JP2002539828A - FLINT polypeptide analog - Google Patents

Abstract

(57) SUMMARY The present invention relates to polypeptide analogs of FLINT, and polydeoxynucleotides encoding FLINT analogs, and methods of using FLINT analogs and polydeoxynucleotides. The FLINT analogs of the present invention include polypeptides having the amino acid sequence of FLINT and modified by amino acid substitutions at one or more positions, and fragments thereof, and Fc fusions containing FLINT and the FLINT analog. Including.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Numerous tumor necrosis factor receptor proteins (“TNFR proteins”) and their protein homologs have recently been isolated. They have many strong biological effects, and the abnormal activity of these proteins has been implicated in a number of disease states.

[0002] One of these TNFR homologs was reported in July 1998 (Gent
WO98 / 30694 by Z. et al., which binds to protein FAS ligands, thereby inhibiting the activation of another TNFR homolog by a FAS ligand (
US Provisional Application Nos. 60 / 112,577, 60 / 112,933 and 60/11.
3,407, which were filed on December 17, 18 and 22, 1998, respectively, and form a part of the present specification). This novel protein is referred to herein as "FAS ligand inhibitory protein" or "FLINT".

[0003] Excessive activation of FAS by FAS ligands is associated with a number of pathological diseases, such as runaway apoptosis (Nature Medical by Kondo et al.).
ine 3 (4): 409-413 (1997) and J. Inlet by Galle et al.
Exp. Med. 182: 1223-1230 (1995)) and inflammatory diseases caused by neutrophil activation (Nature Medicine by Miwa et al.).
4: 1287 (1988)).

“Runaway apoptosis” is a level of apoptosis that is greater than normal, or that occurs at an inappropriate time. Pathological diseases caused by runaway apoptosis include organ failure (eg, liver, kidney and pancreas). Inflammatory diseases associated with excessive amounts of neutrophil activation include, but are not limited to, sepsis, ARDS, SIRS and MODS.

[0005] Compounds, such as FLINT, which inhibit the binding of FAS to FAS ligands or LIGHT to LTβR and / or TR2 / HVEM, treat diseases or disorders that are mechanistically related to these binding interactions. Can be used to prevent or prevent. However, FLI as a useful therapeutic agent
The potential of NT is at least somewhat improved pharmacological properties (eg, greater potency, longer in vitro half-life and greater affinity for FAS ligands) and / or improved pharmacological properties (Eg, reduced aggregation, reduced absorbency at the surface, increased solubility and ease of formulation).

SUMMARY OF THE INVENTION The present invention relates to FLINT polypeptide analogs, polydeoxynucleotides encoding the FLINT analogs, and methods of using the polypeptide analogs and the polydeoxynucleotides. FLINT disclosed herein
Analogs include polypeptides having an amino acid sequence of FLINT modified at one or more positions by amino acid substitutions, deletions or additions (hereinafter, referred to as “modified FLINT polypeptides”) and fragments thereof (hereinafter, referred to as “modified FLINT polypeptides”). Termed "modified FLINT polypeptide fragments"). The FLINT analogs disclosed herein are believed to have improved properties as compared to FLINT. Its improved properties include, for example, greater potency, longer in vivo half-life, reduced aggregation, reduced absorption at the surface, increased solubility, and improved ease of formulation. Improved efficacy can be assessed by the binding assay described in Example 13, and improved pharmaceutical properties can be assessed by the assay described in Example 10.

[0007] One embodiment of the invention is directed to analogs comprising a modified FLINT polypeptide or a fragment thereof.

[0008] In one embodiment, the modified FLINT polypeptide comprises: a) replacing tryptophan at position 53 with aspartic acid; b) replacing threonine at position 88 with proline; c) replacing alanine at position 107 with serine; Substituting aspartic acid, glutamic acid or threonine with d) replacing isoleucine at position 110 with threonine or glutamic acid; or e) replacing the amino acid sequence of SEQ ID NO. The term includes hydrophobic substitution to hydrophilic substitution.

In another embodiment, the modified FLINT polypeptide comprises: a) replacing alanine at position 2 or 12 with asparagine; b) replacing proline at position 25, 38, 126 or 171 with asparagine. C) replacing arginine at position 35 with asparagine; d) replacing serine at position 37 with asparagine and replacing proline at position 38 with any other naturally occurring amino acid; e) position 166 F) Replace leucine at position 172 with asparagine; g) Replace aspartic acid at position 194 with asparagine; h) Replace threonine at position 114 with asparagine and proline at position 115 With any other naturally occurring amino acid; i) replacing arginine at position 218 with a It contains a novel glycosylation site having the amino acid sequence of SEQ ID NO: 1 that has been modified by substitution with spargin.

In yet another embodiment, the modified FLINT polypeptide comprises replacing a charged or hydrophilic residue with a salt-bridge forming residue, wherein the polypeptide comprises: a B) replacing asparagine at position 63 with tryptophan; b) replacing glycine at position 67 with aspartic acid and replacing alanine at position 94 or glycine at position 95 with tyrosine; c) replacing arginine at position 69 with glutamic acid. D) replacing arginine at position 82 with glutamic acid or threonine; e) replacing alanine at position 94 with tyrosine and replacing glycine at position 95 with aspartic acid; f) replacing phenylalanine at position 96 with glutamine. G) replacing alanine at position 101 with threonine; or h) glycine at position 95 The having the amino acid sequence of the modified SEQ ID NO: 1 by replacing aspartate.

In yet another embodiment, the modified FLINT polypeptide further comprises a charged residue: a) replacing arginine at position 10 with glutamine, asparagine, serine or threonine, provided that the substituted amino acid is asparagine In that case, 12
Alanine at position 13 is optionally replaced with serine or threonine); b) glutamic acid at position 13 is replaced with glutamine, asparagine, serine or threonine (provided that the amino acid to be replaced is asparagine;
Glycine at position 5 is optionally substituted with serine or threonine); c) glutamic acid at position 16 is substituted with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine,
Leucine at position 8 is optionally replaced with serine or threonine); d) Arginine at position 17 is replaced with glutamine, asparagine, serine or threonine (provided that the amino acid to be replaced is asparagine, 19)
E) optionally replacing valine at position 31 with serine or threonine; e) replacing arginine at position 31 with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine;
Cysteine at position 13 is optionally replaced with serine or threonine); f) Arginine at position 34 is replaced with glutamine, asparagine, serine or threonine (provided that when the amino acid to be replaced is asparagine, 36
G) optionally replacing arginine at position 35 with glutamine, asparagine, serine or threonine; h) replacing aspartic acid at position 36 with glutamine, asparagine, serine or threonine. Substitution (however, when the amino acid to be substituted is asparagine,
Optionally, replacing proline at position 38 with serine or threonine); i) replacing arginine at position 143 with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine;
Or optionally replacing cysteine at position 145 with serine or threonine); or j) replacing aspartic acid at position 161 with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine; (Replacement of leucine at position 1 with serine or threonine) to a hydrophilic residue having the amino acid sequence of SEQ ID NO: 1.

Optional substitution of amino acids at the two positions removed from replacing the C-terminal asparagine with serine or threonine results in a novel N-linked glycosylation site motif, ie, NXS / T. . A modified FLINT polypeptide having a novel glycosylation site motif is preferably produced from a recombinant mammalian host cell that expresses the gene encoding the polypeptide, such that the N-glycosylated product is To manufacture. Glycosylation site motifs are described in more detail herein below.

In yet another aspect, the invention relates to modified FLINT polypeptides or fragments thereof, as well as physiologically acceptable salts thereof, wherein the polypeptide comprises: a) positions 2, 12, 107 Alanine at position 179 or 209 is replaced with threonine; b) threonine at position 4 or 162 is replaced with alanine; c) valine at position 1 or isoleucine at position 110 is replaced with methionine; d) 13 Glutamic acid is substituted with aspartic acid; e) arginine at position 17 is replaced with tryptophan; f) alanine at position 75 is replaced with proline; g) serine at position 102 is replaced with leucine; h) position 169 Glycine is replaced with alanine; i) glutamic acid at position 183 is replaced with lysine; j 225-position glutamine substitution with arginine; or l) 270 valine at position the amino acid sequence of SEQ ID NO: 1 that has been modified by substituting glycine; k) 237 position valine are substituted with glutamic acid.

In yet another embodiment, the modified FLINT polypeptide comprises: a) replacing alanine at position 12 with asparagine and optionally replacing glutamic acid at position 13 with glutamine; b) replacing arginine at position 34 with asparagine. Substitution, optionally replacing aspartic acid at position 36 with threonine; c) replacing arginine at position 35 with asparagine and optionally replacing serine at position 37 with threonine; d) replacing serine at position 132 with asparagine And optionally replacing serine at position 134 with threonine; e) replacing aspartic acid at position 194 with asparagine and optionally replacing serine at position 196 with threonine; f) arginine at position 35 and asparagine at position 194. G) replacing the acid at position 12 with asparagine; Replacing with sparagine, optionally replacing glutamic acid at position 13 with glutamine, and replacing aspartic acid at position 194 with asparagine and optionally replacing serine at position 196 with threonine; h) arginine at position 34 with asparagine; Substitution, substituting aspartic acid at position 36 with threonine and substituting aspartic acid at position 194 with asparagine;
Optionally replacing serine at position 196 with threonine; i) replacing arginine at position 35 and aspartic acid at position 194 with asparagine and replacing serine at position 37 and / or 196 with threonine; j) position 218 K) replacing glycine at position 26 with aspartic acid and replacing serine at position 132 with asparagine; or l) replacing alanine at position 12 with asparagine and replacing serine at position 132 with glutamine. It contains a novel glycosylation site having the amino acid sequence of SEQ ID NO: 1, which is modified by substitution with asparagine and substitution of serine at position 134 with threonine.

The present invention includes FLINT polypeptide fragments, and modified fragments thereof that are biologically active in vivo or in vitro. Examples of preferred fragments include a polypeptide consisting of amino acids 1 to 218 of SEQ ID NO: 1 as described above, and modified ones thereof. Also included are the physiologically acceptable salts of the modified FLINT polypeptide, and fragments thereof.

The present invention includes the above polypeptide and a fragment thereof, wherein the polypeptide is modified such that glycine at the position corresponding to position 214 of SEQ ID NO: 1 is replaced with alanine.

Another embodiment of the present invention is a modified FLINT polypeptide, a FLINT fragment, or a peptide derivative comprising the modified FLINT polypeptide fragment (hereinafter referred to as a “FLINT peptide derivative”).

Another aspect of the present invention is a FLINT peptide derivative, a FLINT polypeptide or a FLINT polypeptide fragment, or a modified version thereof,
And a suitable pharmaceutical carrier.

Another embodiment of the present invention relates to a method of inhibiting the binding of a FAS ligand to FAS or LIGHT to LTβR and / or TR2 / HVEM in a subject in need of suppression. The method provides a FLINT that is effective in inhibiting its binding.
Administering to the subject an effective amount of the derivative.

[0020] Another embodiment of the present invention is a method of treating a subject using runaway apoptosis. The method comprises administering to the subject an effective amount of a FLINT peptide derivative.

Another embodiment of the present invention provides a compound of structural formula (I):

Embedded image Is a fusion protein indicated by.

Fc is an antibody (preferably a human antibody, preferably IgG, IgG1, IgG2 or IgG).
G4 is more preferred) or an analog thereof. Fc is F
An analog of the c fragment, wherein about 5 to about 15 amino acids are truncated from the N-terminus of both heavy chains in the Fc fragment.

Each X is independently a modified FLINT polypeptide, a modified FLINT
It is a polypeptide fragment, a FLINT fragment or a FLINT peptide derivative. Preferably each X is the same.

[0024] Each modified FLINT polypeptide, modified FLINT polypeptide fragment, FLINT fragment, or FLI represented by X
The NT peptide derivative is covalently linked at its C-terminus to the N-terminus of one of the heavy chains forming the Fc fragment of the antibody.

In yet another embodiment, a nucleic acid encoding a FLINT peptide derivative of the present invention (
For example, a fragment containing residues 1 to 218 of SEQ ID NO: 1).

[0026] Yet another embodiment of the present invention relates to a FLINT peptide derivative, a modified FLI
NT polypeptide, modified FLINT polypeptide fragment, FLIN
T fragments or polydeoxynucleotides encoding the fusion proteins represented by formula (I).

[0027] Yet another embodiment of the present invention is a polydeoxynucleotide derivative. The polydeoxynucleotide derivative includes one of the above-described polydeoxynucleotides of the present invention.

[0028] Yet another embodiment of the present invention relates to a FLINT peptide derivative, a modified FLI
A vector containing a polydeoxynucleotide encoding an NT polypeptide, a modified FLINT polypeptide fragment, or a fusion protein represented by Formula (I).

Yet another embodiment of the present invention is a host cell containing the vector described in the above paragraph.

Another embodiment of the present invention relates to a modified FLINT polypeptide, a modified FINT polypeptide.
LINT polypeptide fragment, FLINT fragment, or FLIN
The present invention relates to a method for producing a T peptide derivative. For example, the method is described in the preceding paragraph under conditions suitable for expressing a FLINT peptide derivative, a modified FLINT polypeptide or a modified FLINT polypeptide fragment, a polydeoxynucleotide encoding a FLINT fragment. Culturing host cells,
Isolating the obtained protein.

Another embodiment of the present invention is directed to diseases such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pulmonary fibrosis (PF) and chronic obstructive pulmonary disease (COPD).
And / or the use of a FLINT derivative in the treatment and / or suppression of

Another embodiment of the present invention relates to a pharmaceutical composition comprising a FLINT analog or Fc-FLI as an active ingredient.
For a formulation having an NT derivative fusion protein, the formulation may be ALI, ARDS,
Formulations suitable for treating and / or controlling COPD and PF, and other diseases that can be treated by FLINT.

Another aspect of the invention involves the use of a FLINT analog in the manufacture of a medicament useful for treating and / or suppressing ALI, ARDS, COPD and PF.

The present invention also relates to methods of treating and / or inhibiting ischemic damage during organ transplantation (including the use of FLINT analogs).

The present invention includes a method of inhibiting and / or treating an individual suffering from ALI, the method comprising administering to the individual a therapeutic amount of a FLINT analog. The invention also relates to a method of treating or inhibiting ARDS in an individual, the method comprising administering to the individual a therapeutic amount of a FLINT analog. In addition, the invention includes a method of treating or suppressing COPD in an individual, the method comprising administering to the individual a therapeutically effective amount of a FLINT analog. In addition, the invention relates to a method of treating or inhibiting pulmonary fibrosis in an individual, the method comprising administering to the individual a therapeutic amount of a FLINT analog.

In another embodiment, the present invention provides a FLI for inhibiting activation of T lymphocytes.
Regarding the use of NT analog.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term “FLINT analog” or “analog”
LINT polypeptide derivatives are meant, including modified FLINT polypeptides, modified FLINT polypeptide fragments and FLINT fragments.

The term “FLINT peptide derivative” or “FLINT derivative” refers to a modified FLINT polypeptide, a FLINT fragment or a modified FLI.
An NT polypeptide fragment is meant or included.

The term “altered FLINT polypeptide” refers to a polypeptide having the amino acid sequence of FLINT that has been altered at one or more positions by amino acid substitutions, deletions, and / or additions.

The term “altered FLINT fragment” refers to a biological activity (eg,
FLIN that retains the ability to bind asL or the ability to bind LIGHT
Means a fragment of T. Preferred fragments are 1-21 of SEQ ID NO: 1.
Contains 8 amino acid residues.

As used herein, the term “FLINT” refers to native FLINT (SEQ ID NO: 3)
) And mature FLINT (SEQ ID NO: 1).

SEQ ID NO: 1 is mature human FLINT (ie, native FLINT minus leader sequence).

SEQ ID NO: 2 is a nucleic acid / cDNA encoding mature human FLINT.

[0044] SEQ ID NO: 3 is natural human FLINT.

[0045] SEQ ID NO: 4 is a human FLINT leader sequence.

One embodiment of the present invention relates to fragments and modified fragments of FLINT, which preferably retain biological activity. Biological activity can be determined by in vitro and / or in vivo effects (eg, F
The ability of a LINT analog to bind FasL and / or the ability to bind LIGHT). Alternatively, the biological activity of FLINT indicates that when administered to a mammal in need of treatment (including humans) in an effective amount, the FLINT analog or fragment thereof will treat a disease or disorder in a mammal, or It may be related to the ability to prevent. Fragments may include certain small regions of the FLINT molecule. For example, sub-region D1 relates to amino acid residues 1-42 of SEQ ID NO: 1, sub-region D2 relates to amino acid residues 43-85 of SEQ ID NO: 1, and sub-region D3 corresponds to 86-amino acid residues of SEQ ID NO: 1. 122 amino acid residues, and D4 corresponds to 1 of SEQ ID NO: 1.
Related to amino acid residues 23-165. A functional FLINT fragment is
Includes one or more of domains D1-D4. The FLINT fragment is in domain D
1 to D4, domain D2 and D3, domain D
Preferably, it contains 2 to D4, contains domains D3 and D4, or contains domain D4.

Preferred FLINT polypeptide fragments are amino acid residues 1 to 218 of SEQ ID NO: 1 (referred to herein as “FLINT metabolites”), or residues 1 to 216 of SEQ ID NO: 1. Consists of The FLINT fragment and preferred FLINT fragments can include a leader sequence (eg, SEQ ID NO: 4).

[0048] FLINT metabolites and other FLINT fragments include FasL and L
Combines with IGHT. LIGHT, a new member of the TNF family, is a membrane-bound ligand that triggers a distinct biological response. L
IGHT can function in immune modulation, which is thought to be involved in herpes virus entry (Jhai et al., Zhai et al.).
Clin. Invest. 102, 1142-1151 (1998) and M
ontgomery et al., Cell, 87, 427-436, 1996). Lytic LIGHT inhibits the growth of many tumor cells and is thought to be associated with the receptors LTβR and TR2, which is also called the herpes virus entry mediator (HVEM). LIGHT is highly expressed on activated lymphocytes and causes immune modulation from hematopoietic cells. For example,
LIGHT stimulates the secretion of INFγ. LIGHT also provides LTβR and T
It also induces apoptosis of tumor cells that express the R2 / HVEM receptor. LIG
HT cytotoxicity is increased by IFNγ, which can be blocked by the addition of soluble LTβR-Fc or TR2 / HVEM-Fc. LIHGH
T is mainly produced by activated T lymphocytes. When LIGHT binds to HVEM on the surface of T cells, it stimulates T cell proliferation (JA Harrop).
J. et al. Biol. Chem. , 273, 27548-27556, 199
8).

