys Arg Lys Thr Gln 85 90 95 Cys Arg Cys Gln Pro Gly Met Phe
Cys Ala Ala Trp Ala Leu Glu Cys 100 105 110 Thr His Cys Glu Leu Leu
Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu 115 120 125 Leu Lys Asp Glu
Val Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys 130 135 140 Ala Gly
His Phe Gln Asn Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro 145 150 155
160 His Thr Arg Cys Glu Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr
165 170 175 Ala Gln Ser Asp Thr Thr Cys Lys Asn Pro Leu Glu Pro Leu
Pro Pro 180 185 190 Glu Met Ser Gly Thr Met Val Asp Lys Thr His Thr
Cys Pro Pro Cys 195 200 205 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 210 215 220 Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 225 230 235 240 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 245 250 255 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 260 265 270 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 275 280
285 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
290 295 300 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 305 310 315 320 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu 325 330 335 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 340 345 350 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 355 360 365 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 370 375 380 Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 385 390 395 400
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 405
410 415 Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425
10421PRTArtificial SequenceChemically synthesized non-glycosylated
LTBR-IgG 10Ala Val Pro Pro Tyr Ala Ser Glu Gln Gln Thr Cys Arg Asp
Gln Glu 1 5 10 15 Lys Glu Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys
Ser Arg Cys Pro 20 25 30 Pro Gly Thr Tyr Val Ser Ala Lys Cys Ser
Arg Ile Arg Asp Thr Val 35 40 45 Cys Ala Thr Cys Ala Glu Asn Ser
Tyr Asn Glu His Trp Asn Tyr Leu 50 55 60 Thr Ile Cys Gln Leu Cys
Arg Pro Cys Asp Pro Val Met Gly Leu Glu 65 70 75 80 Glu Ile Ala Pro
Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys Gln 85 90 95 Pro Gly
Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr His Cys Glu 100 105 110
Leu Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala Glu Leu Lys Asp Glu 115
120 125 Val Gly Lys Gly Asn Asn His Cys Val Pro Cys Lys Ala Gly His
Phe 130 135 140 Gln Gln Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His
Thr Arg Cys 145 150 155 160 Glu Asn Gln Gly Leu Val Glu Ala Ala Pro
Gly Thr Ala Gln Ser Asp 165 170 175 Thr Thr Cys Lys Asn Pro Leu Glu
Pro Leu Pro Pro Glu Met Ser Gly 180 185 190 Thr Met Val Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu 195 200 205 Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 210 215 220 Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 225 230 235
240 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
245 250 255 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 260 265 270 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 275 280 285 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 290 295 300 Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 305 310 315 320 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 325 330 335 Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 340 345 350 Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 355 360
365 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
370 375 380 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 385 390 395 400 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 405 410 415 Ser Leu Ser Pro Gly 420
11426PRTArtificial SequenceChemically synthesized LTBR01, mature
form amino acid sequence 11Ser Gln Pro Gln Ala Val Pro Pro Tyr Ala
Ser Glu Asn Gln Thr Cys 1 5 10 15 Arg Asp Gln Glu Lys Glu Tyr Tyr
Glu Pro Gln His Arg Ile Cys Cys 20 25 30 Ser Arg Cys Pro Pro Gly
Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile 35 40 45 Arg Asp Thr Val
Cys Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His 50 55 60 Trp Asn
Tyr Leu Thr Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val 65 70 75 80
Met Gly Leu Glu Glu Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln 85
90 95 Cys Arg Cys Gln Pro Gly Met Phe Cys Ala Ala Trp Ala Leu Glu
Cys 100 105 110 Thr His Cys Glu Leu Leu Ser Asp Cys Pro Pro Gly Thr
Glu Ala Glu 115 120 125 Leu Lys Asp Glu Val Gly Lys Gly Asn Asn His
Cys Val Pro Cys Lys 130 135 140 Ala Gly His Phe Gln Asn Thr Ser Ser
Pro Ser Ala Arg Cys Gln Pro 145 150 155 160 His Thr Arg Cys Glu Asn
Gln Gly Leu Val Glu Ala Ala Pro Gly Thr 165 170 175 Ala Gln Ser Asp
Thr Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro 180 185 190 Glu Met
Ser Gly Thr Met Val Asp Lys Thr His Thr Cys Pro Pro Cys 195 200 205
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 210
215 220 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 225 230 235 240 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 245 250 255 Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu 260 265 270 Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu 275 280 285 His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 290 295 300 Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 305 310 315 320 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 325 330
335 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
340 345 350 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 355 360 365 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 370 375 380 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 385 390 395 400 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 405 410 415 Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 420 425 12421PRTArtificial SequenceChemically
synthesized LTBR09, mature form amino acid sequence 12Ala Val Pro
Pro Tyr Ala Ser Glu Asn Gln Thr Cys Arg Asp Gln Glu 1 5 10 15 Lys
Glu Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro 20 25
