JP2010521472A - Treatment of autoimmune disorders - Google Patents

Abstract

A method for treating a disease with a soluble inhibitor of the lymphotoxin pathway having improved properties. An improved LTBR-Ig fusion protein and pharmaceutical compositions thereof are also described. In one aspect, the present invention provides a lymphotoxin-β receptor (LT-β-R)-comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig moiety that is 227 amino acids in length. With respect to a composition comprising a population of Ig-fusion proteins, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises a wild-type LT-β-R extracellular domain set forth in SEQ ID NO: 21. Only 5 amino acids have been lost from the N-terminus of the mature form, and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamic acid.

Description

(Related application)
This application claims priority to US Provisional Patent Application No. 60 / 918,518, filed September 15, 2007.

(background)
Protein heterogeneity can be caused by a range of post-translational modifications. Protein heterogeneity often results from various types of post-translational modifications, including carboxylation, hydroxylation, proteolytic processing, sulfation, and glycosylation, of which glycosylation is the most common (Non-patent Document 1). Post-translational modifications potentially result in product production (eg, by affecting the degree of proper protein folding), stability, and a range of pharmacokinetic and pharmacodynamic parameters, and safety And can affect immunogenicity. Post-translational modifications of the therapeutic biologics, or protein-based biopharmaceuticals, can affect protein properties for their therapeutic application.

  N-terminal and / or C-terminal heterogeneity is an example of protein heterogeneity that must be considered in the production of protein-based biopharmaceuticals. N-terminal heterogeneity results from proteolytic processing at the amino-terminal portion of the protein, where such processing can result in a population of proteins having various sizes. Variations in N-terminal proteolysis can occur in proteins that contain a single sequence. Furthermore, the N-terminal glutamine residue can undergo spontaneous cyclization to form pyroglutamic acid. Thus, obtaining a homogeneous protein population that can be used for therapeutic purposes is often a challenge.

Lymphotoxin beta receptor (LTBR) is a member of the tumor necrosis factor receptor (TNFR) family. The receptors are expressed on the cell surface in the parenchyma and stroma of most lymphoid organs, but are not present on T and B lymphocytes. Signaling through LTBR by LTa / beta heterotrimer (LT) are important during lymphoid differentiation, LTBR is also a ligand LIGHT (homologous to L ymphotoxins, exhibits i nducible expression, and competes with herpes simplex virus g It is known to bind lycoprotein D for H VEM, a receptor expressed by T lymphocytes, which had an effect on T cell drive events (both peripheral and thymus). LT and LIGHT are expressed on the surface of activated lymphocytes. Blocking the LT pathway with soluble decoy LTBR has been shown to be effective for treating autoimmune diseases in various animal models.

  Development of dosage forms for soluble forms of LTBR with reduced heterogeneity and optimal administration of these molecules is highly beneficial.

Walsh and Jefferis (2006) Nat Biotech 24 (10): 1241

(Summary of the Invention)
In one aspect, the present invention provides a lymphotoxin-β receptor (LT-β-R)-comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig moiety that is 227 amino acids in length. Concerning a composition comprising a population of Ig-fusion proteins, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises a wild-type LT-β-R extracellular domain as shown in SEQ ID NO: 21. Only 5 amino acids are lost from the N-terminus of the mature form, and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamic acid.

  In one embodiment, the N-terminal amino acid of the modified LT-β-R-Ig fusion protein is a nonpolar amino acid.

  In one embodiment, the nonpolar amino acid is valine (amino acid 6 in the mature form of wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (wild type LT-β-R extracellular of SEQ ID NO: 21). Any of amino acids 5) in the mature form of the domain.

  In one embodiment, at least 95% of the N-terminal amino acid LT-β-R-Ig-fusion protein is valine (amino acid 6 in the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21). Or alanine (amino acid 5 in the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21).

  In one embodiment, the composition is made by expressing in a mammalian cell a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4.

  In one embodiment, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3.

  In one embodiment, the modified Ig portion comprises an Fc region of the IgG1 isotype.

  In one embodiment, the modified Ig moiety comprises the amino acid sequence set forth in SEQ ID NO: 2.

  In one embodiment, the Ig moiety is not glycosylated.

  In one embodiment, the composition is made by expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein shown in SEQ ID NO: 5 in mammalian cells.

  In one embodiment, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 7.

  In one embodiment, the expressing step is performed on a manufacturing scale.

  In one aspect, the invention relates to a composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, Here, the modified LT-β-R extracellular domain is not glycosylated.

  In one embodiment, the non-glycosylated extracellular domain of LTBR comprises amino acids 1-194 of SEQ ID NO: 10.

  In another aspect, the invention relates to a composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion proteins, wherein the fusion protein is 193 amino acids or 194 amino acids in length. Comprising a modified LT-β-R extracellular domain and a modified Ig portion, wherein the population comprises reduced N-terminal pyroglutamate formation and reduced C-terminus compared to wild-type LT-β-R-Ig fusion protein Has non-uniformity.

  In one embodiment, at least 90% of the LT-β-R-Ig-fusion protein comprises a modified LT-β-R extracellular domain that exhibits the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23.

  In one embodiment, the modified Ig portion comprises a mutation in the hinge region.

  In one aspect, the present invention provides a lymphotoxin-β receptor (LT-β-R)-comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig moiety that is 227 amino acids in length. With respect to a pharmaceutical composition comprising a population of Ig-fusion proteins as well as a pharmaceutically acceptable carrier, wherein at least 90% of the LT-β-R-Ig-fusion protein is the wild-type shown in SEQ ID NO: 21 Only 5 amino acids have been lost from the N-terminus of the mature form of the type LT-β-R extracellular domain, and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamic acid.

  In one embodiment, the N-terminal amino acid of the modified LT-β-R-Ig fusion protein is a nonpolar amino acid.

  In one embodiment, the nonpolar amino acid is valine (amino acid 6 in the mature form of wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (wild type LT-β-R extracellular of SEQ ID NO: 21). Any of amino acids 5) in the mature form of the domain.

  In one embodiment, at least 95% of the LT-β-R-Ig-fusion protein has an N-terminal amino acid of valine (amino acid 6 of the mature form of the wild-type LT-β-R extracellular domain of SEQ ID NO: 21). ) Or alanine (amino acid 5 in the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21).

  In one embodiment, the composition is made by expressing in a mammalian cell a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4.

  In one aspect, the invention relates to a method for treating an autoimmune disorder comprising administering to a subject a pharmaceutical composition according to claim 23.

  In one embodiment, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE).

  In one embodiment, the invention relates to a pharmaceutical composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein is , Containing a modified LT-β-R extracellular domain and a modified Ig portion of 193 or 194 amino acids in length, wherein the population is reduced compared to the wild type LT-β-R-Ig fusion protein N-terminal pyroglutamic acid formation and reduced C-terminal heterogeneity.

  In one embodiment, at least 90% of the LT-β-R-Ig-fusion protein comprises a modified LT-β-R extracellular domain that exhibits the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23.

  In one embodiment, the modified Ig portion comprises a mutation in the hinge region.

  In one embodiment, the amino acid sequence is shown in SEQ ID NO: 5.

  In one aspect, the invention relates to a method for treating an autoimmune disorder comprising administering to a subject a pharmaceutical composition according to claim 29.

  In one embodiment, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE).

  In one embodiment, the autoimmune disorder is rheumatoid arthritis.

  In one embodiment, the pharmaceutical composition is administered to the subject once every two weeks at a dose of about 0.6-3 mg / kg.

  In one embodiment, the pharmaceutical composition is administered subcutaneously.

  In one aspect, the present invention relates to an isolated polypeptide comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig moiety that is 227 amino acids in length, wherein The peptide has lost only 5 amino acids from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21, and the polypeptide lacks the N-terminal pyroglutamic acid.

  In one embodiment, the N-terminal amino acid is a nonpolar amino acid.

  In one embodiment, the nonpolar amino acid is valine (amino acid 6 in the mature form of wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (wild type LT-β-R extracellular of SEQ ID NO: 21). Any of amino acids 5) in the mature form of the domain.

  In one embodiment, the polypeptide is made by expressing in a mammalian cell a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4.

  In one embodiment, the invention relates to an isolated nucleic acid molecule that encodes a polypeptide of the invention.

  In one embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 7.

  In one embodiment, the invention relates to a vector comprising a nucleic acid molecule of the invention.

  In one embodiment, the invention relates to a host cell that expresses the vector of the invention.

  In one embodiment, the cell is a Chinese hamster ovary (CHO) cell.

  In one embodiment, the present invention provides a composition comprising a population of lymphotoxin-β receptor (LT-β-R) -Ig-fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety. Wherein at least 90% of the LT-β-R-Ig-fusion protein is only 5% from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21. The amino acid is lost, and the process is a step of expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein shown in SEQ ID NO: 8 in mammalian cells, and obtaining the population from the culture supernatant. And, if necessary, purifying the supernatant, thereby producing a lymphotoxin-β receptor comprising a modified LT-β-R extracellular domain and a modified Ig moiety. Obtaining a composition comprising a population of Scepter (LT-β-R) -Ig-fusion proteins, wherein at least 90% of the LT-β-R-Ig-fusion proteins are SEQ ID NO: 21 Only 5 amino acids have been lost from the N-terminus of the mature form of the wild-type LT-β-R moiety shown in FIG.

  In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 7.

  In one embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 7.

  In one embodiment, the invention relates to a method for treating rheumatoid arthritis in a human subject, the method comprising administering to the subject a dose of an LT-β-R-Ig fusion protein. Where the dose is sufficient to maintain an average concentration of about 0.14 μg / ml to about 3.5 μg / ml in the serum of the subject.

  In another aspect, the invention relates to a method for treating rheumatoid arthritis in a human subject, the method comprising administering to the subject a dose of an LT-β-R-Ig fusion protein. Where the dose is sufficient to maintain an average a minimum average concentration of about 0.6 μg / ml in the serum of the subject.

  In one embodiment, the LTBR-Ig fusion protein comprises the amino acid sequence shown in SEQ ID NO: 5.

  In one embodiment, the concentration is achieved by administering the LT-β-R-Ig fusion protein once every 7-60 days at a dose of about 0.01 to about 5 mg / kg.

  In one embodiment, the present invention relates to a method for treating rheumatoid arthritis in a human subject, wherein the method is directed to the subject about 0.6-3 mg / day at most twice every 7-30 days. administering a kg dose of LT-β-R-Ig fusion protein.

  In one embodiment, the method comprises administering to the subject an LT-β-R-Ig fusion protein at a dose of about 0.6-3 mg / kg once every 7-14 days. .

  In one embodiment, administration is once every 14-30 days.

  In one embodiment, administration is once every 28-60 days.

  In one embodiment, administration is once every 7-30 days.

  In one embodiment, the present invention relates to a method for treating an autoimmune disorder in a human subject, said method comprising an LT-β-R extracellular domain that is 193 amino acids or 194 amino acids in length. administering to the subject a dose of a pharmaceutical composition comprising a population of β-R-Ig fusion proteins, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises Only 5 amino acids have been lost from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21, and the dose is about 0.6 μg / ml in serum to the subject. Is sufficient to maintain a minimum average concentration.

  In one embodiment, the LT-β-R-Ig fusion protein further comprises a modified Ig moiety.

  In one embodiment, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE).

  In one embodiment, the pharmaceutical composition comprises the amino acid sequence shown in SEQ ID NO: 5.

  In one embodiment, administration is twice a month.

  In one embodiment, administration is once a month.

  In one embodiment, administration is subcutaneous.

  In one embodiment, the dose is about 1 mg / kg.

  In one embodiment, the dose is about 3 mg / kg.

  In one embodiment, the dose is administered about 1 mg / kg about every 7-20 days.

  In one embodiment, the dose is administered about 3 mg / kg about every 14-30 days.

  In one embodiment, the dose is administered about 1 mg / kg about every 14 days.

  In one embodiment, the autoimmune disorder is rheumatoid arthritis and the subject is diagnosed with rheumatoid arthritis and before the administration of the LT-β-R-Ig fusion protein. Being treated with drugs.

  In one embodiment, the rheumatoid arthritis drug is selected from the group consisting of DMARD, NSAID, and corticosteroid.

In one embodiment, the human is a DMARD poor responder. In one embodiment, the rheumatoid arthritis drug is a TNF inhibitor.

  In one embodiment, the rheumatoid arthritis drug is adalimumab (Humira®), etanercept (Enbrel®), or infliximab (Remicade®).

  In one embodiment, LT-β-R-Ig is administered in combination with a rheumatoid arthritis drug.

  In one embodiment, the human is evaluated to determine if the response to the rheumatoid arthritis drug is insufficient prior to administration of LT-β-R-Ig.

  In one embodiment, the human is determined to have an inadequate response to the rheumatoid arthritis drug, and then the human is administered LT-β-R-Ig.

  In one embodiment, the human is asymptomatic for a first expression of rheumatoid arthritis and symptomatic for a second expression of rheumatoid arthritis.

  In one embodiment, LT-β-R-Ig is administered instead of the rheumatoid arthritis drug.

  In one embodiment, the administration is in combination with a tumor necrosis factor (TNF) inhibitor.

  In one embodiment, the TNF inhibitor is adalimumab (Humira®), etanercept (Enbrel®), or infliximab (Remicade®).

  In one embodiment, the human is an anti-TNF poor responder.

  In one embodiment, the administration is in combination with a non-steroidal anti-inflammatory agent (NSAID), a corticosteroid, or a disease modifying anti-rheumatic drug (DMARD).

  In one embodiment, administration is in combination with methotrexate.

  In one embodiment, the human is a DMARD poor responder.

FIG. 1 describes the changes made from LTBR01 to LTBR06 to improve non-uniformity. Bold indicates the secretory sequence, and italic / underlined letters indicate the amino acid that is removed in LTBR06 relative to LTBR01 (ie, amino acids 1-4 and the last amino acid (lysine)). Three consensus sites for N-linked glycosylation are located at Asn13, 150 and 276. The amino acid position refers to the full length LTBR, i.e., amino acids 1-4 are those that are removed in LTBR06. FIG. 2 provides an alignment of LTBR01, LTBR05, LTBR06, and LTBR09. The secretory sequence is omitted from the LTBR sequence. FIG. 2 provides an alignment of LTBR01, LTBR05, LTBR06, and LTBR09. The secretory sequence is omitted from the LTBR sequence. FIG. 3 describes LTBR06 compared to wild type and provides a schematic diagram of the protein. FIG. 4 describes the amino acid sequence of the LTBR06 construct (the mature form of LTBR06 shown in SEQ ID NO: 5). LTBR06 is a disulfide-linked and glycosylated dimeric protein. There are 28 cysteine residues and 6 glycosylation sites, the latter glycosylation sites being shown in bold. FIG. 5 is unglycosylated hLTβR hIgG1 (mature form of protein). The asparagine to glutamine mutation in the LTβR extracellular domain is shown in bold. The huLTβR is residues 1 to 204 and the huIgG1 Fc is residues 205 to 431 described above (the glycosylation site of the Fc is intact). FIG. 6 describes a hinge that can be used in the LTBR IgG fusion protein of the invention. FIG. 7 sets forth a table that summarizes the results from the hinge expression analysis. FIG. 8 is a graph showing the percent replacement of RA symptom scores (tender joint count (TJC) and swollen joint count (SJC)) in patients after treatment with LTBR-Fc. It is. FIG. 9 provides a graph showing a decrease in the number of joints that are painful to touch (TJC) in patients after treatment with LTBR-Fc (LTBR06). FIG. 10 provides a graph showing the decrease in the number of swollen joints (SJC) n in patients after treatment with LTBR-Fc (LTBR06). FIG. 11 provides a graph showing the median% change in swollen joint number n (SJC) in patients after treatment with LTBR-Fc (LTBR06). FIG. 12 provides a graph showing ACR20 improvement in patients after treatment with LTBR-Fc (LTBR06). FIG. 13 graphically shows the serum levels of various sialylated LFA3TIP and hu-LTBR-Ig (LTBR05) in mice 24 hours after administration at 100 μg / animal. FIG. 14 illustrates a comparison of LTBR-Fc and representative antibodies, showing that significant differences in efficacy are observed over the long time frame at the same administration. Arrows A and B show representative α and β phases of the antibody or Fc-fusion protein, respectively. For antibodies, the gray line indicated by arrow C indicates a typical lower concentration for potency, whereas arrow D indicates that LTBR-Fc has potency at significantly lower concentrations.

  In order that the present invention may be more readily understood, certain terms are first defined.

  The term “fusion protein” refers to gene expression of a molecule comprising two or more proteins or fragments thereof covalently linked through individual peptide backbones, most preferably a polynucleotide branch encoding those proteins. Refers to molecules produced through In a preferred embodiment, the fusion protein comprises an immunoglobulin domain.

  The general term “immunoglobulin” includes five distinct classes of antibodies that can be distinguished biochemically. All five classes of antibodies are clearly within the scope of the present invention, and the following discussion relates generally to the IgG class of immunoglobulin molecules.

