Internet for Pharmaceutical and Biotech Communities
| Newsletter | Post Jobs | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  IL-16 antagonists
United States Patent: 
7,019,118
Issued: 
March 28, 2006
Inventors: 
Center; David M. (Wellesley Hills, MA); Cruikshank; William W. (Westford, MA); Kornfeld; Hardy (Wellesley Hills, MA)
Assignee: 
Trustees of Boston University (Boston, MA)
Appl. No.: 
929924
Filed: 
August 15, 2001


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

In accordance with the present invention, novel IL-16 antagonists, preferably peptides derived from CD4, have been isolated and synthesized. These peptides possess IL-16 antagonistic properties including the ability to selectively bind to IL-16 and inhibit IL-16-mediated biological activity. The peptides comprise specific portions of the native human CD4 receptor and variations thereof and therefore are non-immunogenic when administered to humans. The present invention also provides compositions containing at least one IL-16 antagonist peptide which can inhibit, suppress or cause the cessation of at least one IL-16-mediated biological activity in mammals, including humans. The present invention provides a method and composition for treating inflammation associated with disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease (IBD) and systemic lupus (SLE) in mammals such as, for example, humans.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to Interleukin-16 (IL-16) antagonists. By "IL-16 antagonist" is meant any molecule that inhibits, suppresses or causes the cessation of at least one IL-16-mediated biological activity by, e.g., interfering with, blocking or otherwise preventing the interaction or binding of IL-16 to an IL-16 receptor, e.g., the CD4 receptor.

More specifically, the present invention provides IL-16 antagonist peptides which substantially correspond to amino acid sequences found in specific portions of the CD4 receptor. The peptides of the present invention correspond to sequences found in the immunoglobulin-like domain 4 (D4) extracellular region of the CD4 and can inhibit the activity of IL-16. Surprisingly, the present inventors have found that such IL-16 inhibiting peptides can be as short as 4 amino acids in length.

As used herein, "peptide" refers to a linear series of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acid residues. The term "synthetic peptide" is intended to refer to a chemically derived chain of amino acid residues linked together by peptide bonds. The term "synthetic peptide" is also intended to refer to recombinantly produced peptides in accordance with the present invention.

The sequences of the peptides of the present invention are derived from and/or correspond to the amino acid sequence of murine CD4 domain 4, however, homologous peptides derived from human, rat and other mammalian CD4 sequences are also encompassed by the invention. It is known that mouse and human CD4 are substantially homologous in amino acid sequence, with the homology being about 63%. It is known that IL-16 is cross reactive between species Keane, et al. (1998) J. Immunol. 160:5945.

By "IL-16 antagonist peptide" is meant a peptide that inhibits, suppresses or causes the cessation of at least one IL-16-mediated biological activity by e.g., binding to IL-16, interfering with, or preventing the binding of IL-16 to the CD4 receptor. An IL-16 antagonist functions in two ways. The antagonist can bind to or sequester IL-16 with sufficient affinity and specificity to substantially interfere with, block or otherwise prevent binding of IL-16 to an IL-16 receptor, thereby inhibiting, suppressing or causing the cessation of at least one IL-16-mediated biological activity, such as T-cells chemotaxis, for example. This type of IL-16 antagonist, also termed a "sequestering antagonist" is a specific feature of this invention. Alternatively, an IL-16 antagonist can compete with IL-16 for the cell surface receptor thereby interfering with, blocking or otherwise preventing the binding of IL-16 to an IL-16 receptor. This type of antagonist, e.g., which binds the receptor but does not trigger signal transduction, is also referred to herein as a "competitive antagonist". The contemplated "competitive antagonists" are, more specifically, described in commonly owned co-pending application Ser. No. 09/368,632, filed on Aug. 5, 1999, entitled "IL-16 Antagonists", the disclosure of which is incorporated herein by reference. The peptide antagonists are useful in the therapy of immunoinflammatory responses. Additionally, analogs, homologs and fragments of the novel peptides provided herein are included within the scope of the term "IL-16 antagonist peptide".