The present invention provides FLINT analogs and / or FLINT metabolites or other FLINs for binding to LIGHT and thereby inhibiting T cell activation.
Further relates to the use of T fragments. The activation of T cells can be chronically inhibited, which is advantageous, for example, in treating autoimmune diseases after organ transplantation and in preventing rejection in systemic inflammatory responses.

FLINT fragments (eg, FLINT metabolites, including modified derivatives thereof) are useful for binding LIGHT and for modulating the effect of LIGHT on immune responses and apoptosis. It is. Therefore,
In another embodiment, a FLINT analog (including a FLINT metabolite or a modified version thereof) is associated with LIGHT and is used to inhibit apoptosis and / or immune modulation.

The FLINT fragments of the present invention can be produced by in vivo or in vitro recombinant methods, or by direct chemical synthesis methods.
FLINT metabolites can be produced in vivo by exogenous administration to mammals. The FLINT fragment is known to one of skill in the art by any of a number of suitable methods, including, for example, chemical synthesis of any portion of SEQ ID NO: 1 or SEQ ID NO: 3, or proteolytic digestion of FLINT. It can be produced by DNA mutagenesis. For example, K. S
truhl's "Reversese biochemistry: Metho"
ds and applications for synthesizing
yeast proteins in vitro ", Meth. Enzymo
l. See 194, 520-535. In a preferred method of recombination, a group of deletion mutations is introduced into the gene or cDNA encoding FLINT (eg, SEQ ID NO: 3) so that varying amounts of the coding region can be derived from either the amino or carboxy terminus of the protein molecule. To be lost. This method can also be used to generate internal fragments of FLINT with the carboxy and amino termini removed. Some suitable nucleases may be replaced by their deletion (eg,
Bal31), or, in the case of single-stranded nucleic acid molecules, mung bean nuclease. For simplicity, FLIN
Preferably, the encoding nucleic acid encoding T is cloned in a single-stranded cloning vector (eg, bacterial phage, M13 or equivalent). If desired, the resulting deletion fragments can be subcloned in any suitable vector for propagation and expression in any suitable host cell.

In a preferred embodiment for in vitro production of FLINT metabolites,
FLINT is treated with a serine protease (eg, thrombin or trypsin) to cleave between the Arg residue at position 218 and the Ala residue at position 219 of SEQ ID NO: 1.

Production of FLINT Metabolites and Protease-Resistant Analogs FLINT undergoes proteolysis in vitro to yield at least two large peptide fragments. One of the fragments consists of residues 1-218 of SEQ ID NO: 1 (or residues 1-247 of SEQ ID NO: 3) (
This is referred to herein as the "FLINT metabolite"), while the other
It consists of residues 19-271 (or residues 248-300 of SEQ ID NO: 3). Cleavage at position 218 in vitro can be achieved by treating native FLINT (SEQ ID NO: 3) or mature FLINT (SEQ ID NO: 1) with a trypsin-like enzyme (eg, thrombin, trypsin or other serine protease). it can. Thus, serine proteases appear to be involved in the hydrolysis of FLINT in vivo.

The term “negatively charged group” or “negatively charged amino acid” means Asp or Glu.

The term “positively charged group” or “positively charged amino acid” refers to His, Ar
means g or Lys.

The term “polar uncharged” or “polar uncharged amino acid” refers to Cys, Th
r, Ser, Gly, Asn, Gln or Tyr.

The term “non-polar” or “non-polar amino acid” refers to Ala, Pro, Met, L
eu, Ile, Val, Phe or Trp.

The term “naturally occurring amino acid” refers to any of the 20 L-amino acids found in proteins.

The term “natural FLINT” refers to SEQ ID NO: 3.

The term “mature FLINT” refers to SEQ ID NO: 1.

The term “FLINT” means natural and mature FLINT from humans, other primates, other mammals and non-mammalian sources.

As used herein, the term “half-life”, as determined by any suitable method, means that about half of FLINT or a FLINT analog is in vitro and / or in vivo at positions 218 and 219 of SEQ ID NO: 1. Means the time required for cleavage by proteolysis at the position. The term is used as a measure of exposure to full-chain FLINT or a FLINT analog (ie, not proteolytic cleavage).

The term “protease resistant” or “resistant” refers to FLINT or FL
A FLINT analog is meant that is more resistant to proteolysis of the residues between positions 218 and 219 of SEQ ID NO: 1 when compared to the INT fragment. Protease-resistant analogs may have one or more amino acid substitutions, deletions, inversions, additions, and / or altered glycosyl sites or patterns relative to or relative to native FLINT, mature FLINT or other FLINT fragments. Is distinguished from FLINT. Preferably, these changes occur in the region of SEQ ID NO: 1 at about positions 214-222.

The term “protease resistant” intends the degree of resistance to proteolysis at position 218 (from complete resistance to partial resistance). Thus, "substantially resistant" analogs exhibit some resistance to proteolysis at position 218, such as when treated with an appropriate protease or exposed to the native FLINT Analogs having a half-life at least about 25% greater than Preferably, a substantially resistant FLINT analog has a half-life resistance that is at least about 2-fold greater than native FLINT.

The susceptibility to proteolysis depends on factors such as the amino acid sequence at or near the site of recognition by certain proteolytic enzymes involved, and the physical and chemical environment in which the protein of the sample is located. . These factors can influence the K _M and / or the rate of protein hydrolysis by proteolytic enzymes. The sequence recognized by thrombin is LVPR /. Other sequences recognized by thrombin are, for example, VDPR /. Other arrays (
For example, VDPR / and others) are also recognized by thrombin. The density of the charge and the steric nature of the enzyme engineered at the active site will determine the extent to which protein hydrolysis occurs. All of these embodiments are intended to be within the scope of the present invention.

As described herein, protease resistance refers to the susceptibility of a FLINT analog to proteolysis at position 218 in vivo or in vitro. For example, the resistance of the analog to a trypsin-like protease (eg, thrombin or trypsin) or other serine protease is compared to the resistance exhibited by FLINT under the same conditions. FLINT analog is F
It preferably exhibits a half-life at least about 5% greater than LINT, or at least 10%, 20%, 30%, 40% or 50% greater than wild-type FLINT.
It preferably has a half-life that is greater by １００100%. It depends on the relative abundance of the full-chain molecule relative to the smaller digestion products (e.g., the 1-218 and 219-271 fragments of SEQ ID NO: 1). Any method suitable for making a qualitative and / or quantitative assessment of the relative amounts (eg, polyacrylamide gel electrophoresis) can be used. Most preferably, the resistant analog has a half-life from about 1 to about 2 times FLINT to about 100 times or more. Applicants have discovered that the FLINT polypeptide is cleaved in vitro between an arginine residue at position 218 and an alanine residue at position 219 of SEQ ID NO: 1, probably by a trypsin-like protease. The product of cleavage of this reaction contains residues 1-218 of SEQ ID NO: 1 (referred to as "FLINT metabolite"). FLINT metabolites can be produced by treating the FLINT polypeptide with a trypsin-like protease (eg, thrombin, trypsin) or other serine protease.

One aspect of the present invention is a method for producing an analog of a FLINT polypeptide that is resistant to proteolysis between positions 218 and 219 of SEQ ID NO: 1 and that retains biological activity. About. Biological activity relates to the ability of the analog to bind to FasL and / or LIGHT, which may include inhibiting apoptosis in vivo and / or in vitro.

Another aspect of the present invention relates to an analog of the FLINT polypeptide that is resistant to proteolysis between positions 218 and 219 of SEQ ID NO: 1 and that retains biological activity. . Biological activity relates to the ability of the analog to bind FaSL and / or LIGHT, which can include inhibiting apoptosis in vivo and / or in vitro.

Preferred FLINT analogs are at least 5%, 10% more than FLINT
It provides a half-life that is greater by 20%, 30%, 40%, or 50% to 100%. This depends on the ratio of the full-length FLINT to the digestion product, which contains the FLINT metabolite and carboxy fragment (ie, residues 219-271 of SEQ ID NO: 1) over time. Most preferably, the FLINT analog has a half-life of at least 2 to 100 times that of FLINT.

A FLINT analog may have one or more primary or secondary structural changes (eg,
Amino acid substitutions, deletions, inversions, additions) or alterations in glycosylation sites or patterns and / or combinations thereof that prevent or reduce proteolysis between positions 218 and 219 of SEQ ID NO: 1. , And / or ratios thereof. These changes can be attributed to thrombin-like recognition sequences (eg, FLI
Or in the vicinity of NTPR), and most preferably at or near the PR dipeptide sequence at positions 217 and 218 of SEQ ID NO: 1. As will be appreciated by those skilled in the art, residues at or near the recognition site may also alter the charge environment of the activation site and / or cause alterations in the region of the activation site due to steric hindrance. Can affect the sensitivity of the substrate protein to proteolysis.

Accordingly, the present invention contemplates FLINT analogs that include amino acid changes in FLINT (preferably the region from about position 214-222 of SEQ ID NO: 1 or a region comparable to that of SEQ ID NO: 3). Here, the analog is resistant to proteolysis at position 218 of SEQ ID NO: 1.

Protease resistance containing substitutions, deletions, insertions, inversions, additions, or changes in glycosylation sites or patterns occurring outside of a preferred window containing the residues at SEQ ID NOs: 214-222 Consider a FLINT analog.
As the skilled artisan will appreciate, numerous substitutions and / or other changes in the sequence or structure of a protein can occur without substantially affecting the biological activity of the protein. For example, substituting a conserved amino acid for one amino acid from another amino acid of the same class of amino acids (eg, a negatively charged residue, a positively charged residue, a polar uncharged residue, and a nonpolar (Residues, or any other class accepted in the art) often has no functional effect. Those changes are within the scope of the present invention.

In one embodiment, one amino acid change is made in this region, or at least two changes are made in this region, or at least three changes are made in this region, or at least four changes are made in this region. Perform in the area.

In one embodiment, the present invention relates to FLINT analog polypeptides and nucleic acids as defined in terms of percent identity similarity for SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3. Sequence identity refers to the comparison of two molecules using standard algorithms well known in the art. Although any suitable sequence comparison algorithm can be used for this purpose, for example, the present embodiment often uses SEQ ID NO: 1 as a reference sequence to define percent identity to a comparator sequence. Known Smith-Waterman (S
mit-Waterman) algorithm. Where sequence identity is used for polypeptides, the entire polypeptide or certain sub-regions thereof may be used for comparison.

The choice of parameter values for matches, mismatches and insertions or deletions is arbitrary. A preferred set of values for use in the Smith-Waterman algorithm is described in the "maximum similarity segments" method, in which a value of 1 for matched residues and-／ for mismatched residues. (Waterman, Bulletin of
Mathematical Biology, 46, 473-500, 19
84). The X of insertions and deletions is weighted as X _k = 1 + k / 3, where k is the number of residues in an insertion or deletion.

For example, a comparator sequence having 20 substitutions and 3 insertions compared to a reference protein sequence of 250 residues is: [(1 × 250) − ((× 20) − (1 + 3/3 )] / 250 = 96% identical.

The FLINT protease resistant analogs of the present invention can be readily tested for biological activity and / or susceptibility to protein hydrolysis, as described herein. Biological activity can be assessed using the in vitro (see Examples 5 and 8) or in vivo (see Example 11) models described herein.

In another embodiment, the present invention relates to a FLINT analog containing one or more amino acid substitutions in the region from position 214 to 222 of SEQ ID NO: 1 and / or the region from position 243 to 251 of SEQ ID NO: 3.

In another embodiment, the present invention relates to a FLINT analog containing an amino acid substitution in the region containing amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Replacing Gly at position 214 with a naturally occurring amino acid other than Gly; b. Replacing Pro at position 215 with a naturally occurring amino acid other than Pro; c. Replacing Thr at position 216 with a naturally occurring amino acid other than Thr; d. Replacing Pro at position 217 with a naturally occurring amino acid other than Pro; e. Replacing Arg at position 218 with a naturally occurring amino acid other than Arg; f. Replacing Ala at position 219 with a naturally occurring amino acid other than Ala; g. Replacing Gly at position 220 with a naturally occurring amino acid other than Gly; h. Replacing Arg at position 219 with a naturally occurring amino acid other than Arg; i. It is selected from the group consisting of substituting Ala at position 222 with a naturally occurring amino acid other than Ala.

In another embodiment, the present invention relates to a FLINT analog containing an amino acid substitution in the region containing amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Replacing Gly at position 214 with a non-Gly positively charged amino acid; b. Replacing Pro at position 215 with a positively charged amino acid that is not Pro; c. Replacing Thr at position 216 with a non-Thr positively charged amino acid; d. Replacing Pro at position 217 with a positively charged amino acid that is not Pro; e. Replacing Arg at position 218 with a positively charged amino acid that is not Arg; f. Replacing Ala at position 219 with a non-Ala positively charged amino acid; g. Replacing Gly at position 220 with a non-Gly positively charged amino acid; h. Replacing Arg at position 221 with a positively charged amino acid that is not Arg; i. Substituting Ala at position 222 with a positively charged amino acid that is not Ala;

In another embodiment, the present invention is directed to a FLINT analog comprising an amino acid substitution in the region comprising amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Replacing Gly at position 214 with a non-Gly negatively charged amino acid; b. Replacing Pro at position 215 with a non-Pro negatively charged amino acid; c. Replacing Thr at position 216 with a non-Thr negatively charged amino acid; d. Pro at position 217 is replaced by a non-Pro negatively charged amino acid. e. Replacing Arg at position 218 with a non-Arg negatively charged amino acid; f. Replacing Ala at position 219 with a non-Ala negatively charged amino acid; g. Replacing Gly at position 220 with a non-GLy negatively charged amine acid; h. Replacing Arg at position 221 with a non-Arg negatively charged amino acid; i. Selected from the group consisting of substituting Ala at position 222 with a negatively charged amino acid that is not Ala.

In another embodiment, the present invention relates to a FLINT analog comprising a substitution of an amino acid in the region containing amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Replacing Gly at position 214 with a non-Gly polar, uncharged amino acid; b. Replacing Pro at position 215 with a non-Pro polar, uncharged amino acid; c. Replacing Thr at position 216 with a non-Thr polar, uncharged amino acid; d. Replacing Pro at position 217 by a non-Pro polar, uncharged amino acid; e. Replacing Arg at position 218 with a non-Arg non-polar, uncharged amino acid; f. Replacing Ala at position 219 by a non-Ala polar, uncharged amino acid; g. Replacing Gly at position 220 with a non-Gly polar, uncharged amino acid; h. Replacing Arg at position 221 with a non-Arg non-polar, uncharged amino acid; i. It is selected from the group consisting of substituting Ala at position 222 with a non-Ala polar and uncharged amino acid.

In another embodiment, the present invention is directed to a FLINT analog comprising a substitution of an amino acid in the region containing amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Replacing Gly at position 214 with a non-polar amino acid that is not Gly; b. Replacing Pro at position 215 with a non-polar amino acid that is not Pro; c. Replacing Thr at position 216 with a non-polar amino acid that is not Thr; d. Replacing Pro at position 217 with a non-polar amino acid that is not Pro; e. Replacing Arg at position 218 with a non-polar amino acid that is not Arg; f. Replacing Ala at position 219 with a non-polar amino acid that is not Ala; g. Replacing Gly at position 220 with a non-polar amino acid that is not Gly; h. Replacing Arg at position 221 with a non-polar amino acid that is not Arg; i. Selected from the group consisting of substituting Ala at position 222 with a non-polar amino acid that is not Ala.

In another embodiment, the present invention relates to a FLINT analog containing an amino acid substitution in the region containing amino acids 214-222 of SEQ ID NO: 1, wherein the amino acid substitution comprises: a. Arg at position 218 is replaced by Gly; b. Arg at position 218 is replaced by Glu; c. Thr at position 216 is replaced by Pro; d. Arg at position 218 is replaced by Ala; e. Arg at position 218 is replaced by Gly; f. Arg at position 218 is replaced by Ser; g. Arg at position 218 is replaced by Val; h. Arg at position 218 is replaced by Tyr; i. Replacing Pro at position 217 with Tyr; j. Thr at position 216 is replaced by Pro, and Arg at position 218 is replaced with G
ln is selected from the group consisting of:

In another embodiment, the present invention relates to a FLINT analog containing SEQ ID NO: 1, wherein Arg at position 34 is replaced by Asn and Asp at position 36 is replaced by Thr. And Arg at position 218 is replaced with Gln
, Glu, Ala, Gly, Ser, Val or Thr.

In another embodiment, the present invention relates to a FLINT analog containing SEQ ID NO: 1, wherein Arg at position 34 is replaced by Asn and Asp at position 36 is replaced by Thr. Replacing Asp at position 194 with Asn and replacing Arg at position 218 with Gln, Glu, Ala, Gly, Ser.
, Val or Thr.

In another embodiment, the present invention relates to a FLINT analog containing one or more amino acid substitutions in SEQ ID NO: 1, wherein the Arg at position 34 in SEQ ID NO: 1 is replaced by Asn. Replaces Asp at position 36 with Thr, 2
Arg at position 18 a. A naturally occurring amino acid that is not Arg; b. A non-Arg positively charged amino acid; c. A non-Arg negatively charged amino acid; d. A polar, uncharged amino acid that is not Arg; e. A non-polar amino acid that is not Arg; and f. Substitution with an amino acid selected from the group consisting of Glu, Gln, Ala, Gly, Ser, Val or Tyr.