  The term “immunoglobulin fusion protein” refers to a functional part of a polypeptide (generally comprising the extracellular domain of a cell surface protein) and an immunoglobulin constant region (eg hinge, CH1, CH2 or CH3 domain or part thereof). Or a fusion with one or more of a combination. In one embodiment, the polypeptide is a member of the TNF family of receptors. The portion of the Ig molecule can be obtained from any of a variety of immunoglobulin isotypes (eg, including IgG1, IgG2, IgM, IgA, etc.). An immunoglobulin fusion protein is referred to herein as an Ig for an Fc fusion protein.

  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 can include a receptor, or a ligand binding portion thereof, and a dimerization domain (eg, an Fc domain).

  As used herein, the term “modified LTBR extracellular domain” refers to an amino acid sequence that differs from the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 21 (wild type hLTBR) at one or more amino acid positions. The mature form of the polypeptide (or protein) it has.

  As used herein, the term “modified Ig moiety” refers to an Fc region known in the art (including the sequence provided herein as SEQ ID NO: 22) at one or more amino acid positions. Refers to polypeptides (or proteins) having different amino acid sequences.

  The term “reduced N-terminal heterogeneity” refers to a decrease in the number of proteins in a population with various N-terminal amino acid residues present in different forms relative to a control protein population. For example, control protein expression resulted in loss of the N-terminal amino acid residue (N-1, N-2, and N-3) ranging from 1 amino acid to 3 amino acids (relative to the putative translated protein). Resulting in a protein population comprising the protein (described as In this case, the protein population comprises three different types (ie, N-1, N-2, and N-3). Thus, a population of proteins with reduced N-terminal heterogeneity can be obtained with less than three different N-terminal amino acid residues (eg, N-1 and N-2, N-2 and N-3, or N- 1 or N-3 type), or a population of proteins with a reduced proportion of one or more of these types of modified molecules. The N-terminal amino acid itself may not be different (eg, for a protein having N-1 N-terminus relative to the wild-type form, the N-terminal amino acid may be alanine and Note that for proteins with the N-terminus of N-3, the N-terminal amino acid may also be alanine). Thus, in one embodiment, N-terminal heterogeneity must deal with overall length differences among proteins within a population (expressed against a putative translated protein). Thus, the term “N- #” refers to the number of amino acids deleted from the amino terminus of the wild type translated protein. For example, if the wild-type predicted protein sequence was AAGTY, the N-1 protein will not include the first A, but instead will begin with AGTY, but both proteins will begin with A. Similarly, N-2 protein begins with GTY.

  In another embodiment, N-terminal heterogeneity can be reduced by reducing the potential number of modified forms in the protein population. For example, the N-terminal glutamine residue can undergo spontaneous cyclization to form pyroglutamic acid. This can lead to further non-uniformities. In one embodiment, pyroglutamic acid formation can be reduced or eliminated compared to that present in the wild-type protein.

  The term “reduced C-terminal heterogeneity” similarly refers to a decrease in the number of proteins in a population with different C-terminal amino acid residues relative to a control protein population. Furthermore, the term “C- #” refers to the number of amino acids deleted from the carboxy terminus of the protein.

  The term “glycosylation” refers to the covalent attachment of one or more carbohydrates to a polypeptide. Typically, glycosylation is a post-translational event that may be present in the intracellular environment of a cell or its extract. The term glycosylation includes, for example, N-linked glycosylation (where one or more sugars are attached to an asparagine residue) and / or O-linked glycosylation (where one or more sugars are Amino acid residues having a hydroxyl group (for example, bound to serine or threonine).

  The phrase “TNF family of receptors” refers to any receptor having a formal TNF family cysteine cross-linking pattern, whether membrane bound or secreted in nature (as is the case with osteoprotegerin), or the TNF family of Refers to any receptor that binds to a defined member of a ligand (eg, Banner et al. 1993). The present invention, in other embodiments, relates to TNF family receptor-Ig fusions obtained by the methods discussed herein, and pharmaceutical preparations comprising the above.

  A “signal peptide” or “signal sequence” is a peptide sequence directed to a newly synthesized polypeptide to which the signal peptide is attached to the endoplasmic reticulum (ER) for further post-translational processing and distribution. A mature form of a protein refers to a protein without a signal sequence.

  As used herein, a “soluble LTBR” is a polypeptide that includes all or part of the extracellular domain of human LTBR (eg, an LTBR immunoglobulin fusion). Preferred soluble LTBRs are sufficient sequences from the extracellular region of LTBR that can bind to a ligand (eg, LT or LIGHT) with at least 10% of the affinity of the molecule of SEQ ID NO: 1, and preferably at least 50%. A soluble molecule containing

  The term “ligand binding domain” or “ligand binding moiety” as used herein refers to any native receptor (eg, cell surface receptor), or at least qualitative ligand binding ability, and preferably the corresponding Refers to any region or derivative thereof that retains the biological activity of the native receptor.

  The terms “about” and “about”, as used herein with reference to a numerical value, generally are unless otherwise indicated or apparent from the context (such numerical values are not Includes numbers that fall within the range of 10% in any direction of the value (greater than or less than the number), except where it exceeds 100% of the possible values.

  As defined herein, the term “conservative substitution” refers to the replacement of one amino acid residue with another biologically similar residue. For example, a conservative amino acid substitution is expected to have little or no effect on its biological activity, particularly if the conservative amino acid substitution represents less than 10% of the total number of residues in the polypeptide or protein. The Preferably, conservative amino acid substitutions show a change of less than 5% of the polypeptide or protein, most preferably less than 2% of the polypeptide or protein (eg when calculated according to SEQ ID NO: 5) Most preferred conservative substitutions represent less 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 both the substituted amino acid and the substituted amino acid are acyclic.

  In particular, other examples of conservative substitutions include the use of one other residue instead of one hydrophobic residue (eg, isoleucine, valine, leucine or methionine), or one polar residue In place of arginine instead of lysine, glutamic acid instead of aspartic acid, glutamine instead of asparagine, etc.

  Genetically encoded amino acids can generally be divided into four families: (1) acidic: aspartic acid, glutamic acid; (2) basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine. , Leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarities: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine can be grouped together as aromatic amino acids. In one embodiment, the amino acid at the N-terminus of the polypeptide of the present invention is a non-polar amino acid (ie, alanine, valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, glycine, except for imino acid (ie, proline) , And cysteine).

  The term “treatment” refers to a disorder described above, either to ameliorate or prevent a condition, symptom, or parameter associated with a disorder, or to a statistically significant extent, or to a degree that can be detected by one skilled in the art. Treatment in an effective amount, manner and / or manner to prevent onset, progression, or worsening (including secondary damage caused by the disorder). Thus, treatment can achieve therapeutic and / or prophylactic benefits. Effective amounts, modes, or embodiments can vary depending on the subject and can be tailored to the subject.

  The term "insufficient response", "insufficient responder" or "-IR" means insufficient efficacy or intolerance for a particular treatment as assessed by a patient or physician with ordinary skill Or patients with unacceptable toxicity. Insufficient efficacy may mean that a predetermined level of response to treatment cannot be met. In the case of rheumatoid arthritis (RA), for example, inadequate efficacy can be attributed to RA clinical parameters (eg, touching and painful joints (TJC), swollen joints (SJC), comprehensive assessment of disease active patients [ PGA visual analog scale (VAS) 0-10 cm], at least 10%, 20% in physicians' comprehensive assessment of disease activity (MDGA VAS 0-10 cm) and C-reactive protein (CRP (mg / dl)) , 25%, 30%, 40%, 50% or more can be defined as unacceptable toxicity results in a medical need or recommendation to discontinue use of the first drug Examples of unacceptable or unacceptable toxicities include liver injury or liver dysfunction, severe alleles A Ghee reaction, a serious depression or suicide ideation, anaphylaxis, or an injection site reaction may be mentioned.

  As used herein, “administered in combination” means that two or more agents (eg, soluble LTBR and a second agent) are administered to a subject simultaneously or within a fixed interval, As a result, it means that there is an overlapping effect of each drug on the patient. Preferably, the administration of the first agent and the second agent are spaced sufficiently close to each other so that a combined effect (eg, an additive or synergistic effect) is achieved. The interval may be an interval of hours, days or weeks. The agent can be simultaneously bioavailable (eg, detectable) in a subject. In preferred embodiments, at least one administration of one of the agents (eg, the first agent) is such that the other agent (eg, the soluble LTBR) is further at a therapeutic level in the subject. Done while in existence.

  The term “once a week” means at most once within a specific 6-8 day period (eg, once every 7 days).

  The term “once every two weeks” means at most once within a specific 12-16 day period (eg, once every 14 days). The term “once a month” means once a month (eg once every 28-31 days). The subject is typically a mammal (e.g., a human, non-human primate (e.g., monkey or ape), dog, cat, rabbit, or animal mammal (e.g., For example, the subject is a human (eg, a human male or female) The subject is at least about 18, 25, 30, 45, 50 years old. 55 years old, 60 years old, or 70 years old.

  As used herein, the term “minimum average concentration” refers to the average minimum concentration of a drug present in the circulation or serum of a subject.

  Various aspects of the invention are described in further detail in the following subsections.

(I. LTBR-Ig fusion protein and composition thereof)
The present invention relates to a composition comprising an LBTR-Ig fusion protein that is improved for therapeutic use in that the population of LTBR-Ig fusion proteins has reduced molecular heterogeneity. The present invention provides compositions (including pharmaceutical compositions) comprising LTBR-Ig fusion proteins, as well as proteins, nucleic acids, vectors, host cells, and methods for making them.

  Lymphotoxin-β receptor-immunoglobulin (LTβR-Ig) fusion protein can block signal transduction between the surface LT ligand and receptor, with consequences for the functional state of follicular dendritic cells (Mackay and Browning) 1998). This block may further result in reduced autoimmune disease in rodent models (Mackay et al., 1998, US patent application Ser. No. 08 / 505,606 (filed Jul. 21, 1995)) and US patent application Ser. No. 029,060 (filed Oct. 26, 1996)).

  In general, the preferred soluble LTBR of the present invention is a fusion protein. Soluble LTBR, as defined herein, is a molecule that contains an LT binding fragment of the extracellular domain of LTBR. For example, a soluble LTBR can comprise all or a substantial portion of the extracellular domain of LTBR (eg, it is residues 40-200, 35-200, 40-210 of SEQ ID NO: 1; 35-220, 32- 225, or 28-225). In one embodiment, the soluble LTBR comprises residues 32-225 of SEQ ID NO: 1. In some embodiments, the soluble LTBR can be modified by covalent attachment of a portion (eg, a heterologous polypeptide (eg, to make an LTBR fusion protein) or a non-polypeptide portion. Such moieties may improve pharmacokinetic and pharmacodynamic parameters (eg, solubility or half-life) LTBR fusion proteins may be antibody constant regions (eg, Fc domains), transferrins, or albumins (eg, , Human serum albumin (HSA) or bovine serum albumin (BSA)) The fusion protein may comprise a linker region between the LTBR sequence and the non-LTBR protein domain. In some embodiments, soluble TBR is modified by covalent attachment to a polymer, such as polyethylene glycol (PEG), although not wishing to be bound by theory or mechanism, such soluble LTBR reduces LTBR activity ( An exemplary LTBR-Fc has an amino acid sequence of SEQ ID NO: 5, 6, 8, 9, or 10.

  The soluble LTBR can include all or a fragment of LTBR fused to one or more heterologous protein domains (eg, a soluble fragment of LTBR) (which domain can increase blood solubility or longevity). Exemplary LTBR moieties are the LTBR sequence of SEQ ID NO: 1, or a sequence that differs from the above sequence by only 1, 2, 3, 5, or 10 amino acid residues. The difference can be any difference (eg, substitution, deletion or insertion), but is preferably a substitution (eg, conservative substitution). A conservative substitution is usually the exchange of one amino acid for another of similar polarity, configuration, or another of the same class (eg, hydrophobic, acidic or basic). Examples of non-LTBR proteins or domains include all or part of an antibody constant region (eg, Fc domain), transferrin, or albumin (eg, human serum albumin (HSA) or bovine serum albumin (BSA)).

  In preferred embodiments, the polypeptides of the present invention are polypeptides (eg, antibodies), preferably IgG immunoglobulins (eg, subtype IgG1, IgG2, IgG3, or IgG4), and preferably subtypes IgG1 or IgG4. An Fc fusion protein comprising In a preferred embodiment, the polypeptide binds to a ligand for LTBR. Amino acid numbering herein for a portion of the Fc region of a polypeptide is described, for example, in Kabat et al., “Sequences of Proteins of Immunological Interest”, U. S. Dept. Corresponds to the Kabat numbering system described by Health and Human Services, 1983 and 1987. In some embodiments, a series of amino acid numbering (eg, for the sequences shown in the sequence listing) is provided.

  In one embodiment, the fusion protein of the invention comprises at least a portion of a hinge region (CH1, CH2, and CH3 regions of an immunoglobulin).

  Heterogeneity within the protein population can arise 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 present invention relates to reduced heterogeneity (reduced N-terminal heterogeneity (eg, with respect to variations in molecular size or molecular morphology (eg, pyroglutamic acid-containing protein)) or reduced C-terminal heterogeneity. Compositions comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion proteins having homogeneity, as well as combinations thereof, are included. The reduction in heterogeneity can be attributed to deletions and / or mutations made within the sequence of the LTBR-Ig protein. As a result, heterogeneity is reduced with respect to the wild type LTBR-Ig fusion protein (ie, LTBR-Ig that has not been mutated and / or deleted). An example of a wild type LTBR-Ig fusion protein is set forth in SEQ ID NO: 11 (mature form of the protein), also referred to as LTBR01. It should be noted that the terms LTβR-Ig and LTBR-Fc are used interchangeably herein.

  In preferred embodiments, the LTβR-Ig fusion protein comprises a modified LTBR extracellular domain and / or a modified Ig portion (eg, the Fc portion of Ig). In one embodiment of the invention, the LTBR-Ig fusion protein comprises either an LTBR extracellular domain variant, a modified Ig moiety, or a combination thereof.

  The amino acid sequence or nucleic acid sequence of wild type LTBR is described as AAH26262 and P36941 in the NCBI database. The wild type human amino acid sequence of LTBR is also set forth as SEQ ID NO: 1. The soluble LTBR can be an LTBR-Fc polypeptide having the sequence of SEQ ID NO: 1, preferably a variant thereof. In a preferred embodiment, the soluble LTBR is an LTBR-Fc polypeptide that differs from the sequence of SEQ ID NO: 1 by no more than 1, 2, 3, 5, or 10 amino acid residues.

(Human LTBR sequence (GenPept ID No. P36941))
SEQ ID NO: 1 is an immature or unprocessed human LTBR sequence, ie, includes a signal sequence. Italic amino acids indicate signal sequences. Amino acids 28-225 are the extracellular region of LTBR.

用語「野生型ＬＴＢＲ−Ｉｇ」とは、本明細書において使用される場合、ヒト野生型ＬＴＢＲの細胞外ドメイン（例えば、配列番号１に示されるＬＴＢＲ配列の細胞外ドメイン）、および当該分野で公知の、例えば、変異、欠失などによって改変されていない任意の免疫グロブリン配列を含む融合タンパク質をいう。例示的な野生型Ｉｇアミノ酸配列は、配列番号２２に提供される。改変を有さない野生型ＬＴＢＲ−Ｉｇ融合タンパク質の例は、配列番号６に記載される。

The term “wild type LTBR-Ig”, as used herein, is the extracellular domain of human wild type LTBR (eg, the extracellular domain of the LTBR sequence shown in SEQ ID NO: 1), and known in the art A fusion protein comprising any immunoglobulin sequence that has not been modified, eg, by mutation, deletion, or the like. 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 without modification is set forth in SEQ ID NO: 6.

  In one aspect, the present invention relates to an LTBR-Ig fusion protein comprising a modified LTBR extracellular domain. For example, the amino acid “SQPQ” can be deleted from the amino terminus of the wild type LTBR protein (mature form) as shown in SEQ ID NO: 4 (the amino acid sequence of the LTBR extracellular domain of LTBR06). As demonstrated in the examples provided below, the deletion of “SQPQ” from the amino terminus of LTBR results in overall heterogeneity (including N-terminal heterogeneity) within the LTBR-Ig protein population. To improve. As described in the Examples, LTBR06 expression is associated with maturation of the wild type LT-β-R extracellular domain, wherein at least 90% of the LT-β-R-Ig-fusion protein is shown in SEQ ID NO: 21. A population of LTBR-Ig fusion proteins is provided that is missing only 5 amino acids from the N-terminus of the form. Thus, expression of LTBR06 results in a population of LTBR-Ig proteins in which 90% of the protein is either N-4 or N-5. Furthermore, LTBR06 has reduced pyroglutamic acid formation.

  In another embodiment, the LTBR-Ig of the invention can comprise a modified Ig portion having a deletion at the c-terminal amino acid (ie, “K”) of the Fc portion. As described in the examples below, deletion of the last amino acid in the Fc portion of the LTBR-Ig fusion protein reduces C-terminal heterogeneity. An example of a modified Ig moiety in which the last amino acid is deleted to improve (ie, reduce) C-terminal heterogeneity is set forth in SEQ ID NO: 2. Examples of LTBR-Ig fusion proteins having a modified Ig portion with the last amino acid (ie, the last lysine) deleted are set forth in SEQ ID NOs: 5, 8, 9, and 12. The modified Ig moiety can include an Fc region of an IgG isotype (including but not limited to an IgG1 isotype).