According to the present invention, preferred IL-16 antagonists include peptides (referred to herein as "IL-16 antagonist peptides") and antibodies.

By "IL-16-mediated biological activity" as used herein is meant chemotaxis of CD4+ cells such as CD4+ T cells, inhibition of retroviral replication (such as inhibition of HIV and SIV in infected PBMCs), upregulation of IL-2R on CD4+ T cells, synergy with IL-2 for CD4+ T cell proliferation, induction of RAG-1 and RAG-2 expression in CD4+ pro-B cells, and inhibition of Mixed Lymphocyte Reaction (MLR). These IL-16 mediated biological activities can be determined using the assays described by Cruikshank et al. (Proc. Natl. Acad. Sci. USA 91: 5109-5113, 1994); Maciaszek et al. (J. Immunol. 158:5, 1997), Zhou, et al. (Nature Medicine 3:659, 1997) and Baier et al. (Nature 378:563, 1995); Parada et al. (J. Immunol. 160:2115, 1998); Szabo et al. (J. Immunol., 161:2248, 1998); and Theodore et al. (J. Immunol. 157:1958, 1996), respectively. The teachings of these references are incorporated herein by reference.

By "homologs" is meant the corresponding peptides from CD4 proteins of other mammalian species substantially homologous at the overall protein (i.e., mature protein) level to human or murine CD4, so long as such homologous peptides retain the IL-16 antagonist activity.

By "analogs" is meant peptides which differ by one or more amino acid alterations, which alterations, e.g., substitutions, additions or deletions of amino acid residues, do not abolish the IL-16 antagonist properties of the relevant peptides.

According to the present invention, an IL-16 antagonist peptide is at least 4 amino acids in length and substantially corresponds to the amino acids comprising the D4 domain of human or murine CD4 surrounding the Leu-Leu motif, i.e., L348-L349 of human CD4 D4 or L347-L348 of murine CD4 D4.

Thus, an analog may comprise a peptide having a substantially identical amino acid sequence to a peptide provided herein and in which one or more amino acid residues have been conservatively or non-conservatively substituted. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another. Likewise, the present invention contemplates the substitution of one polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of one acidic residue such as aspartic acid or glutamic acid for another is also contemplated. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residues such as cyteine, glutamine, glutamic acid, lysine and/or a polar residue for a non-polar residue.

The phrase "conservative substitution" also includes the use of chemically derivatized residues in place of a non-derivatized residues as long as the peptide retains the requisite IL-16 antagonist, inhibition or suppression properties as conventionally measured by the MLR assay (Theodore et al (1996) J. Immunol. 157:1958-1964). Analogs also include the presence of additional amino acids or the deletion of one or more amino acids which do not affect IL-16-mediated biological activity. For example, analogs of the subject peptides can contain an N- or C-terminal cysteine, by which, if desired, the peptide may be covalently attached to a carrier protein, e.g., albumin. Such attachment, it is believed, will minimize clearing of the peptide from the blood and also prevent proteolysis of the peptides. In addition, for purposes of the present invention, peptides containing D-amino acids in place of L-amino acids are also included in the term "conservative substitution." The presence of such D-isomers can help minimize proteolytic activity and clearing of the peptide.

A preferred IL-16 antagonist peptide of the present invention is a tetrameric peptide having the sequence Xaa1-L-L-Xaa2 (SEQ ID NO:1), wherein Xaa1 and Xaa2 can be any amino acid which includes

A=Ala=Alanine

R=Arg=Arginine

N=Asn=Asparagine

D=Asp=Aspartic acid

B=Asx=Asparagine or aspartic acid

C=Cys=Cysteine

Q=Gln=Glutamine

E=Glu=Glutamic acid

Z=Glx=Glutamine or Glutamic acid

G=Gly=Glycine

H=His=Histidine

I=Ile=Isoleucine

L=Leu=Leucine

K=Lys=Lysine

F=Phe=Phenylalanine

P=Pro=Proline

S=Ser=Serine

T=Thr=Threonine

W=Trp=Tryptophan

Y=Tyr=Tyrosine

V=Val=Valine

Preferably, Xaa1 and Xaa2 are those amino acids found in the native sequence of a mammalian CD4. For example Xaa1 can be Cys (human or murine) and Xaa2 can be Ser (human or murine). Homologs and analogs of this tetrameric peptide are also contemplated by the present invention.