30 Pro Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val
35 40 45 Cys Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn
Tyr Leu 50 55 60 Thr Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val
Met Gly Leu Glu 65 70 75 80 Glu Ile Ala Pro Cys Thr Ser Lys Arg Lys
Thr Gln Cys Arg Cys Gln 85 90 95 Pro Gly Met Phe Cys Ala Ala Trp
Ala Leu Glu Cys Thr His Cys Glu 100 105 110 Leu Leu Ser Asp Cys Pro
Pro Gly Thr Glu Ala Glu Leu Lys Asp Glu 115 120 125 Val Gly Lys Gly
Asn Asn His Cys Val Pro Cys Lys Ala Gly His Phe 130 135 140 Gln Asn
Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys 145 150 155
160 Glu Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp
165 170 175 Thr Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met
Ser Gly 180 185 190 Thr Met Val Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 195 200 205 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 210 215 220 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 225 230 235 240 Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 245 250 255 Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln 260 265 270 Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 275 280
285 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
290 295 300 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 305 310 315 320 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn 325 330 335 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 340 345 350 Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr 355 360 365 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 370 375 380 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 385 390 395 400
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 405
410 415 Ser Leu Ser Pro Gly 420 1313PRTArtificial
SequenceChemically synthesized LTBR06 hinge sequence 13Val Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10 1413PRTArtificial
SequenceChemically synthesized Hinge 2211 (D169N) sequence 14Val
Asn Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 1 5 10
1513PRTArtificial SequenceChemically synthesized Hinge 2212 (T198N)
sequence 15Val Asp Lys Asn His Thr Cys Pro Pro Cys Pro Ala Pro 1 5
10 1612PRTArtificial SequenceChemically synthesized Hinge 2221
(valine deletion) sequence 16Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 1 5 10 1717PRTArtificial SequenceChemically synthesized
Hinge 2217 (full length) sequence 17Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro 1814PRTArtificial
SequenceChemically synthesized Hinge 2219 (G4-hinge-G4Fc) sequence
18Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 1 5 10
1916PRTArtificial SequenceChemically synthesized Hinge 2218
(G2-hinge-G2Fc) sequence 19Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro Pro Val Ala Gly Pro 1 5 10 15 207PRTArtificial
SequenceChemically synthesized Hinge 2220 (short hinge - G1Fc)
sequence 20Cys Pro Pro Cys Pro Ala Pro 1 5 21225PRTHomo
sapiensmisc_featureExtracellular domain of human wild type LTBR
amino acid sequence (immature form) corresponding to GenPept ID No.
P36941 21Met Leu Leu Pro Trp Ala Thr Ser Ala Pro Gly Leu Ala Trp
Gly Pro 1 5 10 15 Leu Val Leu Gly Leu Phe Gly Leu Leu Ala Ala Ser
Gln Pro Gln Ala 20 25 30 Val Pro Pro Tyr Ala Ser Glu Asn Gln Thr
Cys Arg Asp Gln Glu Lys 35 40 45 Glu
Tyr Tyr Glu Pro Gln His Arg Ile Cys Cys Ser Arg Cys Pro Pro 50 55
60 Gly Thr Tyr Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val Cys
65 70 75 80 Ala Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr
Leu Thr 85 90 95 Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met
Gly Leu Glu Glu 100 105 110 Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr
Gln Cys Arg Cys Gln Pro 115 120 125 Gly Met Phe Cys Ala Ala Trp Ala
Leu Glu Cys Thr His Cys Glu Leu 130 135 140 Leu Ser Asp Cys Pro Pro
Gly Thr Glu Ala Glu Leu Lys Asp Glu Val 145 150 155 160 Gly Lys Gly
Asn Asn His Cys Val Pro Cys Lys Ala Gly His Phe Gln 165 170 175 Asn
Thr Ser Ser Pro Ser Ala Arg Cys Gln Pro His Thr Arg Cys Glu 180 185
190 Asn Gln Gly Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp Thr
195 200 205 Thr Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser
Gly Thr 210 215 220 Met 225 22227PRTHomo Sapiensmisc_featureHuman
Ig (Fc) domain 22Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 100
105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220
Ser Pro Gly 225 23193PRTArtificial SequenceChemically synthesized
N-5 extracellular domain of LTBR06 23Val Pro Pro Tyr Ala Ser Glu
Asn Gln Thr Cys Arg Asp Gln Glu Lys 1 5 10 15 Glu Tyr Tyr Glu Pro
Gln His Arg Ile Cys Cys Ser Arg Cys Pro Pro 20 25 30 Gly Thr Tyr
Val Ser Ala Lys Cys Ser Arg Ile Arg Asp Thr Val Cys 35 40 45 Ala
Thr Cys Ala Glu Asn Ser Tyr Asn Glu His Trp Asn Tyr Leu Thr 50 55
60 Ile Cys Gln Leu Cys Arg Pro Cys Asp Pro Val Met Gly Leu Glu Glu
65 70 75 80 Ile Ala Pro Cys Thr Ser Lys Arg Lys Thr Gln Cys Arg Cys
Gln Pro 85 90 95 Gly Met Phe Cys Ala Ala Trp Ala Leu Glu Cys Thr
His Cys Glu Leu 100 105 110 Leu Ser Asp Cys Pro Pro Gly Thr Glu Ala
Glu Leu Lys Asp Glu Val 115 120 125 Gly Lys Gly Asn Asn His Cys Val
Pro Cys Lys Ala Gly His Phe Gln 130 135 140 Asn Thr Ser Ser Pro Ser
Ala Arg Cys Gln Pro His Thr Arg Cys Glu 145 150 155 160 Asn Gln Gly
Leu Val Glu Ala Ala Pro Gly Thr Ala Gln Ser Asp Thr 165 170 175 Thr
Cys Lys Asn Pro Leu Glu Pro Leu Pro Pro Glu Met Ser Gly Thr 180 185
190 Met 245PRTArtificial SequenceSynthetic peptide 24Ala Ala Gly
Thr Tyr 1 5 254PRTArtificial SequenceSynthetic peptide 25Ala Gly
Thr Tyr 1 264PRTArtificial SequenceSynthetic peptide 26Ser Gln Pro
Gln 1 2719PRTArtificial SequenceChemically synthesized IgG hinge
peptide 27Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu 1 5 10 15 Gly Gly Pro 2819PRTArtificial SequenceChemically
synthesized hinge peptide 28Val Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro
* * * * *