  The invention also includes an LTBR-Ig fusion protein having both a modified LTBR extracellular domain and a modified Ig moiety. LTBR06 is an LTBR-Ig fusion protein comprising a modified LTBR extracellular domain with the first 4 amino acids removed and having a modified Ig moiety. Here the last amino acid (K) has been deleted. The N- and C-termini of LTBR06 reduce both N- and C-terminal heterogeneity. SEQ ID NO: 8 below describes the amino acid sequence of LTBR06, including the signal sequence. Italic amino acids in the following sequences indicate signal sequences; underlined amino acids indicate sequences obtained from the extracellular region of LTBR; amino acids in bold indicate IgG Fc sequences.

  In one embodiment, the immunoglobulin portion of the Ig fusion protein of the invention comprises at least a portion of an immunoglobulin hinge region. In one embodiment, the modified Fc portion comprises a mutation to valine in at least one hinge region of the Ig upper hinge (eg, a cysteine mutation (amino acid position 220 (also shown as amino acid position 1 of SEQ ID NO: 22)). The subject valine is in bold / underlined / italic below: the underlined sequence is a substantial portion of the extracellular domain of LTBR, and SEQ ID NO: 1 (above ) During proteolytic processing, the signal sequence of the LTBR protein is cleaved, so the final LTBR protein product of LTBR06 is set forth in SEQ ID NO: 5.

配列番号８に記載されるバリン変異に加えて、他の適切なアミノ酸は、使用され得る。例えば、セリン、スレオニン、アラニン、ロイシン、グリシンもしくはイソロイシンを含むアミノ酸は、ＬＴＢＲ０６のバリンの代わりに、および本明細書に記載される他のＬＴＢＲ−Ｉｇ融合タンパク質において使用され得る。

In addition to the valine mutation set forth in SEQ ID NO: 8, other suitable amino acids can be used. For example, amino acids including serine, threonine, alanine, leucine, glycine or isoleucine may be used in place of LTBR06 valine and in other LTBR-Ig fusion proteins described herein.

  In any embodiment, an immunoglobulin hinge can be used to link the LTBR extracellular domain to, for example, the CH1, CH2, and CH3 domains of an immunoglobulin molecule. For example, an IgG hinge region having the sequence CDKTHTCPPCPAPELLGGP can be used. In one embodiment, a hinge region having the sequence VDKTHTCPPCPAPELLGGP can be used. Other exemplary upper hinge and center hinge constructs are shown in FIGS. 6-8 and in SEQ ID NOs 13-20. Thus, the modified Ig portion of the LTBR-Ig fusion protein may comprise upper and middle hinge regions that comprise at least 90% to 95% identity to the hinges set forth in SEQ ID NOs: 13-20. The upper and middle hinge regions shown in SEQ ID NO: 13 were used in the modified LTBR-Ig fusion protein exemplified herein. Thus, an LTBR-Ig fusion protein comprising a modified hinge (eg, those described in SEQ ID NOs: 14-21) is constructed by replacing the sequence shown in SEQ ID NO: 13 with a modified hinge. Optionally, other modified hinge molecules that can be used to link the LTBR extracellular domain to, for example, the CH1, CH2, and CH3 domains of the immunoglobulin molecule are disclosed in 20050163382A1, the contents of which Incorporated herein by reference.

  When an LTBR-Ig fusion protein (eg, LTBR06) is expressed, the resulting protein population is at least partially due to N-terminal and / or C-terminal proteolysis and heterogeneity of pyroglu formation, Includes the entire variety of lengths. An important aspect of the present invention is to eliminate heterogeneity (N-terminal heterogeneity) by deleting the first 4 amino acids of the LTBR-Ig fusion protein (particularly the first 4 amino acids of the LTBR extracellular domain). Is the discovery that can be reduced. Accordingly, in one embodiment, the present invention provides a population of LTβR-Ig-fusion proteins comprising a modified LT-β-R extracellular domain that is 193 amino acids or 194 amino acids in length and a modified Ig portion that is 227 amino acids in length. Wherein at least 90% of the LT-β-R-Ig-fusion protein is the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21. Only 5 amino acids have been lost from and the LT-β-R-Ig-fusion protein lacks pyroglutamic acid. The invention also features a composition comprising a population of LT-β-R-Ig fusion proteins comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig portion, wherein The population has reduced N-terminal pyroglutamic acid formation and reduced C-terminal heterogeneity compared to the wild type LT-β-R-Ig fusion protein.

  The amino terminal amino acid of the modified LTBR-Ig fusion protein may be alanine as shown in SEQ ID NOs: 5 and 12, or alternatively, a nonpolar amino acid. Examples of non-polar amino acids that can be used as the first amino acid of the LTBR-Ig fusion protein include valine (mature form amino acid 6 of the wild type LT-β-R moiety shown in SEQ ID NO: 1) or alanine (SEQ ID NO: Amino acid 5) in the mature form of the wild-type LT-β-R moiety shown in FIG. In addition to non-polar amino acids, serine or threonine can be used. In one embodiment, the composition of the present invention is such that at least 95% of the LT-β-R-Ig-fusion protein is N-terminal valine (amino acid 6 in the mature form of wild type LT-β-R) or Includes a population of LTBR-Ig fusion proteins that can be any of alanine (amino acid 5 in the mature form of wild type LT-β-R).

  In addition to N-terminal and / or C-terminal heterogeneity, heterogeneity can also arise from various glycosylation. To minimize heterogeneity resulting from glycosylation variations within the protein population of the LTBR-Ig fusion protein, the LTBR and / or the Ig can be altered to reduce the amount of glycosylation. In one embodiment, the Ig portion of the LTBR-Ig fusion protein is not glycosylated.

  The present invention also contemplates a composition comprising a population of LTβR-Ig fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, wherein the modified LT-β-R extracellular domain is glycosylated. It has not been. An example of the non-glycosylated extracellular domain of LTBR is provided in the Examples and is also set forth in SEQ ID NO: 10 (see amino acids 1-194 of SEQ ID NO: 10).

  Thus, examples of post-translational modification heterogeneity that can be reduced using the methods and compositions described herein include N-terminal heterogeneity, C-terminal heterogeneity, and glycosylation heterogeneity. Sex. It is within the scope of the present invention to provide LTBR-Ig fusion proteins with reduced heterogeneity in each of these parameters, and combinations thereof. For example, the invention encompasses a composition comprising a population of LT-β-R-Ig fusion proteins, wherein the population is reduced compared to a population of wild-type LT-β-R-Ig fusion proteins. N-terminal heterogeneity and reduced C-terminal heterogeneity.

  Methods for treating autoimmune disorders using the above compositions and LTBR-Ig fusion proteins and pharmaceutical compositions containing them are described in more detail in Sections II and III below. .

  The LTBR-Ig fusion polypeptide of the present invention retains the ligand binding activity of LTBR and is against the LTBR-Ig polypeptide shown in SEQ ID NOs: 5, 6, 8, 9, 10, 11, 12, and 23. A polypeptide having an amino acid sequence with at least 70% homology and each of the variants and derivatives described above. The invention also provides isolated polypeptides set forth in SEQ ID NOs: 3, 4, and 7, which provide either the LTBR extracellular domain of the invention or the Ig variant of the invention. . Preferably, the LTBR-Ig polypeptide (or part thereof) has a homology of greater than 85%, more preferably greater than 90%, more preferably greater than 95% to the aforementioned sequence. Homology, most preferably having an amino acid sequence of greater than 99% homology.

  In one embodiment, the invention features an isolated polypeptide comprising an amino acid sequence that is at least 90% relative to the full length of the mature form of SEQ ID NO: 8 or SEQ ID NO: 23, wherein said ligand binding domain The amino acid sequence has not changed and the polypeptide further comprises a truncated Fc region. In one embodiment, the polypeptide of the invention comprises an amino acid sequence that is at least 95% identical to the polypeptide shown in SEQ ID NO: 8 relative to the full length of the mature form. In one embodiment, the polypeptide of the invention comprises the 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.

  Variants of the polypeptides described herein are also contemplated by the present invention. Accordingly, an amino acid sequence different from the sequences shown 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 at one or more amino acid positions Variants of the polypeptide having and the portions set forth in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 7 are included in the present invention. Such modified polypeptides include the above-described modified polypeptides, as well as conservative substitutions, splice variants, isoforms, homologs from other species, and polymorphisms.

  Alteration of the LTBR-Ig primary amino acid sequence can result in a protein with substantially equivalent activity compared to the unmodified corresponding counterpart polypeptide, and is therefore functionally similar to the parent protein. Can be considered. Such modifications may be deliberate, such as by site-directed mutagenesis, may occur spontaneously, and splice variants, isoforms, homologs from other species, and polymorphisms including. Such functional analogs are also contemplated in accordance with the present invention.

  Furthermore, alterations in the primary amino acid sequence can result in proteins that do not retain the biological activity of the parent protein, including dominant negative forms and the like. Dominant negative proteins can interfere with wild-type proteins by binding to or otherwise sequestering modulators (eg, upstream or downstream components) that functionally and normally interact with the polypeptide. . Such dominant negative forms are also contemplated in accordance with the present invention.

  The LTBR-Ig proteins of the invention can be made according to standard methods known in the art. A nucleic acid molecule encoding LTBR-Ig can be expressed by expressing an LTBR-Ig polypeptide in vivo or by administering an LTBR-Ig-encoding nucleic acid molecule to an animal for in vivo expression. It can be used to express a polypeptide. A nucleic acid molecule encoding LTBR-Ig can be included in a nucleic acid vector (eg, an expression vector or a cloning vector). A nucleic acid molecule encoding LTBR-Ig can be maintained as part of a nucleic acid vector, can be regenerated, transferred or expressed, but is not required. Accordingly, another aspect of the invention is an isolated nucleic acid molecule that encodes an LTBR-Ig protein of the invention described herein. The present invention includes a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 7 (mature form of LTBR06).

  A recombinant expression vector comprising an LTBR-Ig polynucleotide sequence can be introduced and / or maintained in a cell. The cell that hosts the LTBR-Ig vector can be a prokaryotic cell. Alternatively, the LTBR-Ig nucleic acid may be a eukaryotic cell (eg, suitable for secreting the polypeptide into the mature protein, a device suitable for post-translational processing of the polypeptide and / or the extracellular environment of the cell. Eukaryotic cells containing the device). Vectors comprising a nucleic acid molecule encoding the LTBR-Ig fusion protein of the invention are included within the scope of the invention.

  Suitable methods for making the LTBR-Ig proteins of the invention are known in the art, for example, WO 97/03687, WO 98/17313, WO 00/21558, WO 99/38525, WO 00/36092. It is described in. For example, an LTBR immunoglobulin fusion protein can be produced at low temperatures, eg, in cell culture (eg, mammalian cell culture (eg, monkey cos cells or Chinese) to produce an increased amount of appropriately folded fusion protein. It can be expressed in hamster ovary cells) or yeast cell cultures). Host cells that express the LTBR-Ig fusion protein of the invention are included within the scope of the invention, wherein the host cell comprises a vector comprising a nucleic acid encoding the LTBR-Ig fusion protein. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell. The expressed fusion protein can be purified, for example, by affinity chromatography techniques or conventional chromatography techniques. See WO 00/36092. The expression of the LTBR-Ig fusion protein can range from a scale including manufacturing scale.

  The present invention further provides a process for making a composition comprising a population of LTβR-Ig-fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, wherein said LTβR-Ig-fusion At least 90% of the protein has lost only 5 amino acids from the N-terminus of the mature form of the wild-type LT-β-R moiety shown in SEQ ID NO: 1, and the process is performed in SEQ ID NO: 8 in mammalian cells. Expressing a nucleic acid molecule that encodes the LT-β-R-Ig fusion protein shown in FIG. 1, obtaining the population from the culture supernatant, and optionally purifying the supernatant thereby Obtaining a composition comprising a population of LTβR-Ig-fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, wherein L At least 90% of the-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-β-R portion shown in SEQ ID NO: 1. In one embodiment, the process includes expressing a nucleic acid molecule set forth in SEQ ID NO: 7 in a mammalian cell. In one embodiment, the process includes expressing the nucleic acid molecule shown in SEQ ID NO: 7 in a mammalian cell. The present invention is also characterized in that a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR shown in SEQ ID NO: 4 or a nucleic acid sequence shown in SEQ ID NO: 3 is expressed in a mammalian cell. The invention further features a nucleic acid molecule encoding the LTβ-R-Ig fusion protein shown in SEQ ID NO: 8 or a composition made by expressing a nucleic acid sequence shown in SEQ ID NO: 7 in mammalian cells. And

II. Use of the LTBR-IgG of the invention to treat autoimmune disorders
Soluble LTBR is a lymphotoxin (LT) pathway inhibitor useful for treating autoimmune disorders. Autoimmune disorders include, for example, autoimmune arthritis (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 mellitus, uveitis, systemic lupus erythematosus (SLE, or lupus), polychondritis, and graft rejection. The agents and methods described herein are particularly suitable for the treatment of RA.

  Rheumatoid arthritis is indicated by tenderness in the joint. Synovial thickening eventually occurs in the joints where the most changes are made. Stiffness over> 30 minutes when occurring in the morning or after long periods of inactivity is common, as is early afternoon fatigue and fatigue. Deformations, in particular flexion spasticity, can occur rapidly. Carpal tunnel syndrome can result from wrist synovitis. X-rays can reveal soft tissue swelling in the first month of the disease, followed by periarticular osteoporosis, joint space narrowing, and marginal erosion Can do.

  Various tests can be used to assay the efficacy of soluble LTBR for treating autoimmune disorders. In the case of rheumatoid arthritis (RA), for example, inadequate efficacy can be attributed to clinical parameters of RA (eg, touching and painful joints (TJC), swollen joints (SJC), comprehensive assessment of disease active patients [ PGA visual analog scale (VAS) 0-10 cm for pain scale], comprehensive assessment of disease activity physicians (MDGA VAS 0-10 cm), C-reactive protein levels (CRP (mg / dl)) And at least 10%, 20%, 25%, 30%, 40%, 50% or more in red blood cell sedimentation rate (ESR)). C-reactive protein can be produced by the liver during episodes of acute inflammation or infection. CRP levels in blood serum are an indication of the severity of RA. Reduction in CRP levels (eg, 5%, 10%, 15%, 20% or more reduction) is effective for autoimmune disorders (eg, RA) in response to treatment with soluble LTBR (eg, LTBR-Fc) Treatment may be indicated. Erythrocyte sedimentation rate (ESR) is also increased in 90% of RA cases. Thus, a decrease in ESR after treatment with soluble LTBR may also indicate an effective treatment.

  The high potential of soluble LTBR demonstrated herein indicates that a low dosage regimen is equally effective in treating other autoimmune conditions. Autoimmune conditions suitable for treatment as described herein with soluble LTBR (eg, LTBR-Fc) include, for example, autoimmune joint inflammation (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 graft rejection It is done.

(Exemplary dosage regimen)
The present invention is based in part on the discovery that a low dosage or frequency regimen of a soluble form of LTBR (eg, LTBR-Fc) can effectively treat symptoms of autoimmune disorders, particularly rheumatoid arthritis (RA). . Accordingly, in one aspect, the present invention provides a method for treating an autoimmune disease in a patient. The method includes administering to the subject an LTBR blocking agent (eg, a soluble LTBR fusion protein, eg, LTBR-Fc) at low volume and / or frequency.

  As shown in FIG. 14, a comparison of LTBR-Fc with a representative antibody shows significant potency differences over the long time frame at the same administration. This, in addition to antibody-like pharmacokinetics, results in rLTBR-Fc having a high affinity for the target ligand and thus effective at very low doses. Arrows A and B show representative α and β phases for the Fc fusion protein antibody, respectively. For the antibody, the gray line indicated by arrow C shows a typical lower concentration for efficacy, whereas arrow D shows the efficacy of LTBR-Fc at significantly lower concentrations.

  In one embodiment, the soluble LTBR fusion protein is administered to achieve an average concentration between about 0.14 μg / ml and about 3.5 μg / ml. In one embodiment, the soluble LTBR fusion protein is administered to achieve a minimum average concentration of about 0.3 to about 1.0 μg / ml, such as about 0.6 to 0.7 μg / ml. In another embodiment, higher doses of the fusion protein can be administered. For example, the fusion protein can be administered to achieve an average concentration of about 5-15 μg / ml.

  In another embodiment, the soluble LTBR fusion protein is administered at a dose between about 70 and about 200 mg / month to an average subject weighing about 75 kg. In another embodiment, the soluble LTBR fusion protein is administered to an average subject weighing about 75 kg at a dose of about 100 mg / month, about 150 mg / month, or about 175 mg / month.