More preferably, Xaa1LLXaa2 is a tetrameric peptide identical to the native sequence of a human CD4. For example, CLLS (SEQ ID NO:2) is most preferred.

Another preferred IL-16 antagonist peptide of the present invention is a six-residue peptide having the sequence of Xaa1-Xaa2-Xaa3-Leu-Leu-Xaa4 (SEQ ID NO:3), wherein Xaa1-4 can be any amino acid.

Preferably, Xaa1-4 are those amino acids found in the native sequence of a mammalian (e.g. murine and human) CD4 at the relevant position. For example, Xaa1 can be Trp, Xaa2 can be Gln or Ala, Xaa3 can be Cys or Ala and Xaa4 can be Ser.

Even more preferably, Xaa1-Xaa2-Xaa3-L-L-Xaa4 is a 6-mer identical to the native sequence of human or murine CD4. An example of such a 6-mer includes SEQ ID NO:4 WQCLLS. Homologs and analogs of this 6-mer which maintain IL-16 antagonist activity are also contemplated by the present invention. Examples of such homologs and analogs include: WQALLS (SEQ ID NO:5) WACLLS (SEQ ID NO:6) and WQCELS (SEQ ID NO:7).

Still another preferred IL-16 antagonist peptide of the present invention is a 6-mer having the sequence of Xaa1-Val-Xaa2-Val-Xaa3-Xaa4 (SEQ ID NO:8) wherein Xaa1-4 can be any amino acid.

Preferably, Xaa1-4 are those amino acids found in the native sequence of a mammalian (e.g. murine and human) CD4 at the relevant position. For example, Xaa1 can be Val, Xaa2 can be Gln, Xaa3 can be Val and Xaa4 can be Ala.

Even more preferably, Xaa1-Val-Xaa2-Val-Xaa3-Xaa4 is a 6-mer identical to the native sequence of human or murine CD4. An example of such a 6-mer includes SEQ ID NO:9 VVQVVA. Homologs and analogs of this 6-mer are also contemplated by the present invention. Examples of such homologs and analogs include: VKQVVA (SEQ ID NO:10) and VVQKVA (SEQ ID NO:11).

Further, according to the present invention an IL-16 antagonist peptide can be longer than a tetrameric and a 6-mer and composed of up to about 75 amino acids, as long as the antagonist peptide contains as part of the peptide, one or more of the tetrameric or 6-mer sequences described hereinabove, i.e. Xaa1LLXaa2, Xaa1-Xaa2-Xaa3-L-L-Xaa4 or Xaa1-V-Xaa2-V-Xaa3-Xaa4, and preferably Xaa1LLXaa2. Preferably, the antagonist peptide contains less than about 32 amino acids and more preferably less than about 16 amino acids.

Preferred antagonist peptides include those having sequences which coincide with the native sequence of a CD4 starting from Asn302 for human CD4, or the corresponding positions of other mammalian CD4 molecules, such as, for example Thr301 for murine CD4. Examples of such "longer" peptides include GMWQCLLSDSGQVLLE (SEQ ID NO:12), GMWQCLLS (SEQ ID NO:13), TGLWQCLLSEGD (SEQ ID NO:14), VSEEQKVVQVVA (SEQ ID NO:15), NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLE SNIKVLPTWSTPVQPM (SEQ ID NO:16) and TLTCEVMGPTSPKMRLTLKQENQEARVSEEQKVVQVVAPETGLWQCLLSEGDKVKMD SRIQVLSRGVNQTVF (SEQ ID NO:17) and homologs and analogs of the "longer" peptides.