In another embodiment, the present invention is directed to a FLINT analog comprising one or more amino acid substitutions in SEQ ID NO: 1, wherein the Arg at position 34 in SEQ ID NO: 1 is replaced by Asn. Replaces Asp at position 36 with Thr,
Replace Asp at position 94 with Asn, replace Ser at position 196 with Thr, and replace Arg at position 218 with a. A naturally occurring amino acid that is not Arg; b. A non-Arg positively charged amino acid; c. A non-Arg negatively charged amino acid; d. A polar, uncharged amino acid that is not Arg; e. A non-polar amino acid that is not Arg; and f. Substitution with an amino acid selected from the group consisting of Glu, Gln, Ala, Gly, Ser, Val and Thr.

In another embodiment, the present invention relates to a FLINT analog containing one or more amino acid substitutions in SEQ ID NO: 1, wherein the Ser at position 132 in SEQ ID NO: 1 is replaced by Asn, Arg at position 218 a. A naturally occurring amino acid that is not Arg; b. A non-Arg positively charged amino acid; c. A non-Arg negatively charged amino acid; d. Non-Agr polar and uncharged amino acids; e. A non-polar amino acid that is not Arg; and f. Substitution with an amino acid selected from the group consisting of Glu, Gln, Ala, Gly, Ser, Val and Thr.

A FLINT peptide derivative comprises a modified FLINT polypeptide, a modified FLINT polypeptide fragment, or a FLINT fragment and one or more other portions. Examples of FLINT peptide derivatives are shown below.

One example of a FLINT peptide derivative is an oligonucleotide or amino acid with N
A modified FLINT polypeptide or a fragment thereof, added to the -terminus and / or C-terminus. An “oligopeptide” is a chain of 2 to about 20 amino acids joined by peptide bonds at the N- and C-termini. Suitable oligopeptides and amino acids do not significantly reduce the binding of FLINT to the FAS ligand, do not significantly impair the pharmaceutical and pharmacological properties of FLINT, and significantly increase the in vivo half-life of FLINT. It does not decrease. For example, leader sequences found in native FLINT (eg,

Embedded image (SEQ ID NO: 4).

The FLINT peptide derivative has one or more polyethylene glycol groups (hereinafter, referred to as a polyethylene glycol group).
Modified or unmodified FLINT peptides and fragments thereof (called "PEG" groups). The PEG group is a modified FL
It can be linked to an amino or thiol group at the N-terminus or in the side chain of an amino acid in an INT polypeptide or fragment thereof. Suitable PEG
The groups generally have a molecular weight of from 5000 to 20,000 atomic mass units. P
For methods for producing EGylated polypeptides, see Mmutaz and Bachha.
Indian Journal of Biochemistry by wat
and Biophyscs, 28; 346; (1991) and Francc.
International Journal of Hem by iset et al.
atomology 68, 1 (1988) (these constitute a part of the present specification).
Is disclosed.

Yet another example of a FLINT peptide derivative is two or more modified or unmodified FLINT polypeptides, or two or more fragments thereof (eg, a dimerized modified FLINT polypeptide or (A dimerized modified FLINT polypeptide fragment). The dimerization is performed by the PEG polymer chain method (Journal of Sur by Espat et al.).
gitical Research 59: 153 (1995)),
Or domains (eg, O'Shera et al., Science, 254; 5).
39 (1991)) can be achieved by C-terminal fusion combined with another dimerization to induce. Espat and O'Shea
Is a part of the present specification. As another example, a modified FLINT polypeptide dimer, a dimer of a modified FLINT polypeptide, by replacing an amino acid residue in a protein with a cysteine followed by forming an intermolecular disulfide bond. Alternatively, a dimer of a FLINT polypeptide derivative is obtained. Dimerized polypeptides can be obtained, for example, by replacing serine at position 116 or glutamine at position 122 in a modified FLINT polypeptide with cysteine, followed by formation of an intermolecular disulfide bond.

In yet another example, the FLINT peptide derivative is a fusion protein comprising a modified FLINT polypeptide or a fragment thereof fused to another protein or glycosylated on a domain of another protein. It is. A "fusion protein" is a non-naturally occurring hybrid protein molecule containing a translational or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked to a single polypeptide chain. means. The human serum albumin and the C-terminal domain of thrombopoietin are examples of proteins that can be fused with a modified FLINT or modified FLINT fragment. For an example of producing a fusion protein, see European Patent 3
94,827, Nature, 331: 84 (1) by Tranecker et al.
988) and Fares et al., Proc. Natl. Acad. Sci. U
SA 89: 4304 (1992), which are incorporated herein by reference.

An “Fc fragment” of an antibody, as used herein, has the meaning commonly given to that term in the field of immunology. Specifically, the term refers to an antibody fragment obtained by removing the region (Fab fragment) that binds complement and binds two antigens from the antibody. Thus, Fc fragments are generated from nearly equal sized fragments from both heavy chains, both associated by non-covalent interactions and disulfide bonds. Its Fc fragment comprises a hinge region, ranging from _{C H} 2 and _{C H} 3 domain to the C-terminus of the antibody.

[0096] The modified FLINT polypeptides, modified FLINT polypeptide fragments and FLINT peptide derivatives of the present invention may or may not be glycosylated. Glycosylated polypeptides are modified with one or more monosaccharides or oligosaccharides. Monosaccharides are chiral polyhydroxyalkanols or polyhydroxyalkanones, typically present in the form of a hemiacetal. "Oligosaccharides" refer to about 2 usually linked by acetal bonds.
A polymer consisting of up to about 10 monosaccharides. One type of glycosyl group commonly found in glycosylated proteins is N-acetylneuraminic acid. The glycosylated polypeptide is N-glycosylated and / or
Alternatively, it is O-glycosylated, but preferably N-glycosylated.

The term “suppress” or “suppressing” includes its generally accepted meaning, which prevents, prevents, inhibits, slows, halts the progression or severity of a disease or condition. Or including retracting.

The term “N-glycosylated polypeptide” refers to one or more NXS / Ts in which the N atom in the amide of the side chain of asparagine is covalently linked to a glycosyl group.
It means a polypeptide having a motif. "X" means a naturally occurring amino acid residue other than proline. `` Naturally occurring amino acids '' include glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, cysteine, methionine, lysine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, phenylalanine, histidine, tyrosine and tryptophan. It is. The N-glycosylated protein may optionally be
-It may be glycosylated.

The term “O-glycosylated polypeptide” refers to a polypeptide having one or more serine and / or threonine in which a side chain oxygen atom is covalently linked to a glycosyl group. The O-glycosylated protein is optionally
It may be N-glycosylated.

As used herein, the term “treatment” or “treating” means managing and caring for a patient for the purpose of combating a disease, illness or disorder, and treating the occurrence of symptoms or complications. Administering FLINT to prevent, thereby reducing its symptoms or complications.

Preferably, the polypeptide of the present invention is N-glycosylated.

Glycosylated polypeptides can be produced recombinantly by expressing the polypeptide-encoding gene in a suitable mammalian host cell, and the available NXT / This results in glycosylation of the amides of the side chains found in the S motif, and glycosylation of the alcohols in the side chains of available serine or threonine on the surface. A specific method for recombinantly expressing a gene in a mammalian cell is described below. For other methods of producing glycosylated proteins, see EP 640,619 (Elliot and Burn) (
This is incorporated herein by reference. Non-glycosylated polypeptides can be produced recombinantly by expressing the gene encoding the polypeptide in a suitable prokaryotic host cell.

Other FLINT Analogs In another embodiment, the invention relates to FLINs which are expected to alter the properties of the molecule (eg, increase half-life, decrease aggregation, or increase the affinity of binding to FasL).
Regarding the analog of T. To this end, a computer generated model of the FLINT 3-D structure was designed as described in Example 14.

In one class of FLINT analogs, charged residues are replaced by hydrophilic residues. Based on homology and computer models, a number of charged amino acid residues were found to be exposed on the surface of FLINT. In addition, these models suggest that these charged amino acid residues may be associated with the FAS ligand binding FAS ligand.
It is distant from the site of LINT and is therefore thought to contribute minimally to the binding affinity with the FLINT / FAS ligand. Because of their large number of charged amino acid residues on the surface of FLINT, the net charge of FLINT in solution is
It shows that H changes greatly when it changes. By substituting these charged amino acid residues on the surface of FLINT with hydrophilic residues, the gradient of these charges is reduced so that while retaining biological activity, improved pharmaceutical properties FL indicating
Get the INT analog. Examples of improved pharmaceutical properties include reduced aggregation, reduced absorption at the surface, increased solubility, and improved ease of formulation.

In another embodiment, based on homology and computer models, a number of hydrophobic amino acid residues were found to be exposed on the surface of FLINT.
These models show that FLINT binds FAS ligands to hydrophobic amino acid residues.
, Indicating that it minimally contributes to the binding affinity with the FLINT / FAS ligand. Biologically active FLINT analogs with improved pharmaceutical properties can be obtained by replacing these hydrophobic amino acid residues with uncharged, hydrophilic residues. Examples of improved pharmaceutical properties include reduced aggregation, reduced absorption at the surface, increased solubility, and improved ease of formulation.

In another embodiment, based on computational and homology studies, certain amino acid residues in FLINT may interact with specific charged or hydrophilic amino acid residues in FAS ligands. Do you get it. FLINT analogs with improved binding affinity and consequently greater potency can form these amino acids in FLINT into salt bridges or hydrogen bonds with these particular charged or hydrophilic amino acids in FAS ligands It can be obtained by substitution with an amino acid. These computer studies and homology studies have shown that if some other amino acid residue in FLINT is replaced with an amino acid residue having a sterically large side chain, these newly generated or already existing It also shows that some salt bridges or hydrogen bonds can block exposure to solution. By blocking the exposure of these salt bridges or hydrogen bonds to solution, the affinity of these FLINT analogs for FAS ligands can be further increased.

The present invention provides a FLINT peptide derivative, a modified FLINT polypeptide,
It also relates to a modified FLINT polypeptide fragment or a polydeoxynucleotide encoding a FLINT fragment. The present invention also includes a FLINT peptide derivative or a fragment of a polydeoxynucleotide that encodes a modified FLINT polypeptide.

As used herein for nucleic acids, the present invention includes degenerates encoding the same amino acid or the same amino acid sequence and is intended to illustrate the degree of degeneracy of the genetic code. One skilled in the art will recognize that, for example, the codon of aspartic acid may be GAT (RN
It will be readily understood that either GAU for A or GAC can be taken.

The present invention is also intended to include sequences closely related to the sequences disclosed herein, or that encode proteins having similar or identical biological activities. For example, the invention relates to sequences defined for percent identity similarity to SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3. Sequence identity between two molecules can be determined using any standard algorithm well known in the art. For example, consider the well-known Smith-Waterman algorithm when comparing SEQ ID NO: 1 with a comparator sequence. Where sequence identity is used for polypeptides, the entire polypeptide or certain sub-regions thereof may be used for comparison.

The choice of parameter values for matches, mismatches and insertions or deletions is arbitrary. A preferred set of values for use in the Smith-Waterman algorithm is described in the "maximum similarity segments" method, in which a value of 1 for matched residues and-／ for mismatched residues. (Waterman, Bulletin of
Mathematical Biology, 46, 473-500, 19
84). The X of insertions and deletions is weighted as X _k = 1 + k / 3, where k is the number of residues in an insertion or deletion.

For example, a comparator sequence with 20 substitutions and 3 insertions compared to a reference protein sequence of 250 residues would be: [(1 × 250) − (×× 20) − (1 + 3/3 )] / 250 = 96% identical.

The FLINT protease resistant analogs of the present invention can be used as described herein
It can be easily tested for biological activity and / or susceptibility to proteolysis. Biological activity can be assessed using the in vitro models described herein (see Examples 5 and 8) or in vivo models (see Example 11).

In one preferred embodiment, the polydeoxynucleotide has the following modifications: a) replacing the codon encoding tryptophan at positions 157-159 with a codon encoding aspartic acid; b) threonine at positions 262-264. Is replaced with a codon that encodes proline; c) A codon that encodes alanine at positions 319-321 is replaced with a codon that encodes serine, aspartic acid, glutamic acid, or threonine; d) 328-330. Replacing the codon encoding isoleucine at position with a codon encoding threonine or glutamic acid; or e) replacing the codon encoding proline at positions 310-312 with a codon encoding serine. Deoxynucleotide sequence Having.

In another preferred embodiment, the polydeoxynucleotide is modified as follows: a) replacing the codon encoding alanine at positions 4-6 or 34-36 with a codon encoding asparagine; b) 73 Replacing the codon encoding proline at positions -75 with a codon encoding asparagine; c) replacing the codon encoding arginine at positions 103-105 with a codon encoding asparagine; d) replacing codons encoding asparagine; Replacing the codon encoding serine with a codon encoding asparagine, and replacing the codon encoding proline at positions 112-114 with an amino acid encoding any of the other naturally occurring amino acids; e) 112 The codon encoding for proline at positions 114 through 114 is replaced with the one encoding asparagine. F) replacing the codon encoding proline at positions 376-378 with a codon encoding asparagine; g) replacing the codon encoding serine at positions 496-498 with a codon encoding asparagine. H) replacing the codon encoding proline at positions 511-513 with a codon encoding asparagine; i) replacing the codon encoding leucine at positions 514-516 with a codon encoding asparagine; j) 580 Replacing the codon encoding aspartic acid at positions ５８582 with a codon encoding asparagine; k) replacing the codon encoding threonine at positions 340-342 with a codon encoding asparagine, and 343-345. The codon encoding proline is replaced by a naturally occurring amino acid Or 1) replacing the codon encoding arginine at positions 652-654 with a codon encoding asparagine; or 1) replacing the codon encoding arginine at positions 652-654 with a codon encoding asparagine.

In another preferred embodiment, the polydeoxynucleotide has the following modifications: a) the codon encoding asparagine at positions 187-189 is replaced with a codon encoding tryptophan; b) glycine at positions 199-201 Is replaced by a codon encoding asparagine, and a codon encoding alanine at positions 280-282 or a codon encoding glycine at positions 283-285 is substituted with a codon encoding tyrosine; c) The codon encoding arginine at positions 205-207 is replaced with a codon encoding glutamic acid; d) The codon encoding arginine at positions 244-246 is replaced with a codon encoding glutamic acid or threonine; e) 280 Alanine at position 282 The codon encoding tyrosine and the codon encoding glycine at positions 283-285 are replaced with a codon encoding aspartic acid; f) the codon encoding phenylalanine at positions 286-288. G) replacing the codon encoding alanine at positions 301-303 with a codon encoding threonine; or h) replacing the codon encoding glycine at positions 282-285 with aspartic acid. Having the deoxynucleotide sequence of SEQ ID NO: 2 having the following:

In another preferred embodiment, the polydeoxynucleotide has the following modifications: a) The codon encoding arginine at positions 28-30 is replaced with a codon encoding glutamine, asparagine, serine or threonine, with the proviso that In the case of substitution with a codon encoding asparagine, a codon encoding alanine at positions 34 to 36 is optionally substituted with a codon encoding serine or threonine); b) a codon encoding glutamic acid at positions 37 to 39 Is replaced with a codon encoding glutamine, aso-aragine, serine or threonine (however, when replacing with a codon encoding asparagine, the codon encoding glycine at positions 43 to 45 may optionally encode serine or threonine. Codon C) replacing the codon encoding glutamic acid at position 46-48 with a codon encoding glutamine, asparagine, serine or threonine (however, when substituting a codon encoding asparagine, D) optionally replacing the codon encoding leucine with a codon encoding serine or threonine); d) replacing the codon encoding arginine at positions 49-51 with a codon encoding glutamine, aspaaragin, serine or threonine. (However, when the codon encoding asparagine is substituted, the codon encoding valine at the 55-57 position is optionally substituted with a codon encoding serine or threonine); e) arginine at the 91-93 position Gluta , Asparagine, serine or threonine (however, when replacing with a codon encoding asparagine, a codon encoding cysteine at positions 97 to 99 may optionally be replaced with a codon encoding serine or threonine) F) replacing the codon encoding arginine at positions 100-102 with a codon encoding glutamine, asparagine, serine or threonine (however, when substituting a codon encoding asparagine, The codon encoding aspartic acid at position 108 is optionally replaced with a codon encoding serine or threonine); g) the codon encoding arginine at positions 103-105 encodes glutamine, asparagine, serine or threonine. Substituting codon; h) a 106 to 108 of the codon encoding aspartic acid, glutamine,
Substitution with a codon encoding asparagine, serine or threonine (however, a codon encoding proline at positions 112 to 114 is optionally substituted with a codon encoding serine or threonine); i) Arginine at positions 427 to 429; Replacing the encoding codon with a codon encoding glutamine, asparagine, serine or threonine (provided that
If the codon encoding asparagine is substituted, the codon encoding cysteine at positions 433-435 is optionally replaced with a codon encoding serine or threonine); or j) the aspartic acid at positions 481-483 is Substitution with a codon encoding glutamine, asparagine, serine or threonine (however, when substituting with a codon encoding asparagine, leucine at positions 487 to 489 is optionally substituted with a codon encoding serine or threonine) Having the deoxynucleotide sequence of SEQ ID NO: 2 having:

In another preferred embodiment, the polydeoxynucleotide has the following modifications: a) 4-6, 34-36, 319-321, 535-537 or 625-
Replacing the codon encoding alanine at position 627 with a codon encoding threonine; b) replacing the codon encoding threonine at positions 10-12 or 484-486 with a codon encoding alanine; c) 1 Codons encoding valine at positions 3 or isoleucine at positions 328-330 are replaced with codons encoding methionine; d) Codons encoding glutamic acid at positions 37-39 are replaced with codons encoding aspartic acid. E) replacing the codon encoding arginine at positions 49-51 with a codon encoding tryptophan; f) replacing the codon encoding alanine at positions 223-225 with a codon encoding proline; g) 304 Codon encoding serine at position ３０６306, encoding leucine H) replacing the codon encoding glycine at positions 505-507 with a codon encoding alanine; i) replacing the codon encoding glutamic acid at positions 547-549 with a codon encoding lysine J) replacing the codon encoding glutamine at positions 673-675 with a codon encoding arginine; k) replacing the codon encoding glycine at positions 709-711 with a codon encoding glutamic acid; or ) Replace the valine encoding codon at positions 808-810 with a glycine encoding codon;

In another preferred embodiment, the polydeoxynucleotide has the following modifications: a) the codon encoding alanine at positions 34-36 is replaced with a codon encoding asparagine, and optionally glutamic acid at positions 37-39. Is replaced with a codon encoding glutamine; b) a codon encoding arginine at positions 100-102 is substituted with a codon encoding asparagine, and a codon encoding aspartic acid at positions 106-108. Is replaced with a codon encoding threonine; c) the codon encoding arginine at positions 103-105 is replaced with a codon encoding asparagine, and optionally the codon encoding serine at positions 109-111 is replaced with threonine. D) 394- The codon encoding serine at position 96 is replaced with a codon encoding asparagine, and optionally the codon encoding serine at positions 400-402 is replaced with a codon encoding threonine; e) Asparagine at positions 580-582; The codon encoding the acid is replaced with a codon encoding asparagine, and optionally the codon encoding a serine at positions 586-588 is replaced with a codon encoding threonine; f) encoding an arginine at positions 103-105; Codons and 580 to 582
G) replacing the codon encoding aspartic acid with a codon encoding asparagine; g) replacing the codon encoding alanine at positions 34-36 with a codon encoding asparagine, and optionally glutamic acid at positions 37-39. Is replaced with a codon encoding glutamine, and a codon encoding aspartic acid at positions 580-582 is substituted with a codon encoding asparagine, and optionally a codon encoding serine at positions 586-588. Is replaced with a codon encoding threonine; h) the codon encoding arginine at positions 100-102 is replaced with a codon encoding asparagine; and the codon encoding aspartic acid at positions 106-108 encodes threonine. 580-58 The codon encoding aspartic acid at position is replaced with a codon encoding asparagine, and optionally the codon encoding serine at positions 586-588 is replaced with a codon encoding threonine; i) arginine at positions 103-105 And the codon encoding aspartic acid at positions 580-582 is replaced with a codon encoding asparagine, and the codon encoding serine at positions 109-111 and / or 586-588 is replaced with a codon encoding threonine. J) replacing the codon encoding arginine at positions 652-654 with a codon encoding glutamine; k) replacing the codon encoding glycine at positions 76-78 with a codon encoding aspartic acid; And a codon encoding serine at positions 394 to 396 Replacing the codon encoding alanine at positions 34-36 with a codon encoding asparagine, and replacing the codon encoding serine at positions 394-396 with a codon encoding asparagine; Substituting and replacing the codon encoding serine at positions 400-402 with a codon encoding threonine; m) replacing the codon encoding threonine at positions 646-648 with a codon encoding proline, and 652 The codon encoding arginine at position ６５654 is replaced with a codon encoding glutamine; having the deoxynucleotide sequence of SEQ ID NO: 2.