  Embodiments of the invention include autoimmune joint inflammation (including rheumatoid arthritis and Sjogren's syndrome), psoriasis, multiple sclerosis, inflammatory bowel disease (IBD) (including ulcerative colitis and Crohn's disease), insulin dependence Soluble, such as LTBR blockers (eg, LTBR fusion proteins (eg, LTBR-Ig)) for the treatment of diabetes, uveitis, systemic lupus erythematosus (SLE, or lupus), polychondritis, and graft rejection Administration of a regimen of LTBR).

  In one embodiment, the subject is treated with a soluble LTBR (eg, an LTBR immunoglobulin fusion). Exemplary LTBR-Ig fusion proteins are described herein.

  In one embodiment, the subject has one or more symptoms of RA (eg, joint swelling, joint pain, joint stiffness, or difficulty moving). For example, the subject has active RA disease (eg, number of joints that are painful to touch (TJC) or number of swollen joints (SJC) ≧ 5 or synovitis). In another embodiment, the subject is at risk of developing RA (eg, family history, or autoimmune disease).

  In one embodiment, the soluble LTBR can be administered in an amount and / or over time sufficient to alleviate symptoms associated with RA.

  In another embodiment, the soluble LTBR (eg, LTBR-Fc) is administered in an amount sufficient to ameliorate symptoms in one or more RA assessment criteria (eg, criteria described herein). The For example, the soluble LTBR is administered in an amount sufficient to improve the symptom score by at least about 10%, 20%, 25%, 30%, 40%, 50% or more. Symptom scores include, for example, TJC, SJC, comprehensive evaluation of patients with disease activity [PGA visual analog scale (VAS) 0-10 cm], comprehensive evaluation of doctor's disease activity (MDGA VAS 0-10 cm) , C-reactive protein amount (CRP (mg / dl)), or disease activity score, 28 joint version (DAS28). Symptom scores can be reported according to the criteria set forth by the American College of Rheumatology (ACR). ACR score is an indicator of% clinical improvement in RA symptoms. For example, ACR20 is an indicator of 20% clinical improvement in TJC and SJC, and 20% improvement in three of the following five parameters: (i) Comprehensive assessment of patients, (ii) Doctor (Iii) patient pain assessment, (iv) degree of disability, and (v) level of acute phase reactants. ACR scores of ACR50 and ACR70 can also be used to indicate 50% improvement or 70% improvement, respectively.

  In one embodiment, the soluble LTBR (eg, LTBR-Fc) is administered more than once a week, for example, two weeks, or one month. The course of treatment is maintained over a period of time (eg, 1 week or 2 weeks or longer); over 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months; 1 year; or longer. In certain embodiments, the soluble LTBR is administered at a dosage of about 0.03 to 3 mg / kg body weight per administration (eg, about 0.6 to about 1.4 mg / kg per administration). Is done. In another embodiment, the soluble LTBR is a dose of about 0.3-3 (eg, 1 mg / kg body weight per dose (eg, about 0.6 to about 1.4 mg / kg per dose)). In other embodiments, the soluble LTBR is administered at a dose of about 2.5 to 3.5 mg / kg body weight per dose (eg, about 2.8 to 3 mg / kg per dose). In yet other embodiments, the soluble LTBR is about 0.01 to 3 mg / kg body weight per dose (eg, about 0.01 to about 0.05 mg / kg, Administration of 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) Administered in an amount. In some embodiments, the soluble LTBR is about 0.03 mg / kg per dose, about 0.05 mg / kg per dose, about 0.07 mg / kg per dose, and about 0.07 mg / kg per dose. Administered at 0.1 mg / kg, or about 0.2 mg / kg per dose, In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  Table 1: Exemplary doses of LTBR-Fc (mg / kg subject body weight per dose)

一般に、本明細書に開示される低い方の単位用量は、より高い頻度の投与（例えば、１週間に１回）で投与され得る一方で、本明細書に開示される高い方（相対的標準ではなお低いが）の単位用量は、より低い頻度（例えば、１ヶ月に１回）の投与で投与され得る。いくつかの実施形態において、上記可溶性ＬＴＢＲ（例えば、ＬＴＢＲ−Ｆｃ）は、約０．３ｍｇ／ｋｇ／投与〜約３ｍｇ／ｋｇ／投与（例えば、約０．６ｍｇ／ｋｇ／日〜約１．４ｍｇ／ｋｇ／日）の投与量において投与される。例えば、上記用量は、約１ｍｇ／ｋｇ、もしくは約３ｍｇ／ｋｇである。いくつかの実施形態において、上記用量は、１４〜２０日ごとに１回、１ヶ月に１回もしくは２回（例えば、２８〜３１日ごとに１回もしくは２回）において投与される。例えば、上記用量は、１４〜２０日ごとに、例えば、１２〜１６日ごとに、もしくは約２週間ごとに、約１ｍｇ／ｋｇもしくは約３ｍｇ／ｋｇ投与される。あるいは、上記用量は、１ヶ月に１回、例えば、約２８〜３１日ごとに、約１ｍｇ／ｋｇもしくは約３ｍｇ／ｋｇ投与される。

In general, the lower unit dose disclosed herein can be administered at a higher frequency of administration (eg, once a week) while the higher (relative standard) disclosed herein. Unit doses may still be administered with less frequent administration (eg, once a month). In some embodiments, the soluble LTBR (eg, LTBR-Fc) is about 0.3 mg / kg / dose to about 3 mg / kg / dose (eg, about 0.6 mg / kg / day to about 1.4 mg). / Kg / 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 a month (eg, once or twice every 28-31 days). For example, the dose is administered about 1 mg / kg or about 3 mg / kg every 14-20 days, such as every 12-16 days, or about every 2 weeks. Alternatively, the dose is administered about 1 mg / kg or about 3 mg / kg once a month, for example, about every 28-31 days.

  In other embodiments, the soluble LTBR (eg, LTBR-Fc) is at most twice every 20-40 days, eg, at most twice every 25-35 days, or every 28-31 days. At a dose of about 0.6 mg / kg / dose to about 1.4 mg / kg / dose. In other embodiments, the soluble LTBR (eg, LTBR-Fc) is at most twice every 20-40 days (eg, at most twice every 25-35 days, or every 28-31 days). Twice) at a dose of about 2.5 mg / kg / dose to about 3.5 mg / kg / dose.

  In still other embodiments, the soluble LTBR (eg, LTBR-Fc) is about 0.01 mg / kg / dose, eg, once a week, or repeated at least twice every 3-10 days. To about 0.3 mg / kg / dose (eg, about 0.01 mg / kg / day to about 0.25 mg / kg / day). For example, the soluble LTBR is about 0.02 mg / kg. , 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.1 mg / kg, or 0.2 mg / kg or administered once a week Or every 3 to 10 days (eg every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, or every 9 days). In form, soluble above TBR is administered at a dosage shown in Table 1.

  In another embodiment, the soluble LTBR is about 0.01 mg / kg / day to about 0.3 mg / kg / day, for example, once every two weeks, or repeated at least twice every 5-20 days. It is administered at a daily dose (eg, 0.02 mg / kg / day to about 0.25 mg / kg / day). For example, the soluble LTBR is administered at a dose of about 0.03 mg / kg, 0.05 mg / kg, 0.08 mg / kg, 0.1 mg / kg, or 0.2 mg / kg once every two weeks. Or every 5-20 days (eg, every 6 days, every 8 days, every 10 days, every 12 days, every 14 days, every 16 days, or every 18 days) . In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  In another embodiment, the soluble LTBR is about 0.3 mg / kg / day to about 3 mg / kg / day, eg, once every two weeks or repeated at least twice every 5-20 days ( For example, it is administered at a dose of 1 mg / kg / day. In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  In another embodiment, the soluble LTBR is about 0.3 mg / kg / day to about 3 mg / kg / day (eg, once a month, or repeated at least twice every 15-45 days) , 0.4 mg / kg / day to about 3 mg / kg / day). For example, the soluble LTBR is administered at a dosage of about 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg, once a month, or 15- It is administered every 45 days (eg every 16 days, every 18 days, every 20 days, every 25 days, every 30 days, every 35 days or every 40 days). In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  In certain embodiments, the dose of soluble LTBR (eg, 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.

  In certain embodiments, the present invention provides for the administration of particularly low doses of soluble LTBR (eg, LTBR-Fc) for the treatment of autoimmune disorders (eg, RA).

  In certain embodiments, the soluble LTBR is administered intravenously or parenterally, eg, administered subcutaneously or intramuscularly.

  In another embodiment, the soluble LTBR is administered as a monotherapy.

  In one aspect, the present invention provides a method for treating an autoimmune condition (eg, RA) in a subject (eg, human). The method includes administering to the human a dose of soluble LTBR (eg, LTBR-Fc, wherein the dose is about 0.01 mg to about 3 mg LTBR-Fc / kg body weight human (mg / kg ), For example, between about 0.01 mg / kg to about 0.05 mg / kg, In one embodiment, the human has a soluble LTBR (eg, LTBR-Fc) over a course of at least 2 weeks. Administered in a once weekly dose, wherein the dose is about 0.01 mg / kg / day to about 0.3 mg / kg / day once a week (eg, every 3 to 10 days) (Eg, about 0.01 mg / kg / day to about 0.25 mg / kg / day) For example, the soluble LTBR can be administered, for example, every 4 days, every 5 days, every 6 days, 7 8 days every day Or every 9 days, about 0.02 mg / kg / day, 0.03 mg / kg / day, 0.04 mg / kg / day, 0.05 mg / kg / day, 0.06 mg / kg / day, 0 Administered at a dose of 1 mg / kg / day, or 0.2 mg / kg / day In some embodiments, the soluble LTBR is administered at the dose shown in Table 1.

  In another embodiment, the human is administered a dose of soluble LTBR once every two weeks (eg, LTBR-Fc) over a course of at least 4 weeks, wherein the dose is every 5-20 days. Repetitively, from about 0.01 mg / kg / day to about 0.5 mg / kg / day (eg, from about 0.01 mg / kg / day to about 0.3 mg / kg / day). For example, the soluble LTBR can be about 0.03 mg / day, for example, every 6 days, every 8 days, every 10 days, every 12 days, every 14 days, every 16 days, or every 18 days. It is administered at a dosage of kg, 0.05 mg / kg, 0.08 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg or 0.4 mg / kg. In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  In another embodiment, the human is administered a dose of soluble LTBR once a month (eg, LTBR-Fc) over a course of at least 2 months, wherein the dose is about 0.1 mg to about 3 mg soluble LTBR / kg human body weight / day. In one embodiment, the dose is about 0.3 mg / kg / day to about 3 mg / kg administered once a month (eg, every 15 to 45 days). For example, the soluble LTBR can be about 0.5 mg / day, for example, every 16 days, every 18 days, every 20 days, every 25 days, every 30 days, every 35 days, or every 40 days. It is administered at a dose of kg / day, 0.8 mg / kg / day, 1 mg / kg / day, 1.5 mg / kg / day, or 2 mg / kg / day. In some embodiments, the soluble LTBR is administered at the dosage shown in Table 1.

  In some embodiments, patients who respond poorly to treatment did not show clinically acceptable or significant improvement in response to the treatment. In other embodiments, the patient initially showed improvement in response to treatment, but no longer showed improvement as assessed by standard clinical measures for the particular disorder. DMARD-IR subjects may have disease modifying antirheumatic drugs (DMARD) (eg, methotrexate, leflunomide (Arava®), anakinra (R), hydroxychloroquinone (hydroxin) Sulfate (Plaquenil®), antimalarial agent, gold salt, sulfasalazine (Azulfidine®), minocycline (Minocin®), d-penicillamine, cyclosporin A, cyclosporin (Neoral) Registered trademark)), cyclophosphamide and azathioprine (Imuran®)) Was is the subject.

  In one aspect, the invention features a delivery device (eg, a transdermal delivery device (eg, a syringe)) designed for subcutaneous or intramuscular administration, wherein the device comprises a soluble LTBR (eg, , LTBR-Fc) is packaged with or includes at least one unit dose so that an appropriately low amount of the agent is administered to a human. In one embodiment, the device comprises or is packaged with a unit dose of LTBR-Fc so that administration to a human is between about 0.01 mg / kg and 3 mg / kg of LTBR to the human. Deliver Fc (eg, about 0.01 to about 0.05 mg / kg LTBR-Fc, or between about 0.6 mg / kg to 1.4 mg / kg LTBR-Fc. In another embodiment. The device includes or is packaged with a unit dose of LTBR-Fc, so that administration to a human can result in the human being receiving between about 2.5 mg / kg and 3.5 mg / kg of LTBR-Fc. In certain embodiments, the delivery device delivers about 1 mg / kg or about 3 mg / kg of LTBR-Fc to the human. Suitable Exemplary unit doses in humans are provided in Table 2.

In one embodiment, the liquid in the second compartment is water or a buffer. The liquid can contain, for example, a pharmaceutically acceptable carrier (eg, solvent, dispersion medium, antibacterial or antifungal agent, or isotonic or absorption delaying agent). The liquid may also contain a pharmaceutically acceptable salt.
Table 2. Exemplary dose of LTBR-Fc by human body weight (mg)

特定の実施形態において、送達デバイス中に含まれる上記可溶性ＬＴＢＲ（例えば、ＬＴＢＲ−Ｆｃ）の単位用量は、約０．４ｍｇ〜約３７５ｍｇ、約０．４ｍｇ〜約６．２５ｍｇ、約２ｍｇ〜約６．２５ｍｇ、もしくは約４ｍｇ〜約１２．５ｍｇである。一実施形態において、上記デバイスは、凍結乾燥ＬＴＢＲ−Ｆｃを含む。

In certain embodiments, the unit dose of the soluble LTBR (eg, LTBR-Fc) included in the 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 comprises lyophilized LTBR-Fc.

  In one aspect, the invention features a kit that includes between about 0.4 mg to about 375 mg of two or more unit doses of LTBR-Fc. When the unit dose is determined by the human body weight, an appropriately low amount will be administered to the human. Exemplary unit doses suitable for humans of various weights are provided in Table 2.

  In another aspect, the invention provides a device, eg, a transdermal delivery device (eg, having at least two compartments, the first compartment comprising a unit dose of lyophilized LTBR-Fc, and a second The compartment features a syringe containing a liquid to reconstitute the LTBR-Fc prior to administration to a subject. For example, the unit dose is such that administration of the LTBR-Fc delivers LTBR-Fc to a human between about 0.01-3 mg / kg (eg, 1.0 mg / kg). For example, in some embodiments, the unit dose of LTBR-Fc is between about 0.4 mg to about 375 mg of LTBR-Fc (eg, between about 0.4 mg to about 6.25 mg of LTBR-Fc). It is. The unit dose is such that an appropriately low amount is administered to the human as determined by the human body weight. Exemplary unit doses suitable for humans of various body weights are provided in Table 2.

  In another aspect, the invention provides (i) providing the patient with at least two unit doses of LTBR-Fc; and (ii) self-administering the unit dose one dose at a time, eg, subcutaneously. Thus, it features a method for directing a patient with rheumatoid arthritis to treat the RA by directing the patient. In certain embodiments, the delivered dose is between about 0.01 mg to 3 mg LTBR-Fc / kg patient body weight (mg / kg), for example, about 0.6 mg / kg to 1.4 mg / kg. kg, or between about 0.01 mg / kg and 0.05 mg / kg. In one embodiment, the delivered dose is between about 2.5-3.5 mg / kg LTBR-Fc (mg / kg) per kg of the patient's body weight. In certain embodiments, the patient administers the dose once a week for at least 2 weeks; once every 2 weeks for at least 4 weeks; or for 1 month for at least 2 months. Instructed to self-administer. In certain embodiments, the unit dose is about 0.01 to about 3.0 mg / kg LTBR-Fc / administration. In certain embodiments, the unit dose is about 0.01 to about 2.5 mg / kg / dose, about 0.02 to about 0.5 mg / kg / dose, about 0.01 to about 0.3 mg / kg. / Dose, about 0.01 to about 0.2 mg / kg / dose, about 0.01 to about 0.1 mg / kg / dose, or about 0.01 to about 0.05 mg / kg / dose. 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 / dose. is there.

  In one embodiment, the patient is instructed to self-administer the dose once a week for at least 2 weeks, wherein the unit dose is about 0.01 mg / kg per administration. To about 3.0 mg / kg LTBR-Fc (eg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg LTBR- per dose) Fc). For example, the unit dose is about 0.02 mg / kg / dose, 0.03 mg / kg / dose, 0.04 mg / kg / dose, 0.05 mg / kg / dose, 0.06 mg / kg / dose, 1 mg / kg / dose, or 0.2 mg / kg / dose.

  In one embodiment, the patient is instructed to self-administer the dose once every two weeks over a course of at least 4 weeks, wherein the unit dose is about 0.01-1.5 mg / kg. Between (for example, about 0.01 to about 0.3 mg / kg, or about 0.02 mg / kg to about 0.3 mg / kg or about 0.5 to 1.25 mg / kg LTBR-Fc). For example, the unit dose is about 0.03 mg / kg / dose, 0.05 mg / kg / dose, 0.08 mg / kg / dose, 0.1 mg / kg / dose, 0.2 mg / kg / dose, 0.0. 3 mg / kg / dose, 0.4 mg / kg / dose, 0.5 mg / kg / dose, 0.75 mg / kg / dose, or 1.0 mg / kg / dose.