As used herein, the term "substantially corresponds" is meant the degree of amino acid homology of at least about 60% homology, preferably at least about 70%, and more preferably at least about 75%, which degree is the similarity index calculated using the Lipman-Pearson Protein Alignment program with the following choice of parameters: Ktuple=2, Gap penalty=4, and Gap Length Penalty=12.

The term "fragment" refers to any subject peptide having an amino acid sequence shorter than that of any peptide depicted in SEQ ID NOS: 12-17 which contains at least one tetramer or hexamer of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8 (i.e., Xaa1LLXaa2, Xaa1-Xaa2-Xaa3-L-L-Xaa4 or Xaa1-V-Xaa2-V-Xaa3-Xaa4), and which fragment retains the IL-16 mediated antagonist activity of the subject peptides.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of synthetic organic chemistry, protein chemistry, molecular biology, microbiology, and recombinant DNA technology, which are well within the skill of the art. These techniques are applied in connection with peptide synthesis, recombinant production of peptides and peptide mutagenesis, for example. Such techniques are explained fully in the literature. See e.g., Scopes, R. K., Protein Purification Principles and Practices, 2d ed. (Springer-Verlag. 1987), Methods in Enzymology (M. Deutscher, ed., Academic Press, Inc. 1990), Sambrook, et al., Molecular Cloning: A laboratory Manual, 2d ed., (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications), House, Modern Synthetic Reactions, 2d ed., (Benjamin/Cummings, Menlo Park, Calif., 1972).

The peptides of the present invention, homologs, analogs and fragments thereof may be synthesized by a number of known techniques. For example, the peptides may be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc. 85:2149-2154 (1963). Other peptide synthesis techniques may be found in M. Bodanszky, et al. Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can be found in J. Stuart and J. D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also be synthesized by solution methods as described in The Proteins, Vol. II. 3d Ed., Neurath, H. et al., Eds., p. 105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the above-mentioned texts as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present invention can also be prepared by chemical or enzymatic cleavage from larger portions of the CD4 molecule or from the entire CD4 molecule.

Additionally, the peptides of the present invention may also be prepared by recombinant DNA techniques (see e.g. Current Protocols in Molecular Cloning Ausubel et al., 1995, John Wiley & Sons, New York); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York; Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc., New York, N.Y. (1994)). The skilled artisan understands that any of a wide variety of expression systems can be used to provide the recombinant peptides of the present invention. The precise host cell used is not critical to the invention. The IL-16 antagonist peptides can be produced in a prokaryotic host (e.g. E. coli), or in a eukaryotic host (e.g., S. cerevisiae or mammalian cells, e.g. COS1, CHO, NIH3T3, and JEG3 cells, or in the cells of an arthropod, e.g. S. frugiperda). Such cells are available from e.g. the American Type Culture Collection, Manassas, VA. The method of transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g. in Sambrook et al. supra; expression vehicles can be chosen from those provided e.g. in Cloning Vectors: A Laboratory Manual P. H. Powels et al (1985), Supp. 1987.

For most of the amino acids used to build proteins, more than one coding nucleotide triplet (codon) can code for a particular amino acid residue. This property of the genetic code is known as redundancy. Therefore, a number of different nucleotide sequences can code for a particular subject IL-16 antagonist peptide. The present invention also contemplates a deoxyribonucleic acid (DNA) molecule or segment that defines a gene coding for, i.e., capable of expressing, a subject peptide or a subject chimeric peptide from which a peptide of the present invention may be enzymatically or chemically cleaved.

DNA molecules that encode peptides of the present invention can be synthesized by chemical techniques, for example, the phosphotriester method of Matteuccie, et al., J. Am. Chem. Soc. 103:3185(1981). Using a chemical DNA synthesis technique, desired modifications in the peptide sequence can be made by making substitutions for bases which code for the native amino acid sequence. Ribonucleic acid equivalents of the above described DNA molecules may also be used.