[0119] The polydeoxynucleotide fragments of the present invention may comprise 145 to 4 as described above.
It includes a deoxynucleotide at position 95 and encodes a modified FLINT polypeptide fragment. One preferred fragment is a polydeoxynucleotide consisting of amino acids 1-654 of SEQ ID NO: 2, modified as described above.

The present invention also provides the above polydeoxynucleotide and a fragment thereof, wherein the codon encoding glycine at a position corresponding to 640 to 642 of SEQ ID NO: 2 has been modified to be replaced with a codon encoding alanine. Including.

The polydeoxynucleotide derivative includes the polydeoxynucleotide of the present invention or a fragment thereof. The polydeoxynucleotide or fragment has another oligodeoxynucleotide added on the 3 'and / or 5' end. An "oligopolydeoxynucleotide" is a chain of 3 to about 150 deoxynucleotides. Suitable oligodeoxynucleotides include FLIN
A function having the same or increased ability to inhibit FAS / FAS ligand binding as compared to T, and having the same or improved pharmacological and pharmaceutical properties as compared to FLINT One that does not substantially impair the ability of the polydeoxynucleotide to be translated into a sex protein. Suitable oligodeoxynucleotides encode oligodeoxynucleotides encoding start codons, stop codons, promoter sequences, enhancer sequences, and leader sequences, or have improved stability or persistence in host cells or facilitated purification Includes oligopeptides.

The FLINT cDNA can be synthesized by the RT-PCR method using a usual method. Poly A RNA is produced from tissue sources known to express the FLINT gene (eg, lung) using standard methods. First chain FL
INT cDNA synthesis is achieved in a reverse transcriptase reaction using a "downstream" primer from the FLINT sequence. Commercially available kits (eg, GENEAM
P, manufactured by Perkin Elmer). In the subsequent PCR, cD
A forward primer and a reverse primer (SEQ ID NO: 1) specific for FLINT are used to amplify NA.

To determine the length of the amplified fragment (ロ ー 903 base pairs), the amplified sample can be analyzed by agarose gel electrophoresis. The wild-type FLINT cDNA obtained by this method is then used as a template for the introduction of a point mutation (ie, construction of a FLINT analog). For a description of this basic protocol, see "Current Protocols in Mo."
recital biology, "Volume 1, Section 8.5.7 (John Wile
y and Sons, Inc. , Publishers), which form part of the present specification. In this protocol, synthetic oligonucleotides were designed to introduce a point mutation at one end of the amplified fragment. After the first PCR, the amplified fragments, including the mutants, were annealed to each other and extended by mutual primed synthesis. After annealing, a second PCR step using a 5 'forward end primer and a 3' reverse end primer is performed to amplify the completely mutagenized fragment and prepare for subcloning in a suitable vector.

The present invention relates to vectors comprising the isolated polydeoxynucleic acid molecules of the invention, host cells engineered with the recombinant vectors, and modified F cells.
It relates to the production of LINT polypeptides, modified FLINT polypeptide fragments, fusion proteins represented by structural formula (I) and FLINT peptide derivatives by recombinant techniques well known in the art. For example, Sambrook
1989 by K. et al .; 1987-1988 by Ausubel et al., which form part of the present specification.

“Vector” refers to a nucleic acid compound used to introduce exogenous or endogenous nucleic acid into a host cell. The vector comprises a polydeoxynucleotide sequence capable of encoding one or more modified FLINT polypeptides, modified FLINT polypeptide fragments, FLINT fragments, fusion proteins represented by structural formula (I) and FLINT peptide derivatives. Naturally occurring or recombinantly engineered plasmids, cosmids, viruses and bacterial phages are non-standard vectors that are commonly used to obtain recombinant vectors containing at least one isolated nucleic acid molecule. This is a limiting example.

“Host cell” refers to a eukaryotic cell, a prokaryotic cell, a fusion cell, other cells, pseudocells or a membrane-containing material suitable for growing and / or expressing the isolated nucleic acid. Means a construct. Here, the isolated nucleic acid can be introduced into a host cell by any appropriate method known in the art (for example, but not limited to, transformation or transfection), Or a FLINT polypeptide, a modified FLINT polypeptide, a modified FLINT polypeptide fragment, a fusion protein represented by structural formula (I) or FLI
It has been induced according to the present invention to express an endogenous polydeoxynucleic acid encoding an NT peptide derivative. The cell may be part of a tissue or organism, isolated in culture or any other suitable form.

[0127] The polydeoxynucleotide may be optionally associated with a vector containing a marker of choice for growth in a host. Usually, a plasmid vector is introduced into a precipitate (eg, a calcium phosphate precipitate) or into a complex with a charged lipid (eg, lipofectamine). If the vector is a virus, it is packaged in vitro using a suitable packaging cell line and then transduced into host cells.

[0128] The DNA insert may be converted to a suitable promoter, such as the phage lambda PL promoter, phage T7 promoter, E. coli lac, trp and t.
(ac promoter, SV40 early and late promoters, and promoters of retroviral LTRs) or any other suitable promoter. Other suitable promoters are known to those skilled in the art. The expression construct further includes a transcription initiation and termination site in the transcribed region, and a ribosome binding site for translation. Preferably, the coding portion of the mature transcript expressed by the construct includes translation starting at an initiation codon and a termination codon (eg, UAA, UGA or UAG) located approximately at the end of the mRNA to be translated. For expression in mammalian or eukaryotic cells, UGA and UAA are preferred.

Preferably, the expression vector contains at least one selectable marker. These selectable markers include, for example, dihydrofolate reductase, neomycin, zeocin, hygromycin B or puromycin, which are resistant to eukaryotic cell culture. Includes tetracycline or ampicillin resistance genes for cultivation in E. coli and other bacteria or prokaryotes.
Representative examples of suitable host cells include bacteria (eg, E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium) cells, yeast cells (eg, Saccharomyces cerevisiae, Saccharomyces pombe). ) Or Pichia pastoris
s), insect cells (eg, Drosophila S2 Spodoptera)
era) Sf9), animal cells (eg, 293, 293EBNA, CHO, CO
S and COS7, BHK, AV12 and Bowes melanoma cells) and plant cells. Appropriate media and conditions for the above-described host cells are known in the art. Preferred vectors for use in bacteria are
pET15 and pET30 (available from Novagen); pEQ70, p
EQ60 and pEQ-9 (available from Qiagen); pBS vector, P
hagescript vector, Bluescript vector, pNH8A,
pNH16a, pNH18A, pNH46A (these are available from Stratagene); and ptrc99apKK223-3, pKK233-3,
pDR540, pRIT5, which are available from Pharmacia. Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG4
4, pXT1 and pSG (which are available from Stratagene); and pSVK3, pBPV, pMSG and pSVL (which are available from Pharmacia). Other suitable vectors will be readily apparent to one skilled in the art.

The introduction of the vector construct into the host cell can be accomplished by a calcium phosphate transfection method, transfection via DEAE-dextran, transfection via cationic lipids, electroporation, transduction, infection or other methods. it can. These methods are described in a number of standard laboratory manuals (eg, Sambrook, supra, Chapters 1-4 and 16-1).
Chapter 8; described in Ausubel, supra, Chapters 1, 9, 13, 15, and 16 (which form part of this specification).

The modified FLINT polypeptides, modified FLINT polypeptide fragments, FLINT fragments, fusion proteins represented by Structural Formula (I) and FLINT peptide derivatives of the present invention can be obtained by modifying the modified forms (eg, fusion proteins or “labels”). "Proteinized" protein). For example,
Another amino acid region, particularly a hexahistidine (His ₆ ) label, can be added to the carboxy terminus of the polypeptide to facilitate purification. Prior to final production of the polypeptide, those regions can be removed. They can be secreted by heterologous secretion signals, such as mouse Ig in the vector pSecTag2. A number of standard laboratory manuals (eg, 17.29-17.42 and 18.
. 1-18.74; described in Ausubel, supra, Chapters 16, 17, and 18, which form part of the present specification.

Fc fusions The present invention also relates to fusion proteins containing an Fc fragment of an immunoglobulin molecule fused to FLINT or a FLINT analog. Those FLIN
T or FLINT analogs include, for example, FLINT fragments (eg, FLINT metabolites) containing the residues at positions 1-218 of SEQ ID NO: 1 and further include, for example, protease resistant FLINT analogs (eg, R218WQ).

The FcFLINT constructs of the present invention exhibit an increased half-life as compared to native FLINT or a FLINT analog that is not embodied as an Fc fusion. For example, FcR218QFLINT shows a half-life of about 3 hours in CD-1 mice, while a half-life of about 30 minutes in R218Q after intravenous administration (10 μg / mouse). .

One example of a suitable Fc fragment is an Fc fragment obtained by cutting 1 to about 15 amino acids from the N-terminus of two heavy chains (preferably about 5 to about 15 amino acids). Another example of an analog of an Fc fragment is an Fc fragment having one or more point mutations.
c-fragments, which have substantially no increase in the affinity of the analog for complement as compared to the Fc fragment. The amino acid sequence of the Fc analog is F
It has greater than about 95% homology to the sequence of the c-fragment, with greater than about 99% homology being preferred. Examples of preferred point mutations include the replacement of one or more cysteines with a serine, or the replacement of one or more amino acids in the hinge region.
Analogs of the Fc fragment can be produced by the methods disclosed in WO 99/02711 by Strom et al., Which forms part of the present specification.

European patent application EP-A-0464533, which forms part of the present specification, describes the multiple regions of the constant region of an immunoglobulin molecule together with a heterologous human protein or part thereof. Discloses a fusion protein contained in the protein.

The present invention provides a soluble Fc fusion protein having FLINT activity, comprising FLINT, a FLINT analog or a fragment thereof fused to at least one human immunoglobulin constant domain.

“FLINT activity” as used herein refers to anti-apoptotic activity, for example, in an in vitro or in vivo assay.

The immunoglobulin portion of the fusion may be of any subclass (I
IgG, IgM, IgA and IgE), but may be part of an IgG (eg,
gG1, IgG3 or IgG4) are preferred. The constant domains or fragments thereof can be derived from heavy or light chains, or both.

The present invention is directed to mutations, substitutions, or the like of immunoglobulin components that eliminate unwanted properties of natural immunoglobulins (eg, binding to Fc receptors) and / or introduce desired properties (eg, stability). , Additions and / or deletions. For example, An
gal, S.M. King, D .; J. Bodmer, M .; W. , Turner
A. Lawson A .; D. G. FIG. Roberts G .; , Pedley,
B. And Molecular Immunology, by Adair, R.
30, 105-108 (1993), describes an IgG4 molecule in which the residue at position 241 (Kabat numbering) is modified from serine to proline. This modification increases the serum half-life of the IgG4 molecule. Canfield S.C. M. And Morrison S.M. L.
By the Journal of Experimental Medicine,
Nos. 173,1483-1491 describe the modification of the residue at position 248 (numbering of the hippocampus) in IgG3 (leucine to glutamic acid) and the modification in mouse IgG2b (glutamic acid to leucine). Replacement of leucine with glutamate in the former reduces the affinity of immunoglobulin molecules associated with the Fc gamma RI receptor, and replacement of glutamate with leucine in the latter increases its affinity. EP 0307434 describes 24 in IgG
A number of variants are disclosed that contain an L to E mutation at residue 8 (kabat numbering).

The fusion molecules of the present invention preferably lack the hinge region or include a subregion derived therefrom. In preliminary experiments, Applicants found that FLINT-Fc fusion proteins tended to aggregate and produce multimeric species larger than dimers, as judged by Western blot analysis. These aggregated species are inactive in in vitro cell-based apoptosis assays. Subsequent purification of the multimeric aggregate on a protein A column (elution under acidic conditions) failed and the protein was disaggregated. Applicants hypothesized that reducing the conformational mobility of the fusion protein could alleviate the aggregation problem. To test this, a more tonic fusion construct was prepared by progressively deleting the 15 amino acid hinge region. The following constructs were prepared and tested for the in vitro apoptosis assay.

[Table 1]

[0141] The sequence "LVPRGS" present in some constructs encodes a thrombin cleavage site, which is a number of commercially available vectors useful for expressing fusion proteins. Seen in The Fc construct was transferred to 293 EBN
It was expressed transiently in A cells and in the conditioned medium used in the bioassay. These experiments showed that the FL: Fc-hinge and FL: Fc + T hinge constructs were active in inhibiting apoptosis in vitro, while other constructs were less active. Deletion of all or part of the hinge region will prevent the higher aggregation observed when a complete hinge region is present.

In another embodiment, the IgG component is from IgG4, which comprises CH2 and CH3 domains, and a hinge region (eg, 8 of the IgG4 hinge region).
(Including cysteine residues that contribute to the disulfide bond between the heavy chains of residues at positions 11 and 11) (Pinck JR and Milstein C., Nature 216, 941-942, 1967). . Ellison
J. , Baxbaum J .; And Houd L .; DNA, 1,11-
18, 1981, the IgG4 components of which are residues at positions 1-12 of the hinge, residues 1-110 of CH2 and 1-10 of CH3 in IgG4.
It consists of the amino acid corresponding to the residue at position 7.

The fusion of FLINT or a FLINT analog to an Ig constant domain or fragment is by fusion of the C-terminus of one component with the N-terminus of the other component. FLINT
Or its analog is composed of its C-terminus and the N constant of the Ig constant domain or fragment.
It is preferred to fuse with the termini.

In a further aspect, the present invention provides a method for producing a compound according to the present invention, said method comprising expressing a DNA encoding said compound in a recombinant host cell, and Recovering the product.

Expression of Proteins in Host Cells Using the polydeoxynucleotides of the invention, modified FLINT polypeptides, modified FLINT polypeptide fragments, FLINT fragments,
The fusion protein, FLINT peptide derivative, represented by structural formula (I) can be expressed in recombinantly engineered cells (eg, bacterial, yeast, insect or mammalian cells). Because they have been genetically modified by human intervention, their cells are capable of producing proteins in non-native conditions (eg, for quantity, composition, location and / or time).

One skilled in the art will appreciate that the modified FLINT polypeptides of the invention, the modified FLIN
It is expected that one will have knowledge of a number of expression systems that can be used to express T polypeptide fragments, fusion proteins represented by structural formula (I) or FLINT peptide derivatives. No attempt will be made to detail in detail the numerous methods known for the expression of proteins in prokaryotes or eukaryotes.

In brief summary, the expression of an isolated nucleic acid encoding a modified FLINT polypeptide, modified FLINT polypeptide fragment, fusion protein represented by structural formula (I) or a FLINT peptide derivative of the present invention is Typically, this is achieved, for example, by operably linking DNA or cDNA to a promoter, which may be constitutive or inducible, and subsequently incorporated into an expression vector. Will. Vectors can be adapted for replication and integration in prokaryotic or eukaryotic cells. A typical expression vector contains a transcription terminator, a translation terminator, an initiation sequence, and a promoter useful for regulating the expression of DNA encoding a protein of the invention. In order to obtain high levels of expression of the cloned gene, it is desirable to construct an expression vector that contains minimally a promoter that directs transcription, a ribosome binding site for the translation initiation site, and a transcription / translation terminator. One skilled in the art will recognize that modifications can be made to the proteins of the present invention without compromising biological activity. Several modifications can be made to facilitate cloning, expression, and incorporation of the target molecule into the fusion protein. These modifications are well known to those skilled in the art, for example, adding a methionine at the amino terminus to obtain a start site, or a terminal, stop codon or to create an easily located restriction site. Including placing another amino acid (eg, polyhistidine) on the sequence to be purified.