  In one embodiment, the patient is instructed to self-administer the dose once a month, over a course of at least 2 months, wherein the unit dose is about 0.03 to 1 kg body weight of the patient. Between about 3 mg LTBR-Fc (mg / kg). For example, the unit dose is about 0.2 mg / kg, 0.5 mg / kg, 0.8 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg.

  In another aspect, the invention provides (i) instructing the patient to cease therapy to treat RA, and (ii) administering a unit dose of LTBR-Fc to the patient; A method for managing RA in a patient comprising In one embodiment, the unit dose is between about 0.03 and about 3 mg / kg LTBR-Fc, such as 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 and about 0.05 mg / kg LTBR-Fc.

  In another embodiment, the unit dose is between about 0.3 mg / kg and 3 mg / kg LTBR-Fc, and the unit dose is every 20-40 days (eg, every 25-35 days, Or every 28-31 days) at most twice. In another embodiment, the unit dose is between about 0.6 mg / kg to about 1.4 mg / kg LTBR-Fc, every 20-40 days (eg every 25-25 days, or 28 (Every 31 days) given at most twice.

  In another embodiment, the unit dose is between about 0.01 mg / kg to 0.3 mg / kg LTBR-Fc, and the unit dose is every 3-10 days (eg, once a week). ), At most once. In another embodiment, the unit dose is administered from about 0.01 mg / kg to about 0.3 mg / kg LTBR-Fc at most once every 5-20 days (eg, once every two weeks). The In another embodiment, the unit dose is administered every 28-31 days (eg, once a month) at most once from about 0.3 mg / kg to about 3 mg / kg LTBR-Fc.

  In one embodiment, the unit dose is between about 0.01 mg / kg to about 0.25 mg / kg LTBR-Fc, and the unit dose is administered once a week over a course of at least 2 weeks. The For example, the unit dose is about 0.02 mg / kg / dose, 0.03 mg / kg / dose, 0.04 mg / kg / dose, 0.05 mg / kg / dose, 0.06 mg / kg / dose, 1 mg / kg / dose, or 0.2 mg / kg / dose.

  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 once every two weeks over a course of at least 4 weeks. For example, the unit dose is about 0.03 mg / kg / dose, 0.05 mg / kg / dose, 0.08 mg / kg / dose, 0.1 mg / kg / dose, 0.2 mg / kg / dose, 0.0. 3 mg / kg / dose, or 0.4 mg / kg / dose.

  In another embodiment, the unit dose is about 0.03 to about 3 mg LTBR-Fc, and the unit dose is administered once a month over the course of at least 2 months. For example, the unit dose is about 0.2 mg / kg / dose, 0.5 mg / kg / dose, 0.8 mg / kg / dose, 1 mg / kg / dose, 1.5 mg / kg / dose, or 2 mg / kg. / Administration.

  In one embodiment, the patient is instructed to cease NSAID, corticosteroid, or DMARD treatment.

  In one aspect, the invention features a method for managing RA in a patient comprising administering to the patient a unit dose of soluble LTBR (eg, LTBR-Fc), wherein (i) the unit The dose is between about 0.01 mg to about 0.05 mg of the soluble LTBR per kg patient body weight (mg / kg); (ii) the unit dose is at most once every 3-10 days ( For example, once a week) is administered about 0.01 mg / kg to 0.3 mg / kg LTBR-Fc; (iii) the unit dose is every 5-20 days (eg, once every two weeks) ) Administered at most about 0.01 mg / kg to 1 mg / kg LTBR-Fc; (iv) the unit dose is every 20-40 days (eg every 25-35 days or 28- Many (every 31 days) About 0.3 mg / kg to 3 mg / kg LTBR-Fc (for example, about 0.6 to 1.4 mg / kg LTBR-Fc is administered), or (v) the unit dose is Every 20-40 days (eg, every 25-35 days or every 28-31 days), at most once, about 2.5 mg / kg to 3.5 mg / kg LTBR-Fc is administered. By this method, the patient receives a first therapy (eg, NSAID, corticosteroid, or DMARD therapy) to treat RA prior to the administration of the soluble LTBR, and the first therapy Levels are maintained in the patient when at least a first unit dose of the soluble LTBR is administered to the patient. In one embodiment, the level of the first treatment is not maintained in the patient after the first and second unit doses of the soluble LTBR. For example, the level of the first therapeutic agent is, for example, by administering a low dose of the first therapeutic agent, or by stopping administration and causing the first therapeutic agent level to disappear from the body. Reduced. In another embodiment, the patient continues to receive the first treatment during the administration of the soluble LTBR.

(Combination therapy)
The methods and compositions described herein can be used in combination with other treatments, such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and DMARDs.

  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 soluble LTBR for the treatment of autoimmune disorders.

DMARD typically delays the progression of rheumatoid arthritis. Suitable DMARDS include methotrexate, leflunomide (Arava®), anakinra (Kineret®), hydroxychloroquine sulfate (Plaquenil®), antimalarials, gold salts, sulfasalazine (Azulfidine) Registered trademark)), minocycline (Minocin®), d-penicillamine, cyclosporin A, cyclosporine (Neoral®), cyclophosphamide and azathioprine (Imuran®). DMARDs also include TNF inhibitors. Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine produced by macrophages and lymphocytes. TNF inhibitors help to reduce the pro-inflammatory effect of this molecule. The TNF inhibitors, etanercept (Enbrel ^(TM)), infliximab (Remicade ^(R)), and adalimumab (Humira ^(R)) can be mentioned.

  Corticosteroids typically function as anti-inflammatory agents and can be used in combination therapy with soluble LTBR.

  In certain embodiments, soluble LTBR is used as a second-line treatment. For example, patients who have been determined to be DMARD-IR cease receiving treatment with DMARD and begin treatment with soluble LTBR (eg, LTBR-Fc). In some embodiments, patients receiving NSAID and DMARD combination therapy stop receiving the DMARD and begin to receive soluble LTBR in combination with the NSAID. The soluble LTBR can be administered in combination with or separately from the NSAID. For example, a patient receiving a daily dose of NSAID may only receive a dose of soluble LTBR once a week, once every two weeks, or once a month. Alternatively, the patient continues to receive the DMARD and / or NSAID therapy while continuing to receive treatment with the soluble LTBR.

  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 having or at risk for RA. Exemplary second agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, or disease modifying anti-rheumatic drugs (DMARDs).

  In one embodiment, the subject has been treated with an RA drug after being diagnosed with RA and prior to administration of soluble LTBR. The RA drug can be, for example, an NSAID, a corticosteroid, or DMARD. In another embodiment, the subject is evaluated to determine if the response to the RA drug is insufficient prior to administration of the soluble LTBR. In certain embodiments, if the subject is determined to have an inadequate response to the RA drug, the subject is administered soluble LTBR. In one embodiment, the subject is determined to be asymptomatic or fully responsive for the first expression of RA (eg, TJC or SJC). In another embodiment, the subject is asymptomatic for the first expression of RA (eg, TJC or SJC), or is sufficiently responsive and symptomatic, or a second patient with rheumatoid arthritis. Is determined to be an inadequate responder for the expression of (synovitis). Synovitis can be detected by any method known in the art, including magnetic resonance imaging (MRI).

  In one embodiment, the soluble LTBR and the second agent are administered simultaneously. In another embodiment, the soluble LTBR is administered first when it is right and the second agent is administered second when it is right. In another embodiment, the second agent is administered first at the right time and the soluble LTBR is administered second at the right time. The soluble LTBR can replace or augment previously or currently administered therapy. For example, during treatment with LTBR, administration of the second agent can be stopped or reduced (eg, administered at a low level). In other embodiments, administration of the previous treatment is maintained. In some embodiments, the previous treatment is maintained until the level of LTBR achieves a level sufficient to provide a therapeutic effect. The two drugs can be administered in combination.

  In one embodiment, a human undergoing a first treatment for RA (eg, NSAID, corticosteroid, or DMARD) can also be administered soluble LTBR (eg, LTBR-Fc). In one embodiment, when the human is administered the soluble LTBR, the first treatment is stopped. In another embodiment, the human monitors for a first preselected result (eg, an improvement in RA symptoms (eg, a 10%, 20%, 30%, or more reduction in TJC or SJC)). Is done. In one embodiment, treatment with soluble LTBR is reduced or discontinued when the first preselected result is observed. In one embodiment, the human is then treated with a second preselected result (eg, worsening of RA symptoms (eg, 10%, 20% in TJC or SJC) after treatment with the soluble LTBR is discontinued. 30% increase))). If the second preselected result is observed, administration of the soluble LTBR to the human is restored or increased, or administration of the first treatment is restored. Alternatively, the human is administered both soluble LTBR, or an increased amount of soluble LTBR, and the first treatment regimen.

  In one embodiment, a human who has received a first therapy for RA and then is treated with a soluble LTBR (eg, LTBR-Fc) receives the first therapy in the same or reduced amount. to continue. In another embodiment, the treatment with the first therapy overlaps with the treatment with the soluble LTBR for a period of time, but the treatment with the first therapy is subsequently discontinued.

  In one embodiment, the subject is a DMARD poor responder (DMARD-IR). For example, the subject is an anti-TNF poor responder.

  In one embodiment, the method includes evaluating the subject for improvement in RA symptoms. In some embodiments, the assessment is at least 1 hour (eg, at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, or 48 hours, or at least 1 after administration of the soluble LTBR. Day, day 2, day 4, day 10, day 13, day 20 or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more. Can be evaluated in one or more of: before beginning treatment; during treatment; or after one or more elements of treatment have been administered, the step of evaluating further treatment with the same soluble LTBR or A step of assessing the need for further treatment with additional agents may be included, in a preferred embodiment where a preselected result of the assessment is obtained. Additional steps are employed, for example, the subject will be administered another treatment, or another evaluation or test is performed.

(III. Pharmaceutical Composition and Pharmaceutical Administration of the Present Invention)
Soluble LTBR (eg, LTBR-Fc) can be formulated as a pharmaceutical composition, eg, for administration to a subject to treat an autoimmune disorder (eg, RA). Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents that are physiologically compatible. And absorption retardants. The above composition can be prepared as a can include a pharmaceutically acceptable salt (eg, acid addition or base addition salt (see, eg, Berge et al., J. Pharm. Sci. 66: 1-19, 1977)) .

  The soluble LTBR can be formulated according to standard methods. Pharmaceutical formulations are well-established techniques and are described, for example, by Gennaro (eds.), Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams & Wilkins (2000) (ISBN: 06864) Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lipcottcott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); European Association, 3rd edition. (2000) (ISBN: 0917333096X).

  In one embodiment, soluble LTBR (eg, LTBR-Fc) is combined with excipient materials (eg, sodium chloride, dibasic sodium phosphate heptahydrate, monobasic sodium phosphate, and stabilizers). Can be prescribed. The soluble LTBR (eg, LTBR-Fc) can be provided, for example, in an appropriate concentration of buffered solution and stored at 2-8 ° C.

  The pharmaceutical composition can exist in various forms. These include, for example, liquid, semi-solid, and solid dosage forms (eg, liquid solutions (eg, injectable and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories). Agent). The preferred form may depend on the intended mode of administration and therapeutic application. Typically, the pharmaceutical composition described herein is in the form of an injectable or infusible solution.

  Such compositions can be administered by a parenteral mode (eg, intravenous injection, subcutaneous injection, intraperitoneal day or night, or intramuscular injection). The phrases “parenteral administration” and “administered parenterally” as used herein refer to modes of administration other than enteral administration and topical administration (usually by injection), intravenous, muscle Internal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, intrathecal, intravertebral, epidural, intracerebral, Intracranial, intracarotid, and intrasternal injections and infusions include, but are not limited to.

  The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to stable storage at high concentration. A sterile injectable solution is a combination of a required amount of an agent described herein in a suitable solvent with one or a combination of the ingredients listed above, followed by subsequent filter sterilization, if necessary. Can be prepared. Generally, dispersions are prepared by incorporating the agents described herein into a sterile vehicle that contains a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, whereby the agents described herein and further desired To obtain a powder of the ingredients. 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.

  In certain embodiments, the soluble LTBR can be prepared with a carrier that protects the compound against rapid release (eg, controlled release formulations (including implants) and microencapsulated delivery systems). Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known. For example, Sustained and Controlled Release Drug Delivery Systems, J. MoI. R. Robinson, Hen, Marcel Dekker, Inc. , New York, 1978.

  Soluble LTBR (eg, LTBR-Fc), for example, stabilizes and / or circulates (eg, in blood, serum), eg, at least 1.5 times, 2 times, 5 times, 10 times, or 50 times Or in other tissues to improve retention). The modified agent can be evaluated to assess whether it can reach an inflammatory site such as occurs in an autoimmune disorder (eg, RA) (eg, by using a labeled form of the agent).

  For example, the soluble LTBR can be associated with a polymer (eg, a substantially non-antigenic polymer (eg, polyalkylene oxide or polyethylene oxide)). Suitable polymers vary substantially by weight. Polymers having number average molecular 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.

  For example, soluble LTBR can be conjugated to a water soluble polymer, such as a hydrophilic polyvinyl polymer (eg, polyvinyl alcohol or polyvinyl pyrrolidone). A non-limiting list of such polymers includes polyalkylene oxide homopolymers (eg, polyethylene glycol (PEG) or polypropylene glycol), polyoxyethylenated polyols, copolymers and block copolymers thereof, provided that the block The water solubility of the copolymer is maintained. Further useful polymers include polyoxyalkylenes (eg, polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics)); polymethacrylates; carbomers; and branched or unbranched A branched polysaccharide is mentioned.

  When the soluble LTBR (eg, LTBR-Fc) is used in combination with a second agent (eg, DMARD), the two agents can be formulated separately or together. For example, each of the above pharmaceutical compositions can be mixed, for example, just prior to administration and administered together, or can be administered separately, eg, simultaneously or at different times.

  In one embodiment, the pharmaceutical composition comprises a lymphotoxin-β receptor (LT-β-) comprising a modified LT-β-R extracellular domain that is 193 or 194 amino acids in length and a modified Ig moiety that is 227 amino acids in length. R) -Ig-fusion protein population, as well as a pharmaceutically acceptable carrier, wherein at least 90% of the LT-β-R-Ig-fusion protein is wild type as shown in SEQ ID NO: 21 Only 5 amino acids have been lost from the N-terminus of the mature form of the LT-β-R extracellular domain, and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamic acid.

  In another embodiment, the pharmaceutical composition comprises a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein comprises a long Comprising a modified LT-β-R extracellular domain of 193 amino acids or 194 amino acids and a modified Ig portion, wherein the population has a reduced N-terminus compared to the wild-type LT-β-R-Ig fusion protein Has pyroglutamic acid formation and reduced C-terminal heterogeneity.

  In another embodiment, the pharmaceutical composition of the invention comprises the amino acid sequence shown in SEQ ID NO: 5.

(Administration)
Soluble LTBR (eg, LTBR-Fc) can be administered to a subject (eg, a human subject) by a variety of methods. For many applications, the route of administration is one of the following: intravenous injection or intravenous infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection. Several times, administration can be directly to the CNS (eg, intrathecal, intraventricular (ICV), intracerebral or intracranial). The agent can be administered as a fixed dose or in a mg / kg dose.

  The dose can also be selected to reduce or avoid production of antibodies against the agent.

  The route of administration and / or mode of administration of the soluble LTBR can also be associated with individual cases, eg, by monitoring the subject (eg, associated with TJC, SJC, CRP levels and RA or other autoimmune diseases). Standard parameters (e.g., using the criteria described herein) can be customized.

  Dosage regimens may be adjusted to provide the desired response (eg, a therapeutic response or a combined therapeutic effect). In general, a low dose of soluble LTBR (eg, LTBR-Fc) formulated separately or together with an appropriate dose of a second therapeutic agent as needed to provide the subject with the soluble LTBR. Can be used. Exemplary doses of the soluble LTBR are described herein.

  A dosage unit form or “fixed dose” as used herein refers to a physically discrete amount suitable as a unit dosage for the subject to be treated; A unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and, optionally, in combination with other drugs. Appropriate dosing frequencies are described elsewhere herein.

  The pharmaceutical composition may comprise a therapeutically effective amount of the soluble LTBR described herein. Such an effective amount can be determined based on the effect of the administered drug or drug combination, and the effect of the second drug if more than one drug is used. A therapeutically effective amount of an agent depends on factors such as the individual's disease state, age, sex, and weight, and the ability of the compound to elicit a desired response in the individual (e.g., at least one disorder parameter (e.g., Can be varied according to (relaxation of RA parameters), or alleviation of at least one symptom of said disorder (eg RA). A therapeutically effective amount is also such that the therapeutically beneficial effect of the composition is superior to any toxic or deleterious effects of the composition. Typically, a therapeutically effective amount is a low dose as described elsewhere herein.

(Devices and kits)
A pharmaceutical composition comprising a soluble LTBR (eg, 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. As a result, the device may be used and transferred to medical institutions and other medical equipment in emergency situations, for example, by untrained subjects or by emergency medical personnel on site. The device can include, for example, one or more housings for storing pharmaceutical preparations containing soluble LTBR and can be configured to deliver one or more unit doses of the agent.