A nucleic acid molecule comprising a vector capable of replication and expression of a DNA molecule defining coding sequence for a subject polypeptide or subject chimeric polypeptide is also contemplated.

The peptides of the present invention are chemically synthesized by conventional techniques such as the Merrifield solid phase technique. In general, the method comprises the sequential addition of one or more amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.

A preferred method of solid phase synthesis entails attaching the protected or derivatized amino acid to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups including the solid support are removed sequentially or concurrently to yield the final peptide. The lyophilized oligopeptides are resuspended in double distilled H2O at 2 mg/ml as stock solutions and subsequently diluted in M199-HPS for experiments.

Peptides SEQ ID NOS:1-22 and 33-41 have the following sequences:

Xaa1-L-L-Xaa2 SEQ ID NO:1
CLLS SEQ ID NO:2
Xaa1-Xaa2-Xaa3-L-L-Xaa4 SEQ ID NO:3
WQCLLS SEQ ID NO:4
WQALLLS SEQ ID NO:5
WACLLS SEQ ID NO:6
WQCELS SEQ ID NO:7
Xaa1-VaL-Xaa2-Val-Xaa3-Xaa4 SEQ ID NO:8
VVQVVA SEQ ID NO:9
VKQVVA SEQ ID NO:10
VVQKVA SEQ ID NO:11
GMWQCLLSDSGQVLLE SEQ ID NO:12
GMWQCLLS SEQ ID NO:13
TGLWQCLLSEGD SEQ ID NO:14
VSEEQKVVQVVA SEQ ID NO:15
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:16
NPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPM
TLTCEVMGPTSPKMRLTLKQENQEARVSEEQKVVQVV SEQ ID NO:17
APETGLWQCLLSEGDKVKMDSRIQVLSRGVNQTVF
VSEEQK SEQ ID NO:18
VVQVVA SEQ ID NO:19
LSKQKMVSREGT SEQ ID NO:20
VAPETG SEQ ID NO:21
VIQVQA SEQ ID NO:22
ggggggatgtggaattgtctgctgagtgac SEQ ID NO:23
gtcactcagcagacaattccacatccccgc SEQ ID NO:24
atgtggcagtgtatactgagtgactcggga SEQ ID NO:25
tcccgagtcactcagtatacactgccacat SEQ ID NO:26
atgtggcagtgttcgctgagtgactcggga SEQ ID NO:27
tcccgagtcactcagagcacactgccacat SEQ ID NO:28
atgtggcagtgtctgataagtgactcggga SEQ ID NO:29
tcccgactgacttatcagacactgccacat SEQ ID NO:30
atgtggcagtgtctgtcgagtgactcggga SEQ ID NO:31
tcccgagtcactagccagacactgccacat SEQ ID NO:32
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:33
NPEAGMWNCLLSDSGQVLLESNIKVLPTWSTPVQPM
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:34
NPEAGMWQCSLSDSGQVLLESNIKVLPTWSTPVQPM
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:35
NPEAGMWQCLSSDSGQVLLESNIKVLPTWSTPVQPM
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:36
NPEAGMWQCILSDSGQVLLESNIKVLPTWSTPVQPM
NLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVL SEQ ID NO:37
NPEAGMWQCLISDSGQVLLESNIKVLPTWSTPVQPM
TSPKLMLSLKLENKEA SEQ ID NO:38
KVSKREKAVWVLNPEA SEQ ID NO:39
DSGQVLLE SEQ ID NO:40
GMWQ SEQ ID NO:41


including homologs, analogs and fragments which maintain IL-16-antagonist activity; wherein