Alternatively, the polydeoxynucleotide of the present invention can be prepared by modifying the modified FLINT polypeptide, modified FLINT polypeptide fragment, fusion protein represented by structural formula (I), or endogenous DNA encoding a FLINT peptide derivative. And can be expressed by manipulation in a host cell.
These methods are well known in the art and are described, for example, in US Pat. No. 5,580,7.
No. 34, 5,641,670, 5,733,746 and 5,733
, 761, which form part of the present specification.

Expression in Prokaryotes Prokaryotic cells can be used as hosts for expression. The most commonly used prokaryotes are E. coli. It is represented by a number of strains of E. coli. However, strains of other microorganisms can also be used. Commonly used prokaryotic control sequences, which include a promoter for initiation of transcription and optionally define an operator together with a ribosome binding site sequence
Contains commonly used promoters. For example, β-lactamase (penicillinase) and the lactose (lac) promoter system (NAt by Chang et al.)
ure, 198; 1056 (1977)), the tryptophan (trp) promoter system (Nucleic Acids Res, by Goeddel et al.).
. δ: 4057 (1980), T7 phage promoter (Studier, F.
. W. Methods in Enzymology, 185: 60-6.
9 (1990)) and the λ-inducible PL promoter and N-gene ribosome binding site (Shimatake et al., Nature, 292: 128 (
1981)), which form part of the present specification. E. FIG. It is also useful to include a selectable marker in the DNA vector transfected in E. coli. Examples of such markers include genes that specify that they are resistant to ampicillin, tetracycline or chloramphenicol.

The vector can be selected and introduced into a suitable host cell. Bacterial vectors are typically derived from plasmids or phages. Appropriate bacterial cells are infected with the fuzzy vector particles, or the naked phage vector DN
Transfect with A. If a plasmid vector is used, the bacterial cells are transformed with the plasmid vector DNA. Expression systems for expressing the proteins of the present invention include Bacillus subtilis and Salmonella (Palva et al., Gene 22; 229-235).
(1983); Nature 302; 543-545 by Mosbach et al.
(1983), which form part of this specification).

Expression in Eukaryotes A number of eukaryotic expression systems are known by those skilled in the art, for example, yeast, insect cell lines, plant cells and animal cells. As described briefly below, the polydeoxynucleotides of the present invention can be expressed in these eukaryotic systems.

The production of heterologous proteins in yeast is well known. F. Sherman
Et al., Methods in Yeast Genetics, Cold S.
The Spring Harbor Laboratory (1982), which forms part of the present specification, is a well-recognized study that describes a number of methods that can be used to obtain proteins in yeast. . Two widely used yeast strains for eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains and protocols for expression in Saccharomyces and Pichia are well known in the art and can be obtained from commercial sources (eg, Invitrogen). Suitable vectors usually have expression control sequences (eg, a promoter containing 3-phosphoglycerate kinase or alcohol oxidase), an origin of replication, a termination sequence, and those desired.

The modified FLINT polypeptides of the invention, once expressed, the modified FL
An INT polypeptide fragment, a fusion protein represented by structural formula (I) or a FLINT peptide derivative can be isolated from yeast by applying standard protein isolation techniques. Tracking the purification process can be accomplished using SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blot or radioimmunoassay or other immunoassay (eg, ELISA).

A sequence encoding a modified FLINT polypeptide, a modified FLINT polypeptide fragment, a FLINT fragment, a fusion protein represented by Structural Formula (I) or a FLINT peptide derivative of the present invention may be used in cell culture (eg, (Derived from mammals, insects and plants) can also be ligated into a number of expression vectors for use in transfecting. Examples of cell cultures useful for producing the peptide include mammalian cells. Mammalian cell systems can use mammalian cell suspensions, but are often in monolayer form. Numerous host cell lines capable of expressing intact proteins have been developed in the art, for example, AV12, HEK293, BH
Includes K21 and CHO cell lines. Expression vectors for these cells include expression control sequences (eg, origin of replication), promoters (eg, CMV promoter, HSV tk promoter, and pgk (phosphoglycerin kinase) promoter), enhancers (Immunol. Re by Queen et al.).
v. 89; 49 (1986), which form part of this specification, and processing information sites (eg, ribosome binding site, RNA splicing site, polyadenylation site (eg, bovine growth hormone polyA addition site) and transcription. Terminator sequence). Other animal cells useful for the production of the proteins of the invention include, for example, the American Type Culture C
collection Catalog of Cell Lines an
d Available from Hybridoma (7th edition, 1992).

A modified FLINT polypeptide, modified FLINT polypeptide fragment, fusion protein represented by structural formula (I), or FL of the present invention
Suitable vectors for expressing INT peptide derivatives in insect cells are usually
It is derived from F9 baculovirus. Suitable insect cell lines include, for example, mosquito larvae, silkworms, caterpillars, moths and Drosophila cell lines (eg, Schmeider cell lines) (Schmeider,
J. Emryol. Exp. Morphpol. 27: 353-365 (
1997), which form part of this specification).

When using higher animal or plant host cells together with yeast,
A polyadenylation or transcription terminator sequence is typically introduced into the vector. An example of a terminator sequence is a polyadenylation sequence from the bovine growth hormone gene. It may also include sequences for correctly splicing the transcript. An example of a splicing sequence is the VP1 intron from SV40 (Sp1
Rague et al. Virol. 45: 773-781 (1983), which forms part of the present specification). In addition, gene sequences for controlling replication in host cells can be introduced into vectors (including, for example, bovine papillomavirus-type vectors). M. Saveria-Camp
o, Bovine Papilloma virus DNA, a Eukar
yotic Cloning Vector in DNA Cloning
II, a Practical Approach; M. Glover, IRL Press, Arlington, VA, pp. 213-238 (19
85, which forms part of the present specification).

Protein Purification The modified FLINT polypeptide, the modified FLINT polypeptide fragment, the FLINT fragment, the fusion protein represented by Structural Formula (I) or the FLINT peptide derivative can be prepared by a well-known method (for example, ammonium sulfate). Or cells ligated by precipitation with ethanol, extraction with acid, including anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography). For example, it can be recovered from E. coli and purified. High performance liquid chromatography ("HPLC") can also be used for purification. The polypeptides of the present invention may be natural purified products, products of chemical synthesis, prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plant cells, insect cells and mammalian cells. From recombinant methods. Depending on the host used in the recombinant production method, the polypeptides of the invention may be glycosylated or unglycosylated. In addition, the polypeptides of the invention also contain an initially modified methionine residue. The method is described in a number of standard laboratory manuals (e.g., Sambrook, 17.37-17, supra).
. 42; Ausubel at 10, 12, 13, 16, 18, and 20 above.
Sections, which form part of this specification).

[0158] The modified FLINT polypeptides, modified FLINT polypeptide fragments, FLINT fragments and FLINT peptide derivatives comprise FAS
Binding to FAS ligand and LIGHT to LTβR receptor and TR2 /
It is believed to inhibit binding to the HVEM receptor, and thus has a disease and / or condition associated with its binding (eg, FAS is activated to a greater than normal level or is impaired by a FAS ligand. The subject (if appropriately activated) can be treated. FLINT analogs that inhibit FAS / FAS ligand binding show activity in the assay described in Example 1.

Runaway apoptosis is an example of a disease caused by excessive activation of FAS by the FAS / FAS ligand signal transduction pathway, which can be treated with the FLINT analog of the present invention ( U.S. Provisional Patent Application No. 60 / 112,577 (filed December 18, 1998), Naturo by Kondo et al.
e Medicine 3 (4): 409-413 (1997) and Gall.
e. Exp. Med. 182; 1223-1230 (1995), which form a part of the present specification). Runaway apoptosis can cause a pathological condition (eg, organ disease) in a subject. Diseases associated with runaway apoptosis include, for example, acute renal failure (e.g., liver failure, hepatitis, hepatocyte damage and / or liver infection associated with viral infections affecting the liver and bacterial infections affecting the liver). Or other diseases in which hepatocytes undergo massive apoptosis or destruction), renal failure and pancreatic insufficiency. FLINT analogs that inhibit runaway apoptosis are normally active in the Jurkat cell assay described in Example 8 and in the in vivo assay for kidney damage described in Example 11.

The FLINT analogs of the present invention are therapeutically useful for diseases that can normally be treated with FLINT (US Ser. No. 09 / 280,567, and Nature Medicine 4: 1287 by Miwa et al. (1988). )
, Which form part of the present specification). One example is inflammation caused by FAS ligand-induced neutrophil activation. Inflammatory diseases related to neutrophil activation include sepsis, ARDS, SIRS and MODS. FLINT analogs that are active in the in vivo assay described in Example 12 usually inhibit neutrophil activation.

[0161] Other diseases for which FLINT has been found to be therapeutically useful include rheumatoid arthritis (Lancet et al. Lancet 344: 1105-1110 by Elliott et al.
(1994)), acute lung injury, ARDS, fibroproliferative lung disease, fibrotic lung disease, H
IV (J. Clin Invest. 101: 239 by Dockrell et al.).
4-2405 (1998)), ischemia (1988 Bra by Sakurai et al.).
in Res 797: 23-28), brain trauma / injury (19 by Ertel et al.).
97J. Neuroimmunol 80: 93-96), chronic renal failure (Sc)
1998 Lab Invest 78: 813-82 by Helling et al.
4) Transplant versus host disease (GVHD) (Hattori et al. 1998 Blood).
d 11: 4051-4055), skin inflammation (1988 J by Orteu et al.).
. Immunol 161: 1619-1629), vascular leak syndrome (Rafi
1988 J. et al. Immunol 161: 3077-3086), Helicobacter pylori infection (Rudi et al., 1998 J. Clin Inves).
t 102: 1506-1514), goiter (Tamura et al. 1998).
Endocrinology 139: 3646-3653), atherosclerosis (1998 J. Clin Invest by Sata and Walsh).
102: 1682-1689), IDDM (Itoh et al., 1997 J. Am.
Exp. Med. 186: 613-618), osteoporosis (1 by Jilka et al.
998 J.C. Bone Min RES 13: 793-802), Coulomb's disease (1995 Gastroenterolo by van Dulleman et al.).
gy 19: 129-135), transplant (graft) rejection (19 by Lau et al.
96 Science 273: 109-112), sepsis (1998 New Horizons 6: S97-102 by Faist and Kim.),
Pancreatitis (1998 Gut 42: 886-89 by Neoptolemos et al.)
1), cancer (melanoma, colon and esophagus) (1998 by Bennett et al.)
J. Immunol 160: 5569-5675), autoimmune diseases (IBD,
Psoriasis, Down Syndrome (Neuroscience Lett by Seidi et al.)
. 260: 9 (1999)) and multiple sclerosis (19 by D'Souza et al.).
96 J Exp Med 184: 2361-2370).

Concurrent United States Patent Application entitled “TERAPEUTIC APPLICATE
ONS OF FLINT POLYPEPTIDES ", March 30, 1999
Ser. No. 09 / 280,567) discloses other diseases which can be treated using the FLINT analogs of the present invention. For example, Alzheimer's disease; acute respiratory disease syndrome; end stage renal disease (ESRD); mononucleosis; ESV; herpes; ulcerative colon; ulcerative colon; DIC (disseminated intravascular coagulation); eclampsia;
HELLP (thrombocytopenia, pre-eclampsia complicated by hemolysis and impaired liver function); HITS (heparin-induced thrombocytopenia); HUS (hemolytic uremic syndrome)
Preeclampsia; hematopoietic disorders (eg, aplastic anemia); thrombocytopenia (TTP)
Myelodysplasia; and hemolytic fever (eg, due to Ebola fever). The ability of FLINT and FLINT analogs to delay apoptosis in ischemic conditions is also useful for preserving organs and tissues collected for transplantation. Ischemic diseases include, for example, neurological ischemia, limb crush injury, spinal cord injury, myocardial infarction (which includes acute myocardial infarction), subacute and chronic sequelae, and related clinical Syndrome, including but not limited to congestive heart failure. Also, harmless bystander tissue that is damaged during chemotherapy, radiation therapy, toxic drugs, trauma, surgery, and other stresses can be treated with FLINT. One example of those diseases is mucositis, which can be a life-threatening side effect in the treatment of cancer.

Acute Lung Injury and Acute Pulmonary Distress Syndrome Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) represent disease entities that differ only in the severity of hypoxia present at the time of diagnosis. A widely accepted parameter for diagnosis is the ratio of PaO2 to FiO2, according to which ARDS patients show a ratio of 200 mg or less, while ALI patients show a value of 300 mg or less. ARDS is a more intense form of ALI. Many mediators appear to be involved in the pathogenesis of ARDS / ARI, where neutrophils play a prominent role. Although there may be many facilitating factors in the development of direct and indirect ARDS, the main causes are thought to be pulmonary blood and systemic inflammatory responsive syndrome, which accounts for about 40% of cases of ARDS. explain. ARDS
Mortality rates can reach as high as about 40%, with most deaths occurring within the first few weeks. There is currently no improved pharmacological therapy available for ARDS and current treatment is limited to active supportive care.

Evidence exists that ARDS can be mediated by soluble FasL / FAS interactions in humans (Mature-Bello et al., J. Immun.
ol. 163, 2217-2225, 1999). FLINT, by binding to FasL, can inhibit FasL-mediated apoptosis of lung or endothelial cells, thus progressing from acute inflammatory stroke to ALI and from ALI to ARDS. Can be inhibited or prevented.

In one embodiment, the present invention relates to the use of FLINT for inhibiting and / or treating ALI and / or ARDS, wherein the use comprises treating humans in need of such treatment. Administering a clinically effective amount of FLINT.

Chronic Obstructive Pulmonary Disease (COPD) Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of non-accidental death in the United States, following heart disease, cancer and cerebrovascular disease. COPD is an obstructive airway disorder that includes chronic bronchitis, emphysema, bronchiectasis and chronic asthma. COPD progresses slowly, resulting in an irreversible decrease in lung function. Chronic hypoxia and hypercapnia are the end result of the disorder. The mechanism by which COPD disrupts lung function is thought to be involved in unregulated apoptosis. Plasma samples from patients suffering from COPD show higher levels of soluble Fas compared to those of healthy control patients (Resp. Med. By Yasuda et al.).
92, 993-999, 1998). Soluble Fas in COPD patients
May increase Fas-induced apoptosis.

In another embodiment, the present invention relates to the use of FLINT to treat and / or suppress COPD in a patient in need of such treatment by administering a therapeutically effective amount of FLINT. .

Pulmonary Fibrosis (PF) Pulmonary fibrosis, where fibrosis is also known as pulmonary disease, occurs as the end result of an attempted healing process during acute or chronic lung injury. The pathological causes of those lung injuries are due to a number of factors, which first trigger an inflammatory response in the alveoli or surrounding stroma, followed by alveolar / interstitial fibrosis ( I.e., triggering the repair reaction). Fibrosis in other tissues (eg, epidermis or peritoneum) causes macroscopic scarring or adhesion, respectively. In contrast, pulmonary fibrosis results in fibrosis limited to the lungs, which reduces lung volume and oxygen diffusion. Diseases associated with pulmonary fibrosis include, for example, idiopathic fibrosis, connective tissue disease (eg, lupus, scleroderma), drug-induced lung disease (eg, bleomycin), pneumoconiosis (eg, asbestos) Deposition), sarcoidosis, eosinophilic granulomatosis, irritable pneumonia and other diseases associated with severe pulmonary inflammation that can result in acute lung injury and / or acute pulmonary respiratory distress syndrome (eg, trauma, Sepsis, near-drawing
, Gastric aspiration, shock, etc.). Airway fibrosis is also a feature of chronic inflammation of COPD.

The cause of PF may be related to FasL / Fas-triggered apoptosis.
Indeed, in the cause of bleomycin-induced PF in mice, intact F
The asL / Fas system is essential (J. Cli by Kuwano, K. et al.
n. Invst. 104, 13-19 (1999)).

In another embodiment, the present invention relates to the use of a FLINT analog for inhibiting and / or treating PF. For example, a FLINT analog can be administered acutely during an inflammatory attack on the lungs (eg, during treatment with a bleomycin) to prevent PF from occurring.

A “subject” is a mammal in need of treatment, preferably a human, but in need of veterinary treatment (eg, a domesticated animal (eg, dog, ne, etc.))
And domestic animals (eg, cows, sheep, pigs, horses, etc.) and experimental animals (eg, rats, mice, guinea pigs, etc.).

An “effective amount” of a FLINT analog refers to a therapeutic effect or prophylaxis of interest in a patient having a disease or condition associated with abnormal FAS / FAS ligand binding and / or LIGHT-mediated binding. FAS
Or FAS ligand or LIGHT and LTβR and / or TR2 /
An amount sufficient to inhibit one or more processes mediated by binding to HVEM. One example of those processes is runaway apoptosis. Or,
An “effective amount” of a FLINT analog refers to a subject having inflammation caused by FAS ligand-induced neutrophil activation, or any of the other above-mentioned diseases associated with abnormal FAS ligand activity. An amount sufficient to achieve the desired therapeutic and / or prophylactic effect.

“Objective therapeutic and / or prophylactic effects” in a patient with a disease or condition include alleviation of the syndromes associated with those diseases or delayed onset of the syndromes. Alternatively, "the intended therapeutic and / or prophylactic effect" includes increased survival or increased longevity of a patient with the disease. The amount of FLINT analog administered to an individual will vary depending on the type and severity of the disease and the characteristics of the individual (eg, general health, age, sex, weight, and tolerance to drugs). It will also vary depending on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

As a general suggestion, the total pharmaceutically effective amount of the FLINT analog of the present invention administered parenterally with each administration is from about 1 μg / kg / day to 10 mg of body weight of the patient
/ Kg / day, in particular between 2 mg / kg / day and 8 mg / kg / day, more particularly between 2 mg / kg / day and 4 mg / kg / day, even more particularly 2.2 mg / kg / day.
kg / day to 3.3 mg / kg / day and ultimately 2.5 mg / kg / day. As noted above, this may be susceptible to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day.
When given continuously, the FLINT analogs of the present invention typically have 1 FLINT per day.
Administer at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour by injecting 4 times or by continuous subcutaneous injection (eg, via a minipump). Intravenous bag solutions can also be used. The length of treatment is necessary to observe changes, and the interval at which the response occurs after treatment will vary depending on the desired effect.