  For example, the pharmaceutical composition can be administered with a transdermal delivery device such as a syringe (including a hypodermic or multi-chamber syringe). Other suitable delivery devices include stents, catheters, transdermal patches, microneedles, and implantable controlled release devices.

  In other examples, the pharmaceutical composition comprises a needleless subcutaneous injection device (eg, US Pat. Nos. 5,399,163; 5,383,851; 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,790,824; or No. 4,596,556). Examples of well-known implants and modules include: US Pat. No. 4,487,603 (which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate No. 4,486,194 (which discloses a therapeutic device for administering a drug through the skin); US Pat. No. 4,447,233 (which delivers the drug at a precision infusion rate) No. 4,447,224 (which discloses an infusion device capable of varying flow for continuous drug delivery); No. 4,439,196 This discloses an osmotic drug delivery system having a multi-chamber compartment); and 4,475,196 (which discloses an osmotic drug delivery system). Many other devices, implants, delivery systems, and modules are also known.

  Soluble LTBR (eg, LTBR-Fc) can be provided in the kit. In one embodiment, the kit includes (a) a container containing a composition comprising soluble LTBR, and, optionally, (b) informational material. The informational material may be descriptive material, instructional material, marketing material, or other material related to the use of the methods and / or therapeutic benefit agents described herein. In one embodiment, the kit also includes a second agent for treating an autoimmune disorder (eg, DMARD for the treatment of RA). For example, the kit includes a first container containing a composition containing the soluble LTBR and a second container containing the second agent.

  The information material of the kit is not limited in its form. In one embodiment, the informational material may include information on the manufacture of the compound, the molecular weight, concentration, expiration date, batch or manufacturing location information, etc. of the compound. In one embodiment, the informational material is, for example, suitable doses, dosage forms, or to treat a subject who has an autoimmune disorder or is at risk of experiencing an episode associated with the autoimmune disorder. It relates to a method for administering the soluble LTBR (eg, LTBR-Fc) in an administration mode (eg, a dose, dosage form, or mode of administration described herein). The information can be provided in a variety of formats, including printed documents, computer readable material, video recordings or audio recordings, or information that provides links or responses to substantial material.

  In addition to the agent, the composition in the kit can include other components, such as solvents or buffers, stabilizers, or preservatives. The agent can be provided in any form (eg, liquid, dried or lyophilized form), preferably substantially pure and / or sterile. When the drug is provided in a liquid solution, the liquid solution is preferably an aqueous solution. When the drug is provided in dry form, reconstitution is generally by the addition of a suitable solvent. The solvent (eg, sterile water or buffer) can be provided in the kit as needed.

  The kit can include one or more containers for the composition or a composition comprising the agent. In some embodiments, the kit includes a separate container, a divider, or a compartment for the composition and informational material. For example, the composition can be included in a bottle, vial, or syringe, and the informational material can be included 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 included in the form of a label in a bottle, vial or syringe to which the informational material is attached. In some embodiments, the kit includes a plurality (eg, a set) of individual containers, each one or more unit dosage forms of the agent (eg, administrations described herein). Form). The container may contain, for example, a combined unit dose (eg, a unit containing both the soluble LTBR and the second agent) in a desired ratio. For example, the kit includes a plurality of syringes, ampoules, foil packages, blister packs, or medical devices, for example, each containing a single combined unit dose. The kit containers may be airtight, waterproof (eg, impermeable to moisture or changes therein), and / or light-blocking.

  The kit optionally includes a device suitable for administration of the composition (eg, a syringe or other suitable delivery device). The device may be provided pre-loaded with one or both of the drugs, or may be empty but suitable for loading.

Example 1: Construction and characterization of LTBR-IgG construct to reduce heterogeneity
The following examples are many created to solve the molecular heterogeneity found in the expression of LTBR IgG, including N-terminal heterogeneity, C-terminal heterogeneity, pyroglutamate formation, and sialylation The characteristics of various LTBR immunoglobulin fusion proteins are described.

(N-terminal heterogeneity)
Estimating the N-terminus encoded by the full-length mRNA of LTBR (NP — 002333) is based on either a neural network (NN) prediction model trained on eukaryotic cells or a hidden Markov model (HMM). And performed by a program such as SignalP. These two NN and HMM prediction models gave different results for the suggested N-terminus of the protein, which suggested S28 or P30 and Q31, respectively (amino acid numbers refer to numbering including signal sequence) ). Thus, many N-terminal sequence predictions are possible and the optimal N-terminus with limited variability can only be determined by expressing the protein and evaluating its post-translational modifications.

  LTBR01 (LTBR-IgG fusion protein version 01) was the first molecule cloned. The N-terminus of LTBR01 began at S28 based on intact LTBR numbering (ie, including the signal sequence). The LTBR C-terminus was fused with an IgG1 hinge Fc starting at IgG heavy chain C220 (Kabat numbering) at M225. LTBR IgG was included by mutating the IgG1 hinge antibody-derived cysteine (C220) to a structurally similar amino acid valine (V199 in LTBR01). This mutation poses a problem of free unpaired thiols in the LTBR IgG and eliminates the undesired formation; in the IgG1, the heavy chain C220 is usually a disulfide bond with the light chain C107 and the light chain By lacking the chain, the cysteine (C220) remains unpaired in LTBRIgG from the beginning, causing either undesired scrambling of the CRD within the LTBR or aggregation of the LTBRIgG.

  For each LTBR IgG molecule described, the gene was constructed, expressed, purified, and evaluated for variability in N-terminal and C-terminal sequences. The result shows that LTBR01 with the S28 N-terminus of LTBR is heterogeneous to N-, N-3 and N-4 clipping sites and to pyroglutamic acid purified at Q31 ( Numbering indicated that the C-terminal K426 was missing from the Fc domain, based on the full-length LTBR sequence described in FIG.

  Based on these findings, two different constructs (designated LTBR05 and LTBR06) were created to improve heterogeneity between expressed LTBR IgG fusion proteins, particularly with respect to N-terminal proteolysis and C-terminal proteolysis did.

  LTBR05 was constructed to have a C-terminal deletion mutation and cleaved to have a C-terminal lysine that could undergo proteolysis. Analysis of LTBR05 by mass spectrometry (MS) did not confirm further C-terminal truncation upon exclusion of the C-terminal lysine, but confirmed comparable N-terminal variability relative to LTBR01.

  Therefore, LTBR06 was constructed to improve N-terminal heterogeneity. LTBR06 was designed to delete 4 amino acids from its N-terminus. This deletion dramatically reduced N-terminal sequence variability as described in Table 1. LTBR06 had the desired N-terminus (N-4 and N-5 relative to wild type) and more than 90% of the N-1 N-terminus. The deletion of LTBR06 is also described in FIGS.

Table 3 provides an overview of the different constructs created by deleting the N-terminal and C-terminal portions of LTBR and the results from such deletions. LTBR05 had a single amino acid removed from the carboxy terminus of LTBR01 (C-1), and LTBR06 had a single C-1 amino acid deletion and 4 amino acids deleted from the amino terminus ( After signal sequence, referred to as -N-4). Therefore, LTBR06 was manipulated to reduce both N-terminal heterogeneity and C-terminal heterogeneity. A comparison of the mature forms of the different constructs is shown in FIG.
Table 3: Comparison of LTBR05 and LTBR06 vs. LTBR01

ＬＴＢＲ０５を、Ｎ末端不均一性（結果を表４にまとめる）を含め、生化学的に特徴づけた。ＬＴＢＲ０５のアミノ末端は、大部分はピログルタミン酸に変換された（＜Ｑ）Ｎ−１およびＮ−３形態とともに、Ｎ、Ｎ−１、およびＮ−３として存在することが見いだされた。
表４：ＬＴＢＲ０５の生化学的特徴付けのまとめ

LTBR05 was biochemically characterized, including N-terminal heterogeneity (results are 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 being largely converted to pyroglutamic acid (<Q).
Table 4: Summary of biochemical characterization of LTBR05

ＬＴＢＲ０６を、Ｎ末端不均一性およびＣ末端不均一性が、アミノ酸置換に基づいて改善されたか否かを含め、不均一性を改善したか否かを決定するために生化学的に特徴づけた（結果を表５にまとめる）。
表５：ＬＴＢＲ０６の生化学的特徴付けのまとめ

LTBR06 was biochemically characterized to determine whether N-terminal heterogeneity and C-terminal heterogeneity was improved, including whether it was improved based on amino acid substitutions. (The results are summarized in Table 5).
Table 5: Summary of biochemical characterization of LTBR06

エドマン分解を、ＬＴＢＲ０６発現細胞由来のＬＴＢＲ０６に対して行った。表５に記載されるように、ＬＴＢＲ０６タンパク質の集団のうちの５６％が、Ｎ−５であり、４０％がＮ−４であり、約４％がＮ−１１であることが見いだされた。ＬＴＢＲＩｇのＮ−５種の配列は、配列番号２３に記載される。Ｎ−４およびＣ−１欠失を使用して、上記タンパク質集団の不均一性は、ＬＴＢＲ０５（改変体に融合されたＬＴＢＲの非改変細胞外ドメインを示す）と比較して、低下していた。

Edman degradation was performed on LTBR06 from LTBR06 expressing cells. As described in Table 5, 56% of the population of LTBR06 protein was found to be N-5, 40% N-4, and about 4% N-11. The sequence of N-5 species of LTBRIg is set forth in SEQ ID NO: 23. Using N-4 and C-1 deletions, the heterogeneity of the protein population was reduced compared to LTBR05, which represents the unmodified extracellular domain of LTBR fused to the variant. .

Example 2: Glycosylation study of LTBR IgG construct
Glycosylation can also have an impact on protein heterogeneity and activity. Therefore, to further reduce heterogeneity, glycosylation variants were investigated to determine whether variable LTBR domain glycosylation affected binding affinity, expression levels, and folding. LTBR09 was a construct based on LTBR06 with an additional N275Q mutation (amino acid position refers to the mature protein—see FIG. 2 for comparison of sequences and changes between the various constructs).

Table 6 describes the glycosylation retention for each of the three LTBR constructs at Asn13 and Asn150.
Table 6: Glycosylation retention

＊インタクトなＮ末端、Ｎ−１ ＜ＱおよびＮ−３ ＜Ｑの平均として計算された。
＊＊Ｎ−４およびＮ−５の平均として計算された。

* Calculated as the average of intact N-terminus, N-1 <Q and N-3 <Q.
** Calculated as the average of N-4 and N-5.

  As shown in Table 6, glycosylation retention was relatively unchanged within the different constructs for Asn13 and Asn150.

In order to further reduce heterogeneity, glycosylation variants in which the N-terminal glycosylation of LTBR IgG was removed in either the LTBR extracellular domain or the Fc portion of the protein were investigated. Removal of the glycosylation site in the LTBR domain successfully reduced glycosylation and also reduced the expression of this protein by about 10-fold. Table 7 provides results from studies characterizing the effects of various mutant overviews and deletion of glycosylation sites.
Table 7: Additional glycosylation variants

ＬＴＢＲ０３は、図５および配列番号１０に示される。グリコシル化されていないＬＴＢＲ０３は、競合ＦＡＣＳアッセイにおいて野生型ＬＴＢＲＩｇＧ（非改変ＬＴＢＲ細胞外ドメインおよび非改変Ｆｃ領域）に対する結合等価性を示した。

LTBR03 is shown in FIG. 5 and SEQ ID NO: 10. Non-glycosylated LTBR03 showed binding equivalence to wild type LTBR IgG (unmodified LTBR extracellular domain and unmodified Fc region) in a competitive FACS assay.

  Soluble glycosylated LTBR IgG was also generated by first capturing the protein using protein A (initial capture). The captured protein was then run against a phenyl column to remove misfolded and aggregated proteins. After phenyl chromatography, non-glycosylated LTBR IgG was purified to a uniform population using Sephacryl S-200. Soluble LTBR was then generated using Asp N digestion. The digest was then re-exposed to protein A to remove the Fc portion of the fusion protein. The sLTBR is then purified using DEAE (which removed Asp N), and gel filtration / characterization, of which characterization is the optimal refinement step. Finally, a main crystal screen was performed.

(Example 3: Effect of sialylation on LTBR IgG pharmacokinetics)
To study the effect of sialylation on LTBRIgG pharmacokinetics, LTBRIgG sialylation was performed using α-2,6-sialyltransferase from rat liver (Boehringer Mannheim). α-2,6-sialyltransferase is specific for Gaβ1 → 4GlcNAc and sialylates 75-100% of the terminal N-linked galactose residues. LTBRIgG sialylation achieved various levels of sialylated proteins, which were then evaluated for pharmacokinetics. Pharmacokinetic data of low, medium, and highly sialylated LTBRIG (version LTBR05) was analyzed in mouse serum. The results are listed in Tables 8 and 9 below.
Table 8: LTBR-Ig PK data (concentration (μg / ml) sialylation (SA))

blq=定量限界未満
^＊表８に記載される平均中に含められない
表９：表８からの平均ＳＡ

blq = below quantitation limit
^* Not included in the averages listed in Table 8 Table 9: Average SA from Table 8

図１３は、ｈｕＬＴＢＲＩｇＧとＬＦＡ３ＴＩＰ（ヒトＩｇＧ１のＦｃ（ヒンジ、ＣＨ２およびＣＨ３ドメイン）部分に連結されたヒト白血球機能抗原−３（ＬＦＡ−３）の細胞外ＣＤ２結合部分からなるダイマー性融合タンパク質）との間の薬物動態における差異を記載する。図１３に示されるように、ＬＴＢＲＩｇＧは、ＬＦＡ３ＴＩＰとの比較において、シアル化に拘わらず、増大した血清レベルを有した。

FIG. 13 shows huLTBRIgG and LFA3TIP (a dimeric fusion protein comprising an extracellular CD2 binding portion of human leukocyte functional antigen-3 (LFA-3) linked to the Fc (hinge, CH2 and CH3 domains) portion of human IgG1) and The differences in pharmacokinetics between are described. As shown in FIG. 13, LTBRIgG had increased serum levels despite sialylation in comparison to LFA3TIP.

(Example 4: Effect of domain shortening of LTBRIgG)
The TNFRIgG product entanercept has significant misfolding heterogeneity within the protein (situation similar to LTBRIgG). Based on the food intake range shown in the TNFR / TNF co-crystal structure, a shortened version of TNFRFc was used to help reduce the degree of misfolding within the TNFR portion, using the 2.6 domain of TNFR I made it. The truncated TNF receptor molecule maintained full binding activity for the TNFa ligand. Based on the CBR terminal alignment of LTBR domain and TNFR domain 4 and domain 3 as well as several other variations, a shortened version of LTBR IgG was expressed and evaluated for their affinity for LTalb2 (above (See Table 2).

  The binding affinities of various truncations (including D3-1, D2-1 (wt = D1-4)) were determined. None of the purified truncated LTBR IgG molecules retains high affinity for LTa1b2, indicating that this represents a contact between LTBR / LTa1b2. Therefore, TNFR / TNFa was a poor predictor of contact residues between LTBR and LTalb2.

Example 5: Hinge design for LTBR IgG
Many different hinge constructs have been created that can be used to connect LTBR and IgG. The description of the different hinges is set forth in FIG. 6, including the hinge sequence that follows. When used, the following sequence is located after the C-terminal amino acid (ie, M) of the LTBR extracellular domain.
LTBR06 Hinge: VDKTHTCPPCPAP (SEQ ID NO: 13)
Hinge 2211 (D169N): VNKTHTCPPCPAP (SEQ ID NO: 14)
Hinge 2212 (T198N): VDKNHTCPPCPAP (SEQ ID NO: 15)
Hinge 2221 (valine deletion): DKTHTCPPCPAP (SEQ ID NO: 16)
Hinge 2217 (full length): EPKSCDKTHTCPPCPAP (SEQ ID NO: 17)
Hinge 2219 (G4-Hinge-G4Fc): ESCDKYGPPCPPCPAP (SEQ ID NO: 18)
Hinge 2218 (G2-hinge-G2Fc): CCVECPPCPAPPVAGP (SEQ ID NO: 19)
Hinge 2220 (short hinge-G1Fc): CPPCPAP (SEQ ID NO: 20).

  An analysis of expression for each of the hinges in LTBR IgG in comparison to LTBR06 is described in FIG.

  Examples 6-8 describe clinical studies showing the efficacy of LTBR IgG (more specifically, LTBR06) for the treatment of autoimmune disorders.

Example 6: Low dose LTBR-Fc is effective for treating rheumatoid arthritis
A dose of 0.05 mg / kg LTBR-Fc (version LTBR06) was randomized, blinded, placebo-controlled drug elevation to evaluate the safety, tolerability and pharmacokinetics of LTBR-Fc in RA patients Selected as the lowest of five dose cohorts in clinical trials. 0.05 mg / kg was selected as the lowest dose cohort because it was predicted to be an ineffective dose with respect to safety and pharmacokinetic findings. In fact, this was an ineffective dose for the acute phase response in the initial study. Evaluating efficacy was neither a temporary nor secondary objective for the study. Administration was once a week for 4 weeks. All members of the cohort were DMARD-IR. The patient received LTBR-Fc of the study while receiving methotrexate treatment.