A=Ala=Alanine

R=Arg=Arginine

N=Asn=Asparagine

D=Asp=Aspartic acid

B=Asx=Asparagine or aspartic acid

C=Cys=Cysteine

Q=Gln=Glutamine

E=Glu=Glutamic acid

Z=Glx=Glutamine or Glutamic acid

G=Gly=Glycine

H=His=Histidine

I=Ile=Isoleucine

L=Leu=Leucine

K=Lys=Lysine

F=Phe=Phenylalanine

P=Pro=Proline

S=Ser=Serine

T=Thr=Threonine

W=Trp=Tryptophan

Y=Tyr=Tyrosine

V=Val=Valine

X=Xaa=Any amino acid

Consistent with the observed properties of the peptides of the invention, the present peptides can be used to inhibit, suppress, or cause the cessation of at least one Il-16-mediated biological activity. IL-16 functions in the biochemical events associated with the inflammation reaction in animals as an agonist to induce the migration of CD4+ T-cells. Accordingly, the present invention contemplates methods to block, interrupt or otherwise prevent the association of IL-16 to its receptor on CD4 and thereby effectively treat CD4+-cell associated disorders.

IL-16-mediated disorders such as, for example, asthma, rheumatoid arthritis, inflammatory bowel disease (IBD) and systemic lupus are CD4+-cell dependent and therefore treatable with the IL-16 antagonists, preferably IL-16 antagonist peptides, of the present invention. Other CD4+ cell related diseases are also contemplated by the present invention.

In another embodiment of the present invention, one or more IL-16 antagonists, e.g., IL-16 antagonist peptides or antibodies, are included in pharmaceutical compositions.

Preferably, compositions containing the IL-16 antagonist peptides of the present invention are administered intravenously to inhibit, suppress, or cause the cessation of at least one IL-16-mediated biological activity. When administered intravenously, the peptide compositions may be combined with other ingredients, such as carriers and/or adjuvants. The peptides may also be covalently attached to a protein carrier, such as albumin, so as to minimize clearing of the peptides. There are no limitations on the nature of the other ingredients, except that such ingredients must be pharmaceutically acceptable, efficacious for their intended administration and cannot degrade the activity of the active ingredients of the compositions. Examples of other anti-inflammatory ingredients contemplated by the present invention include, but are not limited to anti-CD4 antibodies, anti-TNFα antibody, NSAIDS, steroids, or cyclosporin-A. When employed together with IL-16 antagonists, these agents may be employed in lesser dosages than when used alone.

The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the ultimate solution form must be sterile and fluid. Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization can be accomplished by any art-recognized technique, including but not limited to, filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutano, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.

Production of sterile injectable solutions containing the subject peptides is accomplished by incorporated these compounds in the required amount in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization. To obtain a sterile powder, the above solutions are vacuum-dried or freeze-dried as necessary.

When the peptides of the invention are administered orally, the pharmaceutical compositions thereof containing an effective dose of the peptide can also contain an inert diluent, as assimilable edible carrier and the like, be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.

The subject peptides are thus compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dose.

The peptides should preferably be administered in an amount of at least about 50 mg per dose, more preferably in an amount up to about 500 mg to about 1 gram per dose. Since the peptide compositions of this invention will eventually be cleared from the bloodstream, re-administration of the compositions is indicated and preferred.

The peptides can be administered in a manner compatible with the dosage formulation and in such amount as well be therapeutically effective. Systemic dosages depend on the age, weight and conditions of the patient and on the administration route. For example, a suitable dose for the administration to adult humans ranges from about 0.001 to about 20.0 mg per kilogram of body weight.

As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents the like. The use of such media and agents are well-known in the art. The pharmaceutically acceptable carriers used in conjunction with the peptides of the present invention vary according to the mode of administration. For example, the compositions may be formulated in any suitable carrier for oral liquid formulation such as suspensions, elixirs and solutions. Compositions for liquid oral dosage include any of the usual pharmaceutical media such as, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. In the case of oral solid preparations (capsules and tablets) carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. In addition, carriers such as liposomes and microemulsions may be used.

In a further aspect of the present invention, the pharmaceutical compositions of the present invention are employed for the treatment of IL-16 mediated pathological disorders. Thus, the present invention provides methods of treating an IL-16-mediated disorder in a subject by administering a therapeutically effective amount of a pharmaceutical composition of the present invention.