A pharmaceutical composition comprising a FLINT analog of the present invention may be administered orally, rectally, intracranial,
Parenteral, intracisternal, vaginal, intraperitoneal, topical (eg, by powder, ointment, drops or transdermal patches), transdermal, intrathecal, buccally, Or they can be administered as an oral or nasal spray. "Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, diluent,
Means capsule material or auxiliary agent of any kind of formulation. The term "parenteral" as used herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections, infusions and implants containing the FLINT analog.

The FLINT analogs of the present invention are suitably administered by sustained release systems. Suitable examples of sustained release compositions include semipermeable polymer matrices (eg, films or microcapsules) in the form of a molded product. As a sustained-release matrix, polyactide (US Pat. No. 3,773,919,
EP 58,481), copolymers of L-glutamic acid and γ-ethyl L-glutamate (Sidman, U et al., Biopolymers, 22: 547).
-556), poly (2-hydroxyethyl methacrylate) (R, Language
LJ. Biopolymed. Master Res. 15:16
7-277 (1981) and R.M. Chem Tech. 1
2: 8-105 (1982)), ethylene vinyl acetate (ibid. By R. Lnger) or poly-D-(-)-3-hydroxybutyric acid (EP 13,988). Other sustained release compositions also include modified FLINT polypeptides, FLINT polypeptide fragments and FLINs, eg, liposome entrapped.
Also includes T peptide derivatives. These liposomes are themselves described in DE 3,218,121, Proc. Natl. Acad. Sci. (USA)
82: 3688-3692 (1995); Hwang et al., Proc. Na
tl. Acad. Sci. (USA) 77: 4030-4034 (1980);
EP 52,322; EP 36,676, EP 88,046, EP 143,949, EP 142,641; Patent application 83-118008;
U.S. Pat. Nos. 4,485,045 and 4,554,545 and EP 10
It is produced by the method known from US Pat. No. 2,324. Typically, liposomes are of the small (about 200-800 °) monolayer type, with a lipid content of about 30
More than mole percent cholesterol, the selected ratio of which is optimal TN
Regulated for FR polypeptide therapy.

For parenteral administration, in one embodiment, the FLINT analog of the present invention is purified to a desired degree of purity, using a pharmaceutically acceptable carrier, ie, the dosage used and Normally non-toxic to recipients in concentrations and compatible with the other ingredients in the formulation) to form a unit dose injectable form (eg, solution, suspension or emulsion) Is done. For example, it is preferred that the formulation be free of oxidizing agents and other compounds known to be deleterious to polypeptides.

In general, the formulations are prepared by uniformly and intimately contacting the FLINT analog of the present invention with a liquid carrier or a finely divided solid carrier, or both. The product is then, if necessary, shaped into the desired formulation. The carrier is preferably a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of vehicles for these carriers include water, sarin, Ringer's solution and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

The carrier contains suitably small amounts of additives such as substances that increase isotonicity and chemical stability. The substances are non-toxic to recipients at the dosages and concentrations employed, and include, for example, buffers (phosphate, citrate, succinate, acetic acid and other organic acids or their salts); Oxidizing agents (eg, ascorbic acid); low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptide); proteins (eg, serum albumin, gelatin or immunoglobulin); For example, polyvinylpyrrolidone):
Amino acids (eg, glycine, glutamic acid, aspartic acid or arginine); monosaccharides, disaccharides and other carbohydrates (eg, including cellulose or derivatives thereof, glucose, mannose or dextrin); chelating agents (eg, EDTA); sugars Alcohol (eg, mannitol or sorbitol);
And / or a nonionic surfactant (eg, polysorbate, poloxamers or PEG).

The FLINT analogs of the present invention typically have a pH of about 3 in their vehicles.
-8 at a concentration of about 0.1 mg / mL to 100 mg / mL (preferably 1-10 mg / mL). It will be appreciated that the use of certain excipients, carriers or stabilizers described above will produce salts of the FLINT analogs of the present invention.

[0181] The polypeptides used for therapeutic administration must be sterile.
Sterility is readily accomplished by filtration through sterile filtration membranes (eg, a 0.2 micron membrane). Therapeutic polypeptide compositions are typically placed in a container having a sterile access port, for example, an intravenous solution bag or stoppered vial that can be pierced by a hypodermic injection needle.

FLINT analogs are typically administered in single or multiple dose containers (eg,
Stored in sealed ampoules or vials) as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 mL vial is a sterile filtered 1% (weight / volume) aqueous solution of one of the FLINT analogs of the present invention (5%).
mL) and freeze-dry the resulting mixture. Injection solutions are prepared by reconstituting the lyophilized polypeptide using bacteriostatic water for injection.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such containers may be accompanied by notice in a form mandated by a government body regulating the manufacture, use or sale of the pharmaceutical or biological product, the notice comprising manufacture, use and sale for human administration. Indicates acceptance by a government agency. In addition, the FLINT analogs of the present invention can be used with other therapeutic compounds.

In the case of organ preservation in preparation for collection, FLINT, for example, is useful for prophylactically preventing apoptosis associated with ischemic reperfusion injury to organs once removed from a donor. In one embodiment of this aspect of the invention, the organ donor is administered FLINT prior to organ collection. After organ collection, FLINT is added to a suitable medium for organ transport / storage. Alternatively, the harvested organ is perfused with a medium containing FLINT before transplantation into a recipient.
Suitable media for this purpose are, for example, the media disclosed in EP 0 356 367 A2, which forms part of the present description. The method may also include treating the transplant recipient with FLINT before and / or after the transplant operation.

A typical method involves pretreating an organ donor with an effective amount of a FLINT analog prior to organ collection. Alternatively or additionally, the collected organs are
The solution containing LINT can be perfused or immersed. This method
For example, it can be used for kidney, heart, lung, and other organs and tissues.

The FLINT analogs of the present invention have uses other than therapeutics. For example, these F
LINT analogs can be used to describe how amino acid substitutions can affect positively or negatively, the properties of TNFR proteins (eg, FLINT and their homologs) (eg, 1) the ability of those proteins to bind their ligands, And 2)
(The pharmaceutical properties of those proteins and their homologs) can be used as research tools. Because FLINT is homologous to the TNFR protein, point mutations that affect the properties of FLINT
It is expected that it will have the same effect as when performed on FR proteins or their homologues. Thus, the FLINT analogs of the present invention can be used with models of TNFR protein homology to predict the effect of a point mutation on the properties of the TNFR protein.

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

[0188] Example 1 a number of production of a vector for expressing FLINT analogue in the expression system of prokaryote prokaryotic accommodation cells (e.g., E.Coli) suitable for expressing FLINT or analog thereof in Various expression vectors are easily produced. The vector includes an origin of replication (Ori), a kanamycin resistance gene (Amp), which is useful for selecting cells that have taken up the vector after transformation,
It further includes a T7 terminator sequence operably linked to the T7 promoter and the FLINT coding region. Plasmid pET30a (this is Novogen,
Madison WI) is a suitable parent plasmid. PET30
a is linearized by restriction with the endonucleases NdeI and BamHI. The linearized pET30a is ligated to a DNA fragment having NdeI and BamHI cohesive ends and containing the coding region of the FLINT gene disclosed in SEQ ID NO: 1 or a fragment thereof.

[0189] The FLINT gene used in this construct is slightly modified at the 5 'end (the amino terminus encoding the polypeptide). FLINT site-specific mutants were prepared according to the protocol (refer to the reference: Saiki, RK, et al., Scie).
239: 487-492 (1988), and a comprehensive manual "Current Protocols in Molecula".
r Biology, "Volume 1, Section 8.5.7 (John Wiley and S
ons, Inc. (Based on publishers). These form part of the present specification. Silent mutations can be made in genes (eg, genes to create specific restriction sites that can facilitate the transfer of FLINT analog "cassettes" from prokaryotic expression vectors to eukaryotic expression vectors). Introduce.

Example 2 Prokaryotic Expression of Recombinant FLINT Analog and Its Purification Open Reading Frame Encoding FLINT or Its Analog (
ORF), wherein the ORF is suitably linked to an expression promoter, using a competent E. coli. Coli strain BL21 (DE3) (hsds gal
cIts857 ind1sam7nin5lacUV5-T7gene1) (
This is available from Novagen (Madison WI))
) Was transformed by standard methods. A number of transformants, which are selected because they are resistant to kanamycin, were randomly picked and used to seed LB / kanamycin medium. The culture is grown at 37 ° C. with shaking to a cell density OD ₆₀₀ of 0.8-1.0, after which point FLINT synthesis is reduced to 1 mM I
Induced using PTG. Prior to harvesting the cells, 3 hours prior to inducing the culture, 3 hours
Grow at 7 ° C. FLINT encoded by ORF with vector
Analog products were purified from inclusion bodies, the soluble fraction of the cell lysate. In short, purification consists, respectively, of purification and solubilization of inclusion bodies, and refolding of proteins by cation exchange chromatography and size exclusion chromatography.

Example 3 Construction of Vectors for Expression of FLINT Analogs in Mammals “Internal Ribosome Entry Site” / Enhanced Green Fluorescent Polypeptide (IRES /
An eGFP) PCR fragment is inserted into the mammalian expression vector pGTD to construct an expression vector that is two bicistronic (Biochemicals Jour by Gerlitz, B et al.).
nal 295: 131 (1993)). This new vector (designated pIG1) contains a sequence marker: the GBMT promoter (DT Ber) which is resistant to E1a.
BioTechniques 14: 972 (1992); T.
Nucleic Acids Research 20:54 by Berg et al.
85 (1992)); Specific Bc1 I cDNA cloning site; IRES sequence from encephalomyocarditis virus (EMCV); eGFP (Clontech) coding sequence (Gene 173: 33 (1996) by Cormack et al.); And SV40t antigen splicing site. / Polyadenylation sequence; SV40 early promoter and origin of replication; mouse dihydrofolate reductase (dhfr) coding sequence; and pBR322 ampicillin resistance marker / origin of replication.

Based on the human FLINT cDNA sequence, a Bc1I
Forward and reverse primers with restriction sites were produced.
These primers were used to PCR amplify the FLINT analog cDNA. The resulting 900 bp PCR product was digested with Bc1I and pIG1
And the plasmid pIG1-FLINT was obtained. The human FLINT cDNA orientation and nucleotide sequence were confirmed by restriction digestion and sequencing of the insert duplex. The FLINT in pIG1 was modified by silent mutation so that the "cassette" could be transferred to an analog previously constructed in the prokaryotic vector pET30a. It is itself cleavable hexahistidine (Hi) to facilitate purification of the analog.
s6) It can also be modified by introducing a cassette at the C-terminus.

The references in this embodiment form part of the present specification.

Example 4 Construction of a Vector to Express FLINT Analogs Resistant to Fc-Protease R218Q Fc-hinge / JB02 was constructed from a modified pEAK vector for transient expression in 293EBNA. 7 kb vector. The vector contains a tk promoter, SV40 origin of replication and R218Q Fc.
Contains the gene encoding the fusion, which lacks the hinge region.

The medium from cells transfected with this vector was used for
It was concentrated 10-fold in Amicon ProFlux M12 tangential filtration 10X using nS3Y10 UF membrane. The medium containing R218Q FLINT-Fc was conditioned with 0.5 M NaCl and 5 mM EDTA. The concentrated medium is applied to a protein A HiTrap column (Pharmacia, 5 mL column).
At a flow rate of 5 mL / min. The column was washed with Buffer A (PBS, 1 mM potassium phosphate, 3 mM sodium phosphate;
Wash with 5M NaCl, pH 7.4) and bind the bound polypeptide to 100% buffer B (50 mM citric acid, 0.5 mM NaCl, pH 3.5).
). The eluted material was neutralized with 100 μL of 1 M Tris (pH 8.0) per mL of elution buffer. Fractions containing R218Q Fc are pooled and stored on a Superdex 75 (Pharmacia, 16/60) size column (PBS, 0.5 M NaCl, 10% glycerol, pH 7.4).
Buffered with) at a flow rate of 1 mL / min. R218Q FLINT
Fractions containing -Fc were analyzed by SDS-PAGE. The N-terminal sequence of R218Q FLINT-Fc on the purified polypeptide was confirmed. Addition of EDTA reduces the likelihood of molecule aggregation.

Example 5 R218Q FLINT-Fc Fusion Protein Inhibits Apoptosis When assayed in Jurkat cells in an in vitro cell-based apoptosis assay, R218Q FLINT-Fc (no hinge) binds to wild-type FLINT. It showed comparable activity (FIG. 1).

In addition, when administered 4 hours after sensitization with galactosamine / LPS, R2
The 19Q FLINT-Fc (no hinge) fusion showed a protective effect in an animal model of acute liver failure. The results show the protective effect in 7 out of 10 of the animals, the proportion being identical to the protective effect exhibited by wild-type FLINT.

Example 6 Isolation of FLINT Analog Clones Showing High Production from AV12 RGT8 Transfectants Recombinant plasmids carrying the FLINT gene encode resistance to methotrexate. In addition, the construct contains the same transcript and a gene encoding a fluorescent polypeptide, GFP, immediately 3 ′ of the FLINT gene. Because high levels of GFP expression require high levels of FLINT-GFP mRNA, highly fluorescent clones will have a greater probability of producing high levels of FLINT. AV12 RGT18 cells are tonsfected using the calcium phosphate method with a recombinant pIG1 plasmid containing the FLINT analog. Cells that were resistant to 250 nM methotrexate were selected and stored. The storage of the resistant clones was performed using a fluorescence activated cell sorter (FACS), and the cells having the fluorescence value of the upper 5% of the population were sorted out to obtain single cells.
Store as Three consecutive sorting cycles were performed on the high fluorescent stock. The stock, and individual clones from the second and third cycles, were
LINT production was analyzed by SDS-PAGE. The highest level of FLINT as judged from the stock or Coomassie stained SDS-PAGE gel
Was used for scale-up and purification of FLINT.

Example 7 Quantification of FLINT Analog Developed FLINT ELISA in crude media of transfected cells
During the purification procedure with A, the FLINT analog can be quantified. EL
The ISA uses an anti-FLINT polyclonal antibody as the capture antibody and a biotinylated anti-FLINT TKD-07 as the primary antibody in the “sandwich”.
6A is used. The ELISA was developed with streptavidin-derivatized horseradish peroxidase (SA-HRP), using OPD as substrate and following absorbance at 490 nm. The EL
The effective range of the ISA is in the range of 0.2-20 ng / mL.

Example 8 Determination of the Effect of FLINT Analogs on FasL-Induced Apoptosis of Jurkat Cells FLINT for Measuring Cell Viability (ie, Prevention of Apoptosis)
Bioassays were performed on a 96-well plate format using 100 μL / well of reaction. Over 25 μL of Jurkat cells (5 × 10 ⁴ cells / well) was transiently transfected into 293EBNA cells with recombinant human FasL (25 μL, final concentration is 150 ng / mL) and FLINT analog. Mix with clear (50 μL). Cells were incubated overnight at 37 ° C. MTS tetrazolium compound (20 μL) (US Pat. No. 5,185,185)
No. 450, which was transferred to the University of South Florida and was then replaced by Promega Co.
rportation, Madison, WI have exclusive license)
Was added to each well and incubated at 37 ° C. for 2 hours. The absorbance at 490 nm was recorded on a plate recorder. The results are shown below. All but one analog showed anti-apoptotic activity in this assay.

[Table 2]

[Table 3] PR is protease resistant. CHO is a glycosylated analog.

Example 9 Purification of Large Scale FLINT Analog Polypeptides Large scale production of FLINT analogs (containing a 6 histidine tag) was performed by growing stable clones in several 10 L spinners. After reaching confluence, the cells were incubated for an additional 2-3 days to allow the maximum amount of FLINT analog to be secreted into the medium. The medium containing the FLINT analog was purified using Amicon ProFlux M12 tangent filtration (tangential).
Concentration in the filtration system to 350 mL. The concentrated medium was passed through IMAC (Immobilized Affinity Chromatography (Pharmacia, 5-10 mL column) at a flow rate of 1 mL / min.) Buffer A was run until the absorbance (280 nm) returned to baseline. (PBS, 0.5M Na
Cl, pH 7.4) to remove the bound polypeptide from 0.025 M
Elution was performed with a linear gradient of ~ 0.5 M imidazole (in buffer A) and developed for 60 minutes. The fraction containing the FLINT analog is stored and ultrafree (U
ltrafree) using a centrifugal filter device (Millipore). The collected substance was applied to a 16/60 Superdex 200 size column (manufactured by Pharmacia, which contains PBS, 0.5 M NaCl and 1
(Buffered with 0% glycerol, pH 7.4). Fractions containing the FLINT analog were analyzed by SDS-PAGE. The FLINT N-terminal sequence in the purified polypeptide was confirmed.

Example 10 Biological Characterization of Purified FLINT Analogs FLINT analogs were confirmed in terms of their structural integrity, physical and chemical stability.

Structural analysis of proteins involves assessment of secondary structure by far-ultraviolet CD.
1 mg / mL phosphate buffer solution of FLINT analog (100 μL, pH 7.
4) is a scan of 240 to 180 nm (at a step of 0.5 nm width) at room temperature in a 0.01 cm cell, and a scan of 3 nm on average every 3 seconds with a bandwidth of 1 nm. Near-ultraviolet CD spectroscopy scans 240-350 nm (at 0.5 nm width steps) at room temperature, 1 nm bandwidth, and averages 3 scans every 5 seconds. Natural FLI
Analogs having the same spectrum as that of NT are preferred.

The fluorescence of the endogenous tryptophan was measured using the following parameters. 2
Excitation is performed through a 1 cm cell with an optical path length of 98 nm and a slit width of 2/2 nm. The light emission was collected with an integration time of 1 second. A "blue shift" usually indicates that aromatic residues are buried deeper in the protein structure, and this is often associated with improved pharmaceutical properties.

The quaternary structure of the FLINT analog can be determined by equilibrium sedimentation analysis performed in an ultracentrifuge with a cell width of 3 mm. Analogs having equilibrium sedimentation values comparable to those of native FLINT are preferred.