  Exemplary results are shown in FIG. This figure shows 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) The data about is shown. The number of painful joints (TJC) and the number of swollen joints (SJC) were evaluated for each subject.

  Remarkably, the 0.05 mg / kg cohort showed dramatic (50-70%) improvement in TJC and SJC compared to placebo. Similar improvements were also seen in the next higher dose cohort (0.1 mg / kg). This surprising observation resulted in a complete redesign of the study, which required the addition of an additional dose cohort below 0.05 mg / kg (so that the lower dose range could be tested, And a linear dose response could be seen and the lower dose response was determined), and the release of the highest dose cohort (3 mg / kg) resulted from the study.

Example 7: Clinical study of LTBR-Fc (LTBR06) for treatment of RA (IIA data)
The following example describes the efficacy from a dose range of LTBR-Fc (version LTBR06) and results from a phase IIa clinical study in which patients were administered for the treatment of rheumatoid arthritis (RA).

  This study was a blinded, randomized, placebo controlled dose range study in 47 DMARD-IR RA patients. Screening for the study included identifying patients who had methotrexate (mtx) -IR with active RA. The cohort dose 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 ratio randomized to LTBR-Fc or placebo was 3: 1. Observation was performed for 8 weeks after subcutaneous injection once a week for 4 weeks. Baseline demographics and disease characteristics were similar throughout the placebo and experimental groups (11 placebo patients, 36 LTBR06 patients). The average duration of RA, including all of the above patients, was 3.1 years. 39 (83%) of the patients had prior DMARD treatment and 3 of 6% (3 of 6%) 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 the ACR core set of measurements was 16 SJC, 21 TJC, and 1.33 HAQ.

LTBR-Fc has been found to be effective in treating RA in patients. The results showed a substantial improvement in the majority of ACR center set measurements in a dose-dependent manner. On day 35 (2 weeks after the last dose), the mean improvement was SJC 60% vs 4.6% (LTBR-Fc vs placebo) and TJC 47% vs 6.7% (LTBR-Fc vs placebo). (See FIGS. 10-12). As shown in FIGS. 9-11, the improvement in SJC and TJC was persistent, lasting from 8 weeks to 77 days after the last LTBR-Fc administration. The greatest improvement was reported in the LTBR-Fc 1.0 mg / kg and 3.0 mg / kg groups, and the ACR20 response rates for these two treatment groups were 65% and 85% on day 77, respectively. It was. The improvement in the ACR20 response was also greater than placebo for all dose groups, as shown in FIG. 12 and Table 10.
Table 10: ACR 20/50/70 response rate on day 77 (% of patients)

ＡＣＲ２０（ＡＣＲ５０、ＡＣＲ７０）応答は、ＳＪＣおよびＴＪＣにおける２０％（５０％、７０％）改善として定義され、以下の指標のうちの少なくとも３つにおいて２０％（５０％、７０％）の改善を有する：ＩＧＡ、ＰＧＡ、疼痛−ＶＡＳ、ＨＡＱ、ＣＲＰ（ＣＲＰが見いだされないのであれば、ＥＳＲ）。

ACR20 (ACR50, ACR70) response is defined as a 20% (50%, 70%) improvement in SJC and TJC and has a 20% (50%, 70%) improvement in at least three of the following indicators: : IGA, PGA, Pain-VAS, HAQ, CRP (or ESR if CRP is not found).

  Overall, LTBR-Fc was found to be safe in patients who received treatment. 55% (6/11) of patients receiving placebo and 67% (24/36) of patients receiving LTBR-Fc (LTBR06) had adverse events (AEs) (eg, headache, chills, However, none of them was serious. The most common AE in patients receiving LTBR-Fc (LTBR06) was headache, reported in 19% of patients vs. 9% of patients receiving placebo. No drug-related serious infections or drug-related serious AEs were reported during this study period. One serious infection was reported on day 91 in the 3.0 mg / kg group (acute bronchitis and does not appear to be related to treatment). Temporary mild to moderate influenza-like symptoms were observed in 25% of patients within 24 hours after the first dose of LTBR-Fc. These symptoms responded well to acetaminophen and did not usually recur after subsequent doses (reduced relative to subsequent doses—after the second, third, and fourth doses above) Reported in 8%, 9% and 6% of patients respectively). Reduced immunogenicity was observed at increased doses and had no effect on PK, AE, or efficacy.

  Overall, the results showed that LTBR-Fc is effective in treating RA at doses ranging from 0.01 mg / kg to 3 mg / kg.

Example 8: Clinical study of LTBR-Fc (LTBR06) for treatment of RA
Long-term studies were used to evaluate the safety, tolerance and efficacy of LTBR-Fc (LTBR06) in subjects with RA. Patients from previous studies (eg, the study described in Example 7) can be included in the study. The study design involves administering LTBR-Fc (LTBR06) subcutaneously to RA patients at one of the following doses: 70 mg once every two weeks; 200 mg once every two weeks; 70 mg once a month; 200 mg once a month; or placebo dose. Patients are assessed for improvement in the response and the following efficacy parameter scores are determined: American College of Rheumatology (ACR) Core Set of Measurements, DAS28, Short Form (SF-36), Functional sessential Therapies (FACIT) questionnaires, Sjogren's Assessment. A statistically significant improvement in such parameters when compared to the placebo control group indicates efficacy.
(Equivalent)
All patents, patent applications, and references are hereby incorporated by reference in their entirety. In case of conflict, the present application takes precedence.

  Each of the limitations of the invention can encompass various embodiments of the invention. Accordingly, it will be appreciated that each of the limitations of the invention including any one element or combination of elements can be included in each aspect of the invention.

  The accompanying sequence listing forms part of the present disclosure, the contents of which are summarized in the following table.

（配列表）
配列表
<110> バイオジェン・アイデック・エムエイ・インコーポレイテッド
<120> 自己免疫障害の処置
<130> BGN-717PC
<140> 割り当て未
<141> 出願と同時
<150> 60/918,518
<151> 2007-03-15

配列番号1
GenPept ID No. P36941に対応する全長ヒト野生型ＬＴＢＲアミノ酸配列（非成熟形態）
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 (配列番号1)

配列番号2
バリン変異アミノ酸を含むLTBR06のＦｃ部分
V D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G

配列番号3
LTBR06の細胞外ドメイン−核酸配列
1 ATGCTCCTGC CTTGGGCCAC CTCTGCCCCC GGCCTGGCCT GGGGGCCTCT
51 GGTGCTGGGC CTCTTCGGGC TCCTGGCAGC AGCGGTGCCT CCATATGCGT
101 CGGAGAACCA GACCTGCAGG GACCAGGAAA AGGAATACTA TGAGCCCCAG
151 CACCGCATCT GCTGCTCCCG CTGCCCGCCA GGCACCTATG TCTCAGCTAA
201 ATGTAGCCGC ATCCGGGACA CAGTTTGTGC CACATGTGCC GAGAATTCCT
251 ACAACGAGCA CTGGAACTAC CTGACCATCT GCCAGCTGTG CCGCCCCTGT
301 GACCCAGTGA TGGGCCTCGA GGAGATTGCC CCCTGCACAA GCAAACGGAA
351 GACCCAGTGC CGCTGCCAGC CGGGAATGTT CTGTGCTGCC TGGGCCCTCG
401 AGTGTACACA CTGCGAGCTA CTTTCTGACT GCCCGCCTGG CACTGAAGCC
451 GAGCTCAAAG ATGAAGTTGG GAAGGGTAAC AACCACTGCG TCCCCTGCAA
501 GGCAGGGCAC TTCCAGAATA CCTCCTCCCC CAGCGCCCGC TGCCAGCCCC
551 ACACCAGGTG TGAGAACCAA GGTCTGGTGG AGGCAGCTCC AGGCACTGCC
601 CAGTCCGACA CAACCTGCAA AAATCCATTA GAGCCACTGC CCCCAGAGAT
651 GTCAGGAACC ATG

配列番号4
LTBR06の細胞外ドメイン−アミノ酸配列
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

配列番号5
LTBR06の成熟形態のアミノ酸配列
AVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

配列番号6
LTBR01-アミノ酸
MLLPWATSAP GLAWGPLVLG LFGLLAASQPQAVP PYASENQTCR DQEKEYYEPQ
HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC
DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA
QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

配列番号7
LTBR06-核酸
1 ATGCTCCTGC CTTGGGCCAC CTCTGCCCCC GGCCTGGCCT GGGGGCCTCT
51 GGTGCTGGGC CTCTTCGGGC TCCTGGCAGC AGCGGTGCCT CCATATGCGT
101 CGGAGAACCA GACCTGCAGG GACCAGGAAA AGGAATACTA TGAGCCCCAG
151 CACCGCATCT GCTGCTCCCG CTGCCCGCCA GGCACCTATG TCTCAGCTAA
201 ATGTAGCCGC ATCCGGGACA CAGTTTGTGC CACATGTGCC GAGAATTCCT
251 ACAACGAGCA CTGGAACTAC CTGACCATCT GCCAGCTGTG CCGCCCCTGT
301 GACCCAGTGA TGGGCCTCGA GGAGATTGCC CCCTGCACAA GCAAACGGAA
351 GACCCAGTGC CGCTGCCAGC CGGGAATGTT CTGTGCTGCC TGGGCCCTCG
401 AGTGTACACA CTGCGAGCTA CTTTCTGACT GCCCGCCTGG CACTGAAGCC
451 GAGCTCAAAG ATGAAGTTGG GAAGGGTAAC AACCACTGCG TCCCCTGCAA
501 GGCAGGGCAC TTCCAGAATA CCTCCTCCCC CAGCGCCCGC TGCCAGCCCC
551 ACACCAGGTG TGAGAACCAA GGTCTGGTGG AGGCAGCTCC AGGCACTGCC
601 CAGTCCGACA CAACCTGCAA AAATCCATTA GAGCCACTGC CCCCAGAGAT
651 GTCAGGAACC ATGGTCGACA AAACTCACAC ATGCCCACCG TGCCCAGCAC
701 CTGAACTCCT GGGGGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG
751 GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG TGGTGGTGGA
801 CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC GTGGACGGCG
851 TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC
901 ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA
951 TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCA
1001 TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTG
1051 TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC AGGTCAGCCT
1101 GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG
1151 AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGTTG
1201 GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG
1251 CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC
1301 TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGT

配列番号8
シグナル配列を含むLTBR06アミノ酸配列
1 MLLPWATSAP GLAWGPLVLG LFGLLAAAVP PYASENQTCR DQEKEYYEPQ
51 HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC
101 DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
151 ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA
201 QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
251 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
351 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
401 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

配列番号9
LTBR05,アミノ酸配列の成熟形態
SQPQAVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG


配列番号10
グリコシル化されていないLTBRIgG
1 AVPPYASEQQ TCRDQEKEYY EPQHRICCSR CPPGTYVSAK CSRIRDTVCA
51 TCAENSYNEH WNYLTICQLC RPCDPVMGLE EIAPCTSKRK TQCRCQPGMF
101 CAAWALECTH CELLSDCPPG TEAELKDEVG KGNNHCVPCK AGHFQQTSSP
151 SARCQPHTRC ENQGLVEAAP GTAQSDTTCK NPLEPLPPEM SGTMVDKTHT
201 CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF
251 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN
301 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS
351 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
401 SVMHEALHNH YTQKSLSLSP G

配列番号11
LTBR01,成熟形態アミノ酸配列
SQPQAVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA
QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

配列番号12
LTBR09,成熟形態アミノ酸配列
AVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

配列番号13
LTBR06ヒンジ
VDKTHTCPPCPAP

配列番号14
ヒンジ2211 (D169N)
VNKTHTCPPCPAP

配列番号15
ヒンジ2212 (T198N)
VDKNHTCPPCPAP

配列番号16
ヒンジ2221 (バリン欠失)
DKTHTCPPCPAP

配列番号17
ヒンジ2217 (全長)
EPKSCDKTHTCPPCPAP

配列番号18
ヒンジ2219 (G4-ヒンジ-G4Fc)
ESCDKYGPPCPPCPAP

配列番号19
ヒンジ2218 (G2-ヒンジ-G2Fc)
CCVECPPCPAPPVAGP

配列番号20
ヒンジ2220 (短いヒンジ - G1Fc)
CPPCPAP (配列番号20)

配列番号21
GenPept ID No. P36941に対応するヒト野生型LTBRアミノ酸配列の細胞外ドメイン（非成熟形態）
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 MSGTM

配列番号22
Fcドメイン-アミノ酸
C D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G

配列番号23
LTBR06のＮ−５細胞外ドメイン-アミノ酸配列
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

(Sequence Listing)
Sequence listing
<110> Biogen Idec MEI, Inc.
<120> Treatment of autoimmune disorders
<130> BGN-717PC
<140> Not assigned
<141> Simultaneous with application
<150> 60 / 918,518
<151> 2007-03-15

SEQ ID NO: 1
Full-length human wild-type LTBR amino acid sequence corresponding to GenPept ID No. P36941 (non-mature form)
1 MLLPWATSAP GLAWGPLVLG LFGLLAASQP QAVPPYASEN QTCRDQEKEY YEPQHRICCS
61 RCPPGTYVSA KCSRIRDTVC ATCAENSYNE HWNYLTICQL CRPCDPVMGL EEIAPCTSKR
121 KTQCRCQPGM FCAAWALECT HCELLSDCPP GTEAELKDEV GKGNNHCVPC KAGHFQNTSS
181 PSARCQPHTR CENQGLVEAA PGTAQSDTTC KNPLEPLPPE MSGTMLMLAV LLPLAFFLLL
241 ATVFSCIWKS HPSLCRKLGS LLKRRPQGEG PNPVAGSWEP PKAHPYFPDL VQPLLPISGD
301 VSPVSTGLPA APVLEAGVPQ QQSPLDLTRE PQLEPGEQSQ VAHGTNGIHV TGGSMTITGN
361 IYIYNGPVLG GPPGPGDLPA TPEPPYPIPE EGDPGPPGLS TPHQEDGKAW HLAETEHCGA
421 TPSNRGPRNQ FITHD (SEQ ID NO: 1)

SEQ ID NO: 2
Fc part of LTBR06 containing a valine mutant amino acid
VDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPQMHTP

SEQ ID NO: 3
LTBR06 extracellular domain-nucleic acid sequence
1 ATGCTCCTGC CTTGGGCCAC CTCTGCCCCC GGCCTGGCCT GGGGGCCTCT
51 GGTGCTGGGC CTCTTCGGGC TCCTGGCAGC AGCGGTGCCT CCATATGCGT
101 CGGAGAACCA GACCTGCAGG GACCAGGAAA AGGAATACTA TGAGCCCCAG
151 CACCGCATCT GCTGCTCCCG CTGCCCGCCA GGCACCTATG TCTCAGCTAA
201 ATGTAGCCGC ATCCGGGACA CAGTTTGTGC CACATGTGCC GAGAATTCCT
251 ACAACGAGCA CTGGAACTAC CTGACCATCT GCCAGCTGTG CCGCCCCTGT
301 GACCCAGTGA TGGGCCTCGA GGAGATTGCC CCCTGCACAA GCAAACGGAA
351 GACCCAGTGC CGCTGCCAGC CGGGAATGTT CTGTGCTGCC TGGGCCCTCG
401 AGTGTACACA CTGCGAGCTA CTTTCTGACT GCCCGCCTGG CACTGAAGCC
451 GAGCTCAAAG ATGAAGTTGG GAAGGGTAAC AACCACTGCG TCCCCTGCAA
501 GGCAGGGCAC TTCCAGAATA CCTCCTCCCC CAGCGCCCGC TGCCAGCCCC
551 ACACCAGGTG TGAGAACCAA GGTCTGGTGG AGGCAGCTCC AGGCACTGCC
601 CAGTCCGACA CAACCTGCAA AAATCCATTA GAGCCACTGC CCCCAGAGAT
651 GTCAGGAACC ATG

SEQ ID NO: 4
LTBR06 extracellular domain-amino acid sequence
AVPPYASENQTCRDQEKEYYE PQHRICCSRCPPGTYVSAKCS RIRDTVCATCAENSYNEHWNY LTICQLCRPCDPVMGLEEIAP CTSKRKTQCRCQPGMFCAAWA LECTHCELLSDCPPGTEAELK DEVGKGNNHCVPCKAGHFQNT SSPSARCQPHTRCENQGLVEATM

SEQ ID NO: 5
Amino acid sequence of mature form of LTBR06
AVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

SEQ ID NO: 6
LTBR01-amino acid
MLLPWATSAP GLAWGPLVLG LFGLLAASQPQAVP PYASENQTCR DQEKEYYEPQ
HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC
DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA
QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