The term "therapeutically effective amount" means the dose required to treat an IL-16 mediated disorder.

By "an IL-16-mediated disorder" is meant a pathological disorder, the onset, progression or the persistence of the symptoms of which requires the participation of IL-16 molecules. Particularly, IL-16-mediated disorders contemplated by the present invention include asthma, rheumatoid arthritis, inflammatory bowel disease, Graves, disease, multiple sclerosis, lupus and bullous pemphigoid.

The term "treatment" or "treat" refers to effective inhibition, suppression or cessation of the IL-16 activity so as to prevent or delay the onset, retard the progression or ameliorate the symptoms of the disorder.

The term "subject" refers to any mammalian subject. Preferably, the subject is a human.

The present invention thus provides methods of interfering with, blocking or otherwise preventing the interaction or binding of IL-16 with an IL-16 receptor by employing the IL-16 antagonists contemplated by the present invention.

The IL-16 antagonist peptides of the present invention (or homologs, analogs or fragments) can be used to raise single-chain antibodies (SAb) or humanized monoclonal antibodies useful in the invention. The peptides can be coupled to a carrier protein such as KLH as described in Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. The KLH-antagonist peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, donkeys and the like or preferably into rabbits. Antibodies may be purified by peptide antigen affinity chromatography.

A single-chain antibody (SAb) is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Such single-chain antibody variable fragments (Fvs) can be fused to all or a portion of the constant domains of the heavy chain of an immunoglobulin molecule, if necessary. The use of sAb avoids the technical difficulties in the introduction of more than one gene construct into host cells. Single chain antibodies and methods for their production are known in the art. See, e.g., Bedzyk et al. (1990) J. Biol. Chem., 265:18615; Chaudhary et al. (1990) Proc. Natl. Acad. Sci., 87:9491; U.S. Pat. No. 4,946,778 to Ladner et al.; and U.S. Pat. No. 5,359,046 to Capon et al.

Monoclonal antibodies can be prepared using IL-16 antagonist peptides and standard hybridoma technology (see e.g. Kohler et al., (1975) Nature 256:495; Hammerling et al., (1981) In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y.). For example, monoclonal antibodies to IL-16 antagonist peptides (homologs, analogs or fragments thereof) can be raised in Balb/C or other similar strains of mice by immunization with purified or partially purified preparations of IL-16 antagonist peptides. The spleens of the mice can be removed, and their lymphocytes fused to a mouse myeloma cell line. After screening of hybrids by known techniques, a stable hybrid will be isolated that produces antibodies against IL-16 antagonist peptides. Such activity can be demonstrated by the ability of the antibody to prevent the binding of radiolabelled IL-16 to the CD4 receptor. The monoclonal antibody can then be examined for its ability to inhibit the biological activity of IL-16, e.g. cell migration. Once produced, monoclonal antibodies are tested for specific IL-16 recognition by Western blot or immunoprecipitation analysis (by methods described in Ausubel et al., supra). Antibodies which antagonize IL-16/CD4 receptor binding or IL-16 mediated CD4 receptor function are considered to be useful antagonists in the invention.

The monoclonal antibodies of the present invention can be humanized to reduce the immunogenicity for use in humans. One approach is to make mouse-human chimeric antibodies having the original variable region of the murine mAb, joined to constant regions of a human immunoglobulin. Chimeric antibodies and methods for their production are known in the art. See, e.g., Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Taniguchi et al., European patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533, (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Robinson et al., International Patent Publication #PCT/US86/02269 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988). These references are incorporated herein by reference. Generally, DNA segments encoding the H and L chain antigen-binding regions of the murine mAb can be cloned from the mAb-producing hybridoma cells, which can then be joined to DNA segments encoding CH and CL regions of a human immunoglobulin, respectively, to produce murine-human chimeric immunoglobulin-encoding genes.

 

Claim 1 of 6 Claims

1. An antibody directed against an isolated IL-16 antagonist peptide consisting of CLLS (SEQ ID NO:2).
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]