Analysis of physical stability involves examining the tendency of FLINT analogs to aggregate as a reflection of their surface properties. Physical stability assays are described in the following paragraphs.

Dynamic Light Scattering Assay (DLS): Protein solution was prepared by a) PBS, pH7.
4, b) PBS, 0.5M NaCl or c) PBS, pH 7.4 and 3
When diluted in mg / mL m-cresol, it contains 0.1-5 mg / mL protein. The pH was adjusted to 7.4 (± 0.05) with HCl / NaOH and filtered into 6 × 50 mm borasilica Type-I glass tubes. The average light scattering intensity weighted by particle size was
collected on an avem BI900 instrument. The instrument comprises a 90 degree goniometer, a digital correlator and a Lexel Model 3 adjusted to a 488 nm line.
Consists of a 500 argon ion laser. The experimentally determined autocorrelation function C (t) was analyzed by the cumulant method to obtain the hydraulic diameter. The time before or before the particle size changed significantly was determined by fitting a straight line to the pre-growth and growth data points. The intersection is defined as a delay time. Reduced light scattering by the analog compared to native FLINT at the same concentration and temperature suggests that the analog aggregates to a lesser extent than the native protein.

Indicative Scanning Calorimeter (DSC): Physical stability affecting the melting point (Tm) of proteins was measured by DSC (Indicative Scanning Calorimeter). Usually FLINT
The analog was scanned at a scan rate of 60 ° / hour and the filtering parameters in the range of 5 ° to 100 ° in 16 seconds. A higher melting point usually indicates physical stability.

The chemical stability of the FLINT analog diluted to 0.5 mg / ml was followed by reverse phase HPLC analysis (RP-HPLC) and size exclusion chromatography. The reverse phase method detects at 214 nm and at 40 ° C. Zorbax
Consists of a FLINT optimized acetonitrile / TFA gradient system using a 300SB-C8 column. The size exclusion method consists of a Superdex-75 column (3.2 x 300 mm) (mobile phase is PBS, pH 7.4, room temperature). Changes on the reverse phase chromatogram usually indicate that it is chemically unstable.

Example 11 In Vivo Testing of FLINT Analogs for Treatment of Liver Injury Sensitization with low doses of lipopolysaccharide (LPS) induces acute and massive liver damage in rodents Can be used as a model for liver damage (Infection and Immunit by Tsuji H et al.).
y, 65 (5): 1892-1898 (1997)). The activity of the FLINT analog of the present invention on acute liver failure was measured using a modification of the method reported by Tsuji et al., Supra. Simply put, BALB / c mouse (
Harlan), D (+)-Gactoctosamine (Sigma, 39F-0539)
(6 mg) of PBS (100 μL) and lipopolysaccharide (3 μg) (E. coli)
026: B6, LPS) (Difco, 3920-25-2) in PBS (10
(0 μL), an intravenous injection was made outside the tail. After sensitization with LPS, the animals were injected intraperitoneally with FLINT (200 μg). Suitable controls include hamster IgG at 0, 2, 4, and 6 hours, respectively.
(500 μg, Cappel, 30926), mAb against mouse TNF,
TN3-19.12 (500 μg, as described by Sheehan KCF et al.
Immunol. 1989, 142: 3884) and anti-mouse Fas ligand (500 μg, PharMingen, MO24301). Mice survival was measured 24 and 48 hours after LPS injection.

In one experiment, R218Q FLINT Fc was compared to FLINT produced in AV12 or CHO cells. In this experiment, R218Q FLI
The NT Fc construct was effective as FLINT in preventing animals from acute liver injury (FIG. 2).

Example 12 In Vivo Testing of FLINT Analogs for the Treatment of Pulmonary Shock In order to investigate the effects of FLINT analogs in a pulmonary shock model, a modification of the method described in Example 10 was performed. LPS (200 μl)
g) was used to give FLINT analog (200 μg) at various time points after sensitization. Without treatment, it is expected that those doses of LPS will cause the mice to die.

Example 13 FLINT Analog / FAS Ligand Binding Assay Binding of a FLINT analog to a Fas ligand was determined by real-time biomolecular interaction analysis.
and a binding affinity (e.g., kinetics, specificity, cooperativity, specific binding pattern, etc.)
Concentration) can be measured. To track the interactions of biomolecules, Biacore uses surface plasmon optical phenomena. Detection relies on changes in the weight concentration of macromolecules on the surface specific to the organism.

Materials and Methods: Biacore® 2000 instrument (Biacore AB, Rapsgatan 7
, S-754, 50 Uppsala, Sweden) Sensor chip CM5 (Biacore) Amine coupling kit (Biacore) Washing buffer: HBS-EP (Biacore) Guanidine isothiocyanate solution (GibcoBRL) Fas ligand (Kamiya Biomedical Company, 9)
10 Industry Drive, Seattle, WA98188) FLINT analog Fas / Fc chimera (R & D system)

Solidification protocol: FasL or FLINT analog was loaded into a flow chamber (Sensor C).
immobilized by the primary amine groups of lysine residues on the carboxymethyl dextran polymer bound to the gold surface in HIP CM5). Immobilization is performed using an amine coupling kit (Biacore) according to the manufacturer's protocol.
Briefly, a solution of FasL or FLINT analog (100 μL, 10-25 μg / mL in sodium acetate buffer, 20 mM, pH 5.0) is loaded onto an activated CM5 chip. After the coupling reaction, excess reactive groups on the surface are quenched with 1 M ethanolamine hydrochloride (pH 8.5). The chip is then washed with a sodium acetate buffer (20 mM, pH 5.0) to remove non-covalent substances.

Interaction Analysis: To analyze interactions with FasL and FLINT analogs, FLIN
The solution containing the T analog is passed through the chip on which the Fas ligand is immobilized. F
The amount of FLINT analog associated with asL was measured using the surface plasmon signal (response unit is RU). Typically, a solution of the FLINT analog (
(Different concentrations in HBS-ET buffer) at a flow rate of 5 μL for 10 minutes onto the sensor chip. Next, the chip was washed with HBS-ET buffer (10 mM Hep).
es, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%
Wash for 2 minutes at a flow rate of 5 μL / min using Surfactant P20).

To further analyze the interaction of the FLINT analog with FasL, FasL
Is passed through the chip on which the FLINT analog is immobilized. FLIN
The amount of FasL associated with the T analog was determined by the surface plasmon signal (response unit: RU
Is determined). The protocol of loading, washing and regeneration of the FLINT bioactive chip is as described for the FasL chip above.

Determination of Affinity Constants Experimental data is evaluated using the Biacore Evaluation 3.0.2 software (Biacore) to determine binding parameters.

Example 14 FLINT Analog with Surface N-Glycosylation Site Available A three-dimensional FLINT model was obtained by homology modeling. SEQFOL
D Program (Molecular Simulations Inc. San
FLINT was obtained from Protein Databank using 1tnr (Brookhaven National Laboratory, FL).
1tnr. From Upton NY. pdb) was found to have significant sequence homology. The identity of FLINT with its tumor necrosis factor receptor I was found to be about 30% or more of the 133 amino acids of FLINT at the N-terminus. BLAS
Using the T program, the identity was calculated to be about 36% and the homology was about 50%.

The MODELER program (Molecular Simulations)
Inc. (Available from San Diego, CA), several models were built and the model with the best profile score was selected as the final model. The profile score of the best model is about 28.
It was 3. On the other hand, the profile score of the crystal structure of the template was 29.2. The model can also depend on the template structure for the appropriate disulfide bond.

As in the case of FLINT described above, TNF-β was selected as having the best homology with FasL, and a model was constructed. FasL is about 30% with TNF-β
Have the same identity. And the profile score of the model was 29.2, while the profile score of TNF-β was 39.5.

A three-dimensional model for FLINT and FasL was constructed using tumor necrosis factor (p55) in complex formation with TNF-β (reported as 1tnr.pdb).
Were compounded together in a manner similar to the known crystal structure of This model was minimized to eliminate non-bonded short contacts. The final model of the complex was used for further analysis.

Positions 12 and 34 (along with the substitution of residue 36 with threonine),
By substituting the amino acid residues at positions 35, 132 and 194 with asparagine, a FLINT analog having a novel N-glycosyl site can be obtained.

[0224] The computer modeling and homology modeling described above provide a novel N-
Glycosylation sites are predicted to be exposed on the surface of the molecule. Thus, the new site appears to be glycosylated. The modeling shows that the novel glycosyl site is located sufficiently far away from the site where FLINT binds to the FAS ligand, so that the affinity of the analog for the FAS ligand is not adversely affected by the glycosylation of the FLINT analog It seems.

Example 15 Analysis of FLINT Analogs Containing Alternate Glycosylation Sites FLINT analogs can be analyzed using mass spectrometry and HPLC profile measurements on fluorescently labeled oligosaccharides. In the case of HPLC profile measurement of fluorescently labeled oligosaccharides, FLINT analog (50 to 100
μg) is reduced by DTT. This material is then alkylated with iodoacetic acid. N-linked oligosaccharides from the analog are removed by N-glycosidase F. The protein is precipitated by acetic acid. Next, the cleaved oligosaccharide is labeled with 2-aminobenzamide, and HPLC profile measurement is performed on a weak ion exchanger. A similar method is described in Molecular B
Glycoprotein Analyzes, published in ology series 14,
is in Bioedicine (Elizabeth F. Hunsel)
1 supervised, 1993) (which forms part of the present specification).

Example 16 Production of FLINT Metabolites FINT was purified from AV12 RGT18 cells transfected with a recombinant vector having FLINT cDNA (SEQ ID NO: 1 or SEQ ID NO: 3). This material was added in a weight ratio (ratio of thrombin to FLINT) of 1:10.
With thrombin at room temperature for 3 hours and a 20 mM MOPS (0.
It was dialyzed against 1% CHAPS, pH 6.5) and passed through a SP Sepharose column at a flow rate of 1 mL / min. Allow the column to return until the absorbance returns to baseline.
Washing was performed using buffer A (20 mM MOPS, 0.1% CHAPS, pH 6.5). The bound metabolite (amino acids 1-218 of SEQ ID NO: 1) was eluted using a linear gradient (0-300 mM NaCl in buffer A)
Developed for 1 min, then eluted with a linear gradient (0.3-0.5 M NaCl in buffer A). Fractions were analyzed by SDS-PAGE and mass spectrometry.
Fractions containing only FLINT metabolites (1-218) were pooled and concentrated by Millipore ultrafree centrifugal filters. The concentrated metabolite (1-218
) Was analyzed again by SDS-PAGE and mass spectrometry to confirm the purity. N-terminal sequencing confirmed identity with the purified material as FLINT metabolites (1-218).

Example 17 FLINT Metabolites Perform a Competitive ELISA That Binds LIGHT and FLINT Metabolites Are Flag-tagged LIGHT
Whether to bind to T was measured. Plates were coated with 2 μg / mL FLINT. In some wells, sLIGHT was used as a control with BSA / PBS, FLINT metabolite, FLINT or HVEM-
Incubation was performed with Fc. After a 30 minute incubation, the mixture was added to the FLINT coated wells. Some free sL
IGHT will bind to FLINT coated on the plate.
The amount of sLIGHT bound to FLINT was measured using 1 μg / mL of FLAG antibody. Detection was performed using 1: 1000 strep / HRP. The data showed that, like FLINT, its metabolites bound LIGHT.

[0228] The medium containing Example 18 mass scale R218Q FLINT-Fc (hinge none) polypeptide of refining and biophysical confirmed R218Q FLINT-Fc (hingeless), Amico
Concentrate in Amicon ProFlux M12 tangential filtration 10X using nS3Y10 UF membrane. The medium containing R218Q FLINT-Fc (hinge pulled) was conditioned with 0.5 M NaCl and 5 mM EDTA. The concentrated medium was passed through a Protein A HiTrap column (Pharmacia, 5 mL column) at a flow rate of 5 mL / min. The column was washed with buffer A (PBS (1 mM potassium phosphate, 3 mM sodium phosphate), 0.5 M NaCl, pH 7.4) until the absorbance returned to baseline, and the bound poly The peptide was added to 100% buffer B (50 mM citric acid, 0.5 mM NaCl, p
H 3.5). The eluted substance was added per mL of elution buffer,
Neutralized with 100 μL of 1 M Tris (pH 8.0). R218Q FL
The fractions containing INT-Fc were pooled and stored in Superdex 75 (Pharmacia,
A 16/60) size column, which was buffered with PBS, 0.5 M NaCl, 10% glycerol, pH 7.4, was passed at a flow rate of 1 mL / min.
Fractions containing R218Q FLINT-Fc were analyzed by SDS-PAGE. The N-terminal sequence of R218Q FLINT-Fc on the purified polypeptide was confirmed.

Equilibrium sedimentation analysis indicated that R218Q FLINT-Fc was a dimer with a molecular weight of 130 kilodaltons. By differential scanning calorimetry, R218Q F
The LINT-Fc construct was shown to have increased conformational stability and a melting point of about 10 ° C. higher than that of wild-type FLINT (64 ° C. versus 54 ° C.). No change in mean hydraulic diameter at 37 ° C. for 24 hours at で 1 mg / mL protein was observed by differential light scattering analysis.

Example 19 Molecular Cloning of FLINT R218Q-Fc The construction of two FLINT R218Q-Fc fusions is described in the Examples below. Plasmids pJB02 and pJB03 were modified pEAK vectors (Edge BioSystems, Gaithersburg, MD)
Which is used for high production of proteins in transiently transfected 293EBNA cells.

The starting plasmid containing FLINT R218Q, pJB03 / R2
18Q was digested with the restriction endonuclease Eco47III and
The 624 bp fragment containing most of the 3 'portion of the T sequence was gel purified.

Embedded image

Plasmids pJB02 / Flint: Fc (+ thrombin-hinge) and p
JB03 / FLINT: Fc (-hinge) was digested with Eco47III,
The 6.5 kb scaffold was isolated, treated with phosphatase, and gel purified.

Embedded image

[0233] DNA sequencing was performed on the recombinant plasmid to confirm the presence of the correct insert.

Embedded image

Embedded image

Example 20 Expression of R218Q FLINT Immunoadhesin The R218Q and WT FLINT / Fc constructs were expressed in 293EBNA cells transiently transfected for purification. Transfect is
Roller bottles in 700 cm ^2, 293EBNA cells (which the monolayer is confluent, DMEM F12 (3: 1) , 5% FBS, 20mM HEPES, 2
(grown in mM L-glutamine) using 50 μg / ml G418. The transfected cocktail was mixed with plasmid DNA (65 μg) and Roche Molecular Biomedi per roller bottle.
Cals transfection reagent FuGene was used in Gibco optimized media. Forty-eight hours after transfection, the monolayer was rinsed with PBS and replaced with serum-free medium (Hybritech). Cells, CO ₂ levels, followed for glutamine levels and glucose levels, 10 days, was collected every 24 hours. Typical expression levels were 2-5 μg / ml.

Equivalents Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents for the particular embodiments of the invention described herein. . Those equivalents are intended to be encompassed by the following claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the results of the assay in Jurkat cells in an in vitro cell-based apoptosis assay.

FIG. 2 shows the results of experiments comparing R218Q FLINT Fc with FLINT produced in AV12 or CHO cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C07K 19/00 C07K 14/47 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/02 A 1/21 C12N 15/00 ZNAA 5/10 5/00 A C12P 21/02 A61K 37/02 (31) Priority claim number 60/140, 156 (32) Priority Date June 21, 1999 (June 21, 1999) (33) Priority claim country United States (US) (31) Priority claim number 60 / 160,566 (32) Priority date October 20, 1999 (1999.10.20) (33) Priority claim country United States (US) (31) Priority claim number 60 / 183,398 (32) Priority date February 18, 2000 (2000.18.18) ( 33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY) DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, P , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW Fredrick J. Cohen 18940 Autumn Drive, Newtown, PA, USA (72) Inventor Patricia Ann Gonzales-Dewitt 46601 East One Hundred, Noblesville, Indiana 12121, Ninety First Street (72) Inventor John Edward Hale United States 46038 Fishers, Indiana, Forest Drive 7644 (72) Inventor Radomira Mikanovic United States 46236 Indianapolis, Indiana White Oak Trail 7126 (72) Inventor Christie Michelle Newton 46259 Meadow Bend Circle, Indianapolis, Indiana, No. 7908 (72) Inventor Timothy Wayne Noblitt, United States 46038 Charleston Parkway, Fishers, Indiana 11515 (72) Inventor Radha Krishnan Ratnacharam United States 46032 Carmel, Indiana Lattice Court 3793 (72) Inventor Shen-Hun Rainbow Chan United States 46033 Indianamerica Carmel, Riley Muse 4963 (72) Inventor Derrick Ryan Witcher United States 46038 Indiana Fishers, Parrot Court No. 10898 (72) Inventor Victor John Lowbleski, Wood Pond South Roundboutout 1466, Carmel, Indiana 46460, USA F-term (reference) 4B024 AA01 CA04 FA02 GA11 HA17 4B064 AG20 CA21 DA01 4B065 AB01 BA02 CA44 4C084 AA02 AA07 BA01 BA08 BA22 BA23 CA53 CA59 DC50 NA14 ZA592 ZA662 ZB212 ZC022 4H045 AA10 AA20 AA30 BA42 CA40 DA50 EA23 EA25 FA16

Claims (52)