SEQ ID NO: 7
LTBR06-nucleic acid
1 ATGCTCCTGC CTTGGGCCAC CTCTGCCCCC GGCCTGGCCT GGGGGCCTCT
51 GGTGCTGGGC CTCTTCGGGC TCCTGGCAGC AGCGGTGCCT CCATATGCGT
101 CGGAGAACCA GACCTGCAGG GACCAGGAAA AGGAATACTA TGAGCCCCAG
151 CACCGCATCT GCTGCTCCCG CTGCCCGCCA GGCACCTATG TCTCAGCTAA
201 ATGTAGCCGC ATCCGGGACA CAGTTTGTGC CACATGTGCC GAGAATTCCT
251 ACAACGAGCA CTGGAACTAC CTGACCATCT GCCAGCTGTG CCGCCCCTGT
301 GACCCAGTGA TGGGCCTCGA GGAGATTGCC CCCTGCACAA GCAAACGGAA
351 GACCCAGTGC CGCTGCCAGC CGGGAATGTT CTGTGCTGCC TGGGCCCTCG
401 AGTGTACACA CTGCGAGCTA CTTTCTGACT GCCCGCCTGG CACTGAAGCC
451 GAGCTCAAAG ATGAAGTTGG GAAGGGTAAC AACCACTGCG TCCCCTGCAA
501 GGCAGGGCAC TTCCAGAATA CCTCCTCCCC CAGCGCCCGC TGCCAGCCCC
551 ACACCAGGTG TGAGAACCAA GGTCTGGTGG AGGCAGCTCC AGGCACTGCC
601 CAGTCCGACA CAACCTGCAA AAATCCATTA GAGCCACTGC CCCCAGAGAT
651 GTCAGGAACC ATGGTCGACA AAACTCACAC ATGCCCACCG TGCCCAGCAC
701 CTGAACTCCT GGGGGGACCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAG
751 GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG TGGTGGTGGA
801 CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC GTGGACGGCG
851 TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC
901 ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA
951 TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCA
1001 TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTG
1051 TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC AGGTCAGCCT
1101 GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG
1151 AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGTTG
1201 GACTCCGACG GCTCCTTCTT CCTCTACAGC AAGCTCACCG TGGACAAGAG
1251 CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC
1301 TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGT

SEQ ID NO: 8
LTBR06 amino acid sequence including signal sequence
1 MLLPWATSAP GLAWGPLVLG LFGLLAAAVP PYASENQTCR DQEKEYYEPQ
51 HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC
101 DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA
151 ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA
201 QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
251 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
351 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
401 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

SEQ ID NO: 9
LTBR05, mature form of amino acid sequence
SQPQAVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG


SEQ ID NO: 10
Non-glycosylated LTBRIgG
1 AVPPYASEQQ TCRDQEKEYY EPQHRICCSR CPPGTYVSAK CSRIRDTVCA
51 TCAENSYNEH WNYLTICQLC RPCDPVMGLE EIAPCTSKRK TQCRCQPGMF
101 CAAWALECTH CELLSDCPPG TEAELKDEVG KGNNHCVPCK AGHFQQTSSP
151 SARCQPHTRC ENQGLVEAAP GTAQSDTTCK NPLEPLPPEM SGTMVDKTHT
201 CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF
251 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN
301 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS
351 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
401 SVMHEALHNH YTQKSLSLSP G

SEQ ID NO: 11
LTBR01, mature form amino acid sequence
SQPQAVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTAAPG
QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK
DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV
YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

SEQ ID NO: 12
LTBR09, mature form amino acid sequence
AVP PYASENQTCR DQEKEYYEPQ HRICCSRCPP GTYVSAKCSR IRDTVCATCA ENSYNEHWNY LTICQLCRPC DPVMGLEEIA PCTSKRKTQC RCQPGMFCAA WALECTHCEL LSDCPPGTEA ELKDEVGKGN NHCVPCKAGH FQNTSSPSAR CQPHTRCENQ GLVEAAPGTA QSDTTCKNPL EPLPPEMSGT MVDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG

SEQ ID NO: 13
LTBR06 hinge
VDKTHTCPPCPAP

SEQ ID NO: 14
Hinge 2211 (D169N)
VNKTHTCPPCPAP

SEQ ID NO: 15
Hinge 2212 (T198N)
VDKNHTCPPCPAP

SEQ ID NO: 16
Hinge 2221 (valine deletion)
DKTHTCPPCPAP

SEQ ID NO: 17
Hinge 2217 (full length)
EPKSCDKTHTCPPCPAP

SEQ ID NO: 18
Hinge 2219 (G4-Hinge-G4Fc)
ESCDKYGPPCPPCPAP

SEQ ID NO: 19
Hinge 2218 (G2-Hinge-G2Fc)
CCVECPPCPAPPVAGP

SEQ ID NO: 20
Hinge 2220 (short hinge-G1Fc)
CPPCPAP (SEQ ID NO: 20)

SEQ ID NO: 21
Extracellular domain of human wild-type LTBR amino acid sequence corresponding to GenPept ID No. P36941 (non-mature form)
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 MSGTM

SEQ ID NO: 22
Fc domain-amino acid
CDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPQMHTP

SEQ ID NO: 23
LTBR06 N-5 extracellular domain-amino acid sequence
AVPPYASENQTCRDQEKEYYE PQHRICCSRCPPGTYVSAKCS RIRDTVCATCAENSYNEHWNY LTICQLCRPCDPVMGLEEIAP CTSKRKTQCRCQPGMFCAAWA LECTHCELLSDCPPGTEAELK DEVGKGNNHCVPCKAGHFQNT SSPSARCQPHTRCENQGLVEGT APLETAPL

Claims (82)

A composition comprising a population of lymphotoxin-β receptor (LT-β-R) -Ig-fusion proteins, the composition comprising a modified LT-β-R extracellular domain of 193 amino acids or 194 amino acids in length and Comprising a modified Ig portion that is 227 amino acids in length, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21 A composition wherein only 5 amino acids have been lost from the N-terminus of the mature form and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamic acid. The composition of claim 1, wherein the N-terminal amino acid of the modified LT-β-R-Ig fusion protein is a nonpolar amino acid. The non-polar amino acid may be valine (amino acid 6 in the mature form of wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21). The composition according to claim 2, which is any one of amino acids 5). At least 95% of the LT-β-R-Ig-fusion protein has an N-terminal amino acid of valine (amino acid 6 in the mature form of wild-type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (sequence The composition according to claim 1, which is any one of amino acids 5) of the mature form of the wild type LT-β-R extracellular domain of number 21. 5. A composition according to any one of claims 1, 3 or 4 produced in mammalian cells by expressing a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4. object. 6. The composition of claim 5, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3. 6. The composition of claim 5, wherein the modified Ig moiety comprises an Fc region of IgG1 isotype. 6. The composition of claim 5, wherein the modified Ig moiety comprises the amino acid sequence set forth in SEQ ID NO: 2. 6. The composition of claim 5, wherein the Ig moiety is not glycosylated. 6. The composition of claim 5, wherein the composition is produced by expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein shown in SEQ ID NO: 5 in a mammalian cell. 11. The composition of claim 10, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 7. The composition according to claim 11, wherein the developing step is performed on a production scale. A composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, wherein the modified LT- The composition, wherein the β-R extracellular domain is not glycosylated. 14. The composition of claim 13, wherein the non-glycosylated extracellular domain of LTBR comprises amino acids 1-194 of SEQ ID NO: 10. A composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion protein, wherein the fusion protein is a modified LT-β-R cell of 193 amino acids or 194 amino acids in length A composition comprising an ectodomain and a modified Ig moiety, wherein the population has reduced N-terminal pyroglutamate formation and reduced C-terminal heterogeneity compared to a wild-type LT-β-R-Ig fusion protein object. 16. The composition of claim 15, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises a modified LT-β-R extracellular domain that exhibits the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23. object. 16. The composition of claim 15, wherein the modified Ig portion comprises a mutation in the hinge region. A population of lymphotoxin-β receptor (LT-β-R) -Ig-fusion proteins comprising a modified LT-β-R extracellular domain of 193 or 194 amino acids in length and a modified Ig moiety of 227 amino acids in length; and A pharmaceutical composition comprising a pharmaceutically acceptable carrier, wherein at least 90% of said LT-β-R-Ig-fusion protein is a wild type LT-β- shown in SEQ ID NO: 21. A composition wherein only 5 amino acids have been lost from the N-terminus of the mature form of the R extracellular domain and the LT-β-R-Ig-fusion protein lacks the N-terminal pyroglutamate. 20. The composition of claim 19, wherein the N-terminal amino acid of the modified LT-β-R-Ig fusion protein is a nonpolar amino acid. The non-polar amino acid may be valine (amino acid 6 in the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (amino acid form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21 The composition according to claim 20, which is any one of amino acids 5). At least 95% of the LT-β-R-Ig-fusion protein has an N-terminal amino acid of valine (amino acid 6 in the mature form of wild-type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (sequence 20. The composition of claim 19, which is any of amino acids 5) of mature form of wild type LT-β-R extracellular domain of number 21. 23. A composition according to any of claims 19, 21 or 22 produced by expressing in a mammalian cell a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4. 24. A method for treating an autoimmune disorder comprising administering a pharmaceutical composition according to claim 23 to a subject. 25. The method of claim 24, wherein the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE). A pharmaceutical composition comprising a population of lymphotoxin-β receptor-immunoglobulin (LT-β-R-Ig) -fusion protein and a pharmaceutically acceptable carrier, the fusion protein having a length of 193 amino acids or A 194 amino acid modified LT-β-R extracellular domain and a modified Ig portion, wherein the population has reduced N-terminal pyroglutamate formation and reduction compared to a wild-type LT-β-R-Ig fusion protein A pharmaceutical composition having a C-terminal heterogeneity. 27. The pharmacology of claim 26, wherein at least 90% of the LT-β-R-Ig-fusion protein comprises a modified LT-β-R extracellular domain showing the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 23. Composition. 27. The pharmaceutical composition of claim 26, wherein the modified Ig moiety comprises a mutation in the hinge region. A pharmaceutical composition comprising the amino acid sequence shown in SEQ ID NO: 5. 30. A method of treating an autoimmune disorder comprising administering to a subject a pharmaceutical composition according to claim 29. 32. The method of claim 30, wherein the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE). 32. The method of claim 30, wherein the autoimmune disorder is rheumatoid arthritis. 36. The method of claim 32, wherein the pharmaceutical composition is administered to the subject once every two weeks at a dose of about 0.6-3 mg / kg. 32. The method of claim 30, wherein the pharmaceutical composition is administered subcutaneously. An isolated polypeptide comprising a modified LT-β-R extracellular domain having a length of 193 or 194 amino acids and a modified Ig moiety having a length of 227 amino acids, wherein the polypeptide is SEQ ID NO: 21 An isolated polypeptide that has lost only 5 amino acids from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in Figure 5 and lacks the N-terminal pyroglutamic acid. 36. The isolated polypeptide of claim 35, wherein the N-terminal amino acid is a nonpolar amino acid. The non-polar amino acid may be valine (amino acid 6 in the mature form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21) or alanine (amino acid form of the wild type LT-β-R extracellular domain of SEQ ID NO: 21 37. The isolated polypeptide of claim 36, which is any of amino acids 5). 36. The isolated polypeptide of claim 35, produced by expressing in a mammalian cell a nucleic acid molecule comprising a nucleotide sequence encoding the extracellular domain of LTBR set forth in SEQ ID NO: 4. 40. An isolated nucleic acid molecule that encodes the polypeptide of any one of claims 35-39. 40. The isolated nucleic acid molecule of claim 39, wherein the nucleic acid molecule is composed of the nucleotide sequence set forth in SEQ ID NO: 7. 41. A vector comprising the nucleic acid molecule of claim 40. 42. A host cell that expresses the vector of claim 41. 43. The cell of claim 42, wherein the cell is a Chinese hamster ovary (CHO) cell. A process for making a composition comprising a population of lymphotoxin-β receptor (LT-β-R) -Ig-fusion proteins comprising a modified LT-β-R extracellular domain and a modified Ig moiety, comprising: At least 90% of the LT-β-R-Ig-fusion protein has lost only 5 amino acids from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain shown in SEQ ID NO: 21, The process comprises expressing a nucleic acid molecule encoding the LT-β-R-Ig fusion protein shown in SEQ ID NO: 8 in mammalian cells, obtaining the population from the culture supernatant, and optionally Purifying the supernatant, thereby a lymphotoxin-β receptor (LT-Β-R) -Ig-fusion tag comprising a modified LT-β-R extracellular domain and a modified Ig moiety. Obtaining a composition comprising a population of proteins, wherein at least 90% of said LT-β-R-Ig-fusion protein comprises a wild-type LT-β-R moiety as shown in SEQ ID NO: 21. Process that has lost only 5 amino acids from the N-terminus of its mature form. 45. The process of claim 44, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 7. 45. The process of claim 44, wherein the nucleic acid molecule consists of the nucleotide sequence set forth in SEQ ID NO: 7. A method for treating rheumatoid arthritis in a human subject comprising administering to the subject a dose of an LT-β-R-Ig fusion protein, wherein the dose comprises: A method which is sufficient to maintain an average concentration of about 0.14 μg / ml to about 3.5 μg / ml in the serum of the subject. A method for treating rheumatoid arthritis in a human subject comprising administering to the subject a dose of an LT-β-R-Ig fusion protein, wherein the dose comprises: A method that is sufficient to maintain a minimum average concentration of about 0.6 μg / ml in the serum of the subject. 49. The method of claim 48, wherein the LTBR-Ig fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 5. 49. The method of claim 48, wherein the concentration is achieved by administering the LT-β-R-Ig fusion protein at a dose of about 0.01 to about 5 mg / kg once every 7-60 days. A method for treating rheumatoid arthritis in a human subject comprising administering to said subject at a dose of about 0.6-3 mg / kg at most twice every 7-30 days. Administering a R-Ig fusion protein. 52. The method of claim 51 comprising administering a dose of about 0.6-3 mg / kg LT-β-R-Ig fusion protein once every 7-14 days. 52. The method of claim 51, wherein administration is once every 14-30 days. 52. The method of claim 51, wherein administration is once every 28-60 days. 52. The method of claim 51, wherein the administration is once every 7-30 days. A method for treating an autoimmune disorder in a human subject comprising: LT-β- comprising a modified LT-β-R extracellular domain having a length of 193 amino acids or 194 amino acids in the subject. Administering a dose of a pharmaceutical composition comprising a population of R-Ig fusion proteins, wherein at least 90% of the LT-β-R-Ig-fusion protein is shown in SEQ ID NO: 21. Only 5 amino acids have been lost from the N-terminus of the mature form of the wild-type LT-β-R extracellular domain to maintain a minimum average concentration of about 0.6 μg / ml in the serum of the subject Is sufficient for the method. 57. The method of claim 56, wherein the LT-β-R-Ig fusion protein further comprises a modified Ig moiety. 57. The method of claim 56, wherein the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease, or systemic lupus erythematosus (SLE). 57. The method of claim 56, wherein the pharmaceutical composition comprises the amino acid sequence set forth in SEQ ID NO: 5. 60. The method of claim 59, wherein administration is twice a month. 60. The method of claim 59, wherein administration is once a month. 60. The method of claim 59, wherein administration is subcutaneous. 60. The method of claim 59, wherein the dose is about 1 mg / kg. 60. The method of claim 59, wherein the dose is about 3 mg / kg. 60. The method of claim 59, wherein the dose is administered about 1 mg / kg about every 7-20 days. 60. The method of claim 59, wherein the dose is administered about 3 mg / kg about every 14-30 days. 60. The method of claim 59, wherein the dose is administered about 1 mg / kg about every 14 days. 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-β-R-Ig fusion protein. 60. The method according to Item 59. 69. The method of claim 68, wherein the rheumatoid arthritis drug is selected from the group consisting of DMARD, NSAID, and corticosteroid. 69. The method of claim 68, wherein the human is a DMARD poor responder. 69. The method of claim 68, wherein the rheumatoid arthritis drug is a TNF inhibitor. 69. The method of claim 68, wherein the rheumatoid arthritis drug is adalimumab (Humira (R)), etanercept (Enbrel (R)), or infliximab (Remicade (R)). 69. The method of claim 68, wherein LT-β-R-Ig is administered in combination with the rheumatoid arthritis drug. 69. The method of claim 68, wherein the human is evaluated to determine if the response to the rheumatoid arthritis drug is insufficient prior to administration of LT-β-R-Ig. 75. The method of claim 74, wherein the human is determined to have an inadequate response to the rheumatoid arthritis drug, and then the human is administered LT-β-R-Ig. 69. The method of claim 68, wherein the human is asymptomatic for a first expression of rheumatoid arthritis and symptomatic for a second expression of rheumatoid arthritis. 69. The method of claim 68, wherein LT-β-R-Ig is administered instead of the rheumatoid arthritis drug. 69. The method of claim 68, wherein the administration is in combination with a tumor necrosis factor (TNF) inhibitor. 79. The method of claim 78, wherein the TNF inhibitor is adalimumab (Humira®), etanercept (Enbrel®), or infliximab (Remicade®). 69. The method of claim 68, wherein the human is an anti-TNF poor responder. 69. The method of claim 68, wherein the administration is in combination with a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, or a disease modifying anti-rheumatic drug (DMARD). 69. The method of claim 68, wherein administration is in combination with methotrexate. 82. The method of claim 81, wherein the human is a DMARD poor responder.

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