[Claims] 1. A FLINT analogue, comprising a polypeptide having the biological activity of FLINT or a fragment thereof, the polypeptide comprising: a) replacing tryptophan at position 53 with aspartic acid; b. C) replacing threonine at position 88 with proline; c) replacing alanine at position 107 with serine, aspartic acid, glutamic acid or threonine; d) replacing isoleucine at position 110 with threonine or glutamic acid; or e) position 104 FLINT analogs having the amino acid sequence of SEQ ID NO: 1 modified by substituting proline with serine, and pharmaceutically acceptable salts of the polypeptides and fragments thereof. 2. A FLINT analogue, comprising a polypeptide having the biological activity of FLINT or a fragment thereof, wherein the polypeptide comprises: a) replacing alanine at position 2 or 12 with asparagine. B) replacing proline at position 25, 38, 126 or 171 with asparagine; c) replacing arginine at position 35 with asparagine; d) replacing serine at position 37 with asparagine and Replacing proline with another naturally occurring amino acid; e) replacing serine at position 166 with asparagine; f) replacing leucine at position 172 with asparagine; g) replacing aspartic acid at position 194 with asparagine; Or h) replacing threonine at position 114 with asparagine and FLINT analogs having the amino acid sequence of SEQ ID NO: 1 modified by replacing phosphorus with other naturally occurring amino acids, and pharmaceutically acceptable salts of the polypeptides and fragments thereof. 3. A FLINT analog, comprising a polypeptide having the biological activity of FLINT or a fragment thereof, wherein the polypeptide comprises: a) replacing asparagine at position 63 with tryptophan; b) Replace glycine at position 67 with aspartic acid and replace alanine at position 94 or glycine at position 95 with tryptophan; c) replace arginine at position 69 with glutamic acid; d) replace arginine at position 82 with glutamic acid or threonine. E) replacing alanine at position 94 with tyrosine and replacing glycine at position 95 with aspartic acid; g) replacing alanine at position 101 with threonine: or h) replacing glycine at position 95 with asparagine SEQ ID NO: 1 modified by substitution with an acid FLINT analogs having a amino acid sequence, and pharmaceutically acceptable salts of the polypeptides and fragments thereof. 4. A FLINT analog comprising a polypeptide having FLINT biological activity or a fragment thereof, wherein the polypeptide comprises: a) arginine at position 10 as glutamine, asparagine, serine or threonine. (However, when the amino acid to be substituted is asparagine, 12
Alanine at position 13 is optionally replaced with serine or threonine); b) glutamic acid at position 13 is replaced with glutamine, asparagine, serine or threonine (provided that the amino acid to be replaced is asparagine;
Glycine at position 5 is replaced with serine or threonine); c) glutamic acid at position 16 is replaced with glutamine, asparagine, serine or threonine (provided that when the amino acid to be substituted is asparagine, 1
Leucine at position 8 is optionally replaced with serine or threonine); d) Arginine at position 17 is replaced with glutamine, asparagine, serine or threonine (provided that the amino acid to be replaced is asparagine, 19)
E) optionally replacing valine at position 31 with serine or threonine; e) replacing arginine at position 31 with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine;
Cysteine at position 13 is optionally replaced with serine or threonine); f) Arginine at position 34 is replaced with glutamine, asparagine, serine or threonine (provided that when the amino acid to be replaced is asparagine, 36
G) optionally replacing arginine at position 35 with glutamine, asparagine, serine or threonine; h) replacing aspartic acid at position 36 with glutamine, asparagine, serine or threonine. Substitution (however, when the amino acid to be substituted is asparagine,
Optionally, replacing proline at position 38 with serine or threonine); i) replacing arginine at position 143 with glutamine, asparagine, serine or threonine (provided that the amino acid to be substituted is asparagine;
Cysteine at position 145 is optionally replaced with serine or threonine); or j) replacing aspartic acid at position 161 with glutamine, asparagine, serine or threonine (provided that the amino acid to be replaced is asparagine, 163) FLINT analogs having the amino acid sequence of SEQ ID NO: 1 modified by replacing leucine at position 1 with serine or threonine, and pharmaceutically acceptable salts of the polypeptides and fragments thereof. 5. A FLINT analog, comprising a polypeptide having FLINT biological activity or a fragment of the polypeptide, wherein the polypeptide comprises: a) position 2, 12, 107, 179 or Alanine at position 209 is replaced with threonine; b) threonine at position 4 or 162 is replaced with alanine; c) valine at position 1 or isoleucine at position 110 is replaced with methionine; d) glutamic acid at position 13 is asparagine. E) replacing arginine at position 17 with tryptophan; f) replacing alanine at position 75 with proline; g) replacing serine at position 102 with leucine h) replacing glycine at position 169 with alanine I) replacing glutamic acid at position 183 with lysine; j) gluta at position 225 K) replacing valine at position 237 with glutamic acid; or 1) a FLINT analog having the amino acid sequence of SEQ ID NO: 1 which has been modified by replacing valine at position 270 with glycine; Pharmaceutically acceptable salts of the polypeptide and fragments thereof. 6. A FLINT analog, comprising a polypeptide having FLINT biological activity or a fragment thereof, the polypeptide comprising: a) replacing alanine at position 12 with asparagine; Glutamic acid at position 13 is replaced with glutamine; b) arginine at position 34 is replaced with asparagine, and aspartic acid at position 36 is replaced with threonine; c) arginine at position 35 is replaced with asparagine, optionally at position 37. D) replacing serine at position 132 with asparagine and optionally replacing serine at position 134 with threonine; e) replacing aspartic acid at position 194 with asparagine and optionally replacing position 196 with asparagine. Replacing serine with threonine; f) arginine at position 35 and Replacing aspartic acid at position 194 with asparagine; g) replacing alanine at position 12 with asparagine, optionally replacing glutamic acid at position 13 with glutamine, and replacing aspartic acid at position 194 with asparagine, optionally replacing 196 Replacing serine at position 34 with threonine; h) replacing arginine at position 34 with asparagine, replacing aspartic acid at position 36 with threonine, replacing aspartic acid at position 194 with asparagine, and optionally replacing serine at position 196 with asparagine. I) replacing arginine at position 35 and aspartic acid at position 194 with asparagine and replacing serine at position 37 and / or 196 with threonine;
Or j) a FLINT analog having the amino acid sequence of SEQ ID NO: 1 modified by replacing arginine at position 218 with glutamine, and pharmaceutically acceptable salts of the polypeptides and fragments thereof. 7. The FLINT fragment of any one of claims 1 to 6, wherein the fragment comprises residues 1-218 of SEQ ID NO: 1. 8. A fragment of a FLINT polypeptide which is biologically active in vivo and / or in vitro. 9. The biological activity comprises: a. Binding to FasL in vivo or in vitro; b. Binding to LIGHT in vivo or in vitro; or c. 9. The FLINT fragment of claim 8, wherein the FLINT fragment is selected from the group consisting of treating a disorder that can be treated with FLINT. 10. The biologically active FLINT polypeptide according to claim 8, wherein the polypeptide comprises residues 1-218 of SEQ ID NO: 1. 11. The biologically active polypeptide fragment of claim 8, wherein the polypeptide consists essentially of residues 1-218 of SEQ ID NO: 1. 12. The FLINT polypeptide fragment of claim 8, wherein the fragment comprises residues 1-216 of SEQ ID NO: 1. 13. The FLINT polypeptide fragment of claim 11, which is produced by digesting FLINT with an enzyme. 14. The FLINT polypeptide fragment according to claim 13, which is produced by digesting FLINT with an enzyme using a serine protease. 15. The FLINT polypeptide fragment of claim 14, wherein the protease is thrombin or trypsin. 16. The FLINT fragment according to claim 10, which is produced by a recombinant method. 17. Structural formula: embedded image Wherein Fc is the Fc fragment of the antibody. Each X is independently claim 1, 2, 3, 4, 5, 6, 7, 10, 11 or 12;
The peptide derivative according to the above. And each polypeptide indicated by X is covalently linked at its C-terminus to one N-terminus of the polypeptide forming the Fc fragment of the antibody]. 18. Structural formula: embedded image Wherein Fc is the Fc fragment of the antibody. Each X is a protease resistant FLINT analog. And each polypeptide indicated by X is covalently linked at its C-terminus to one N-terminus of the polypeptide forming the Fc fragment of the antibody]. 19. The method according to claim 1, wherein the Fc is an Fc fragment derived from a human antibody.
8. The protein according to 7. 20. The Fc is a human antibody-derived Fc fragment.
9. The protein according to 8. 21. The Fc lacking the hinge region.
A protein according to claim 1. 22. The protein of claim 10, wherein the analog comprises SEQ ID NO: 1 wherein arginine at position 218 has been replaced by glutamine. 23. The protein of claim 18, wherein the analog has an amino acid sequence that is at least about 50% identical to residues 214-222 of SEQ ID NO: 1. 24. The analog comprises an Arg at position 218 a. A non-Arg, naturally occurring amino acid; b. Any positively charged amino acid that is not Arg; c. Any non-Arg, negatively charged amino acid; d. A polar, uncharged amino acid that is not Arg; e. A non-polar amino acid that is not Arg; and f. 19. The protein according to claim 18, comprising the amino acid sequence of SEQ ID NO: 1 substituted by an amino acid selected from the group consisting of Glu, Gln, Ala, Gly, Ser, Val, and Tyr. 25. A nucleic acid encoding any one of the FLINT analogs according to claim 1. 26. A nucleic acid encoding any one of the FLINT fragments according to claims 7 to 12. 27. A nucleic acid encoding the fusion protein according to claim 17. 28. A nucleic acid encoding the fusion protein according to claim 18. 29. A vector containing the nucleic acid according to claim 25. 30. A vector containing the nucleic acid according to claim 26. A vector containing the nucleic acid according to claim 27. A vector comprising the nucleic acid according to claim 28. 33. A host cell transformed with the vector according to claim 29. 34. A host cell transformed with the vector according to claim 30. 35. A host cell transformed with the vector according to claim 31. 36. A host cell transformed with the vector according to claim 32. 37. A polydeoxynucleotide or a fragment thereof, the polydeoxynucleotide comprising: a) replacing the codon encoding tryptophan at positions 157-159 with a codon encoding aspartic acid; b) 262-264. C) replacing the codon encoding threonine with a codon encoding proline; c) replacing the codon encoding alanine at positions 319-321 with a codon encoding serine, aspartic acid, glutamic acid or threonine; d). Modifying the codon encoding isoleucine at positions 328-330 with a codon encoding threonine or glutamic acid; or e) modifying the codon encoding proline at positions 310- 312 with a codon encoding serine. did A polydeoxynucleotide or fragment thereof, having the nucleotide sequence of SEQ ID NO: 2, wherein the fragment comprises 145-495 deoxynucleotides of the polydeoxynucleotide. 38. A polydeoxynucleotide or a fragment thereof, the polydeoxynucleotide comprising: a) replacing the codon encoding alanine at positions 4-6 with a codon encoding asparagine; b) positions 73-75. C) replacing the codon coding for proline with a codon coding for asparagine; c) replacing the codon coding for arginine at positions 103-105 with a codon coding for asparagine; d) coding for serine at positions 109-111 Is replaced with a codon encoding asparagine, and a codon encoding proline at positions 112-114 is replaced with a codon encoding any of the other naturally occurring amino acids; e) positions 112-114. Codon encoding proline and codon encoding asparagine F) replacing the codon encoding proline at positions 376-378 with a codon encoding asparagine; g) replacing the codon encoding serine at positions 496-498 with a codon encoding asparagine. H) replacing the codon encoding proline at positions 511-513 with a codon encoding asparagine; i) replacing the codon encoding leucine at positions 514-516 with a codon encoding asparagine; j) 580 Replacing the codon encoding aspartic acid at positions ５８582 with a codon encoding asparagine; k) replacing the codon encoding threonine at positions 340-342 with a codon encoding asparagine, and 343-345. Replace the codon encoding proline with any of the naturally occurring amino acids Or 1) having the nucleotide sequence of SEQ ID NO: 2 modified by replacing the codon encoding arginine at positions 652-654 with a codon encoding asparagine; The polydeoxynucleotide or a fragment thereof, wherein the fragment comprises 145 to 495 deoxynucleotides of the polydeoxynucleotide. 39. A polydeoxynucleotide or a fragment thereof, the polydeoxynucleotide comprising: a) replacing the codon encoding asparagine at positions 187-189 with a codon encoding tryptophan; b) positions 199-201. Is substituted with a codon encoding aspartic acid, and a codon encoding alanine at positions 280-282 or a codon encoding glycine at positions 283-285 is substituted with a codon encoding tyrosine. C) replacing the codon encoding arginine at positions 205-207 with a codon encoding glutamic acid; d) replacing the codon encoding arginine at positions 244-246 with a codon encoding glutamic acid or threonine; e. ) 280-2 The codon encoding alanine at position 82 is replaced with a codon encoding tyrosine, and the codon encoding glycine at positions 283-285 is substituted with a codon encoding aspartic acid; f) phenylalanine at positions 286-288; G) is replaced with a codon encoding glutamine; g) a codon encoding alanine at positions 301 to 303 is substituted with a codon encoding threonine; or i) glycine encoding positions 283 to 285 is encoded. Having the nucleotide sequence of SEQ ID NO: 2 modified by substituting a codon for aspartic acid with a codon encoding aspartic acid, the fragment comprising 145 to 495 deoxynucleotides of the polydeoxynucleotide, That fragment G. 40. A polydeoxynucleotide or a fragment thereof, the polydeoxynucleotide comprising: a) replacing the codon encoding arginine at positions 28-30 with a codon encoding glutamine, asparagine, serine or threonine ( However, in the case of substitution with a codon encoding asparagine, the codon encoding alanine at positions 34 to 36 is optionally substituted with a codon encoding serine or threonine); b) encoding glutamic acid at positions 37 to 39 Is substituted with a codon encoding glutamine, asparagine, serine or threonine (however, when the codon encoding asparagine is substituted, the codon encoding glycine at positions 43 to 45 is optionally replaced with serine or threonine. The C) replacing the codon encoding glutamic acid at positions 46 to 48 with a codon encoding glutamine, asparagine, serine or threonine (however, when substituting a codon encoding asparagine) , Optionally replacing leucine at positions 52-54 with a codon encoding serine or threonine); d) replacing the codon encoding arginine at positions 49-51 with a codon encoding glutamine, asparagine, serine or threonine. (However, when the codon encoding asparagine is substituted, the codon encoding valine at the 55-57 position is optionally substituted with a codon encoding serine or threonine); e) arginine at the 91-93 position Codons that encode glutamine and ass Substitution with a codon encoding lagin, serine or threonine (however, when substituting a codon encoding asparagine, a codon encoding cysteine at positions 97 to 99 is optionally substituted with a codon encoding serine or threonine) F) replacing the codon encoding arginine at positions 100-102 with a codon encoding glutamine, asparagine, serine or threonine (however, when substituting a codon encoding asparagine, positions 106-108) G) optionally replacing the aspartic acid with a codon encoding serine or threonine); g) replacing the codon encoding arginine at positions 103-105 with a codon encoding glutamine, asparagine, serine or threonine; h) 106 ~ A codon encoding aspartic acid at position 108, glutamine;
Substitution with a codon encoding asparagine, serine or threonine (however, when substituting a codon encoding asparagine, a codon encoding proline at positions 112 to 114 is optionally substituted with a codon encoding serine or threonine) I) replacing the codon encoding arginine at positions 427-429 with a codon encoding glutamine, asparagine, serine or threonine (provided that
(When replacing with a codon encoding asparagine, cysteines at positions 433 to 435 are optionally substituted with a codon encoding serine or threonine.)
Or j) replacing the codon encoding aspartic acid at positions 481-483 with glutamine,
Substitution with a codon encoding asparagine, serine or threonine (however, when substituting with a codon encoding asparagine, a codon encoding leucine at positions 487 to 489 is optionally substituted with a codon encoding serine or threonine) Or a fragment thereof comprising the nucleotide sequence of SEQ ID NO: 2, wherein the fragment comprises 145 to 495 deoxynucleotides of the polydeoxynucleotide. 41. A polydeoxynucleotide or a fragment thereof, the polydeoxynucleotide comprising: a) alanine at positions 4-6, 34-36, 319-321, 535-537 or 625-627. Replacing the encoding codon with a codon encoding threonine; b) replacing the codon encoding threonine at positions 10-12 or 484-486 with a codon encoding alanine; c) valine at positions 1-3 Alternatively, the codon encoding isoleucine at positions 328-330 is replaced with a codon encoding methionine; d) the codon encoding glutamic acid at positions 37-39 is substituted with a codon encoding aspartic acid; e) 49- The codon encoding arginine at position 51 is replaced with tryptophan F) Replace the codon encoding alanine at positions 223-225 with a codon encoding proline; g) Replace the codon encoding serine at positions 304-306 with a codon encoding leucine. H) replacing the codon encoding glycine at positions 505-507 with a codon encoding alanine; i) replacing the codon encoding glutamic acid at positions 547-549 with a codon encoding lysine; j) 673 Replacing the codon encoding glutamine at positions ６675 with a codon encoding arginine; k) replacing the codon encoding glycine at positions 709-711 with a codon encoding glutamic acid; or m) 808- Codon encoding valine at position 810 is replaced with a codon encoding glycine Has a nucleotide sequence of SEQ ID NO: 2 which is modified by its fragments containing 145-495 deoxynucleotides of the polydeoxynucleotide, polydeoxynucleotide or fragment thereof. 42. A vector containing the polydeoxynucleotide according to any one of claims 37, 38, 39, 40 or 41. 43. Use of a FLINT analog for treating a disease or condition in a patient in need of such treatment. 44. The binding of FasL and Fas, or LIGHT and LTβR.
And / or a method of treating a patient suffering from a disease or condition associated with binding to TR2 / HVEM, comprising administering a therapeutically effective amount of a FLINT analog. 45. An analog comprising FLI containing residues 1-218 of SEQ ID NO: 1.
45. The method of claim 44, which is an NT fragment. 46. The method of claim 44, wherein said analog comprises a protease resistant FLINT-Fc fusion protein. 47. The method of claim 44, wherein the disease is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis, chronic obstructive pulmonary disease, ulcerative colitis, or Crohn's disease. 48. Use of a FLINT analog in the manufacture of a medicament useful for treating a disease or disorder associated with abnormal apoptosis. 49. Use of a FLINT analogue according to claim 48, wherein the disorder is selected from the group consisting of acute lung injury, acute respiratory distress syndrome, pulmonary fibrosis, chronic obstructive pulmonary disease, ulcerative colitis or Crohn's disease. . 50. A pharmaceutical composition comprising a FLINT analog together with one or more pharmaceutically acceptable diluents, carriers or excipients. 51. The pharmaceutical composition according to claim 50, wherein the analog comprises the FLINT fragment of SEQ ID NO: 1 from 1 to 218. 52. The pharmaceutical composition according to claim 50, wherein the analog comprises a protease resistant FLINT-Fc fusion protein.