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Title:  Synthetic peptides and pharmaceutical compositions comprising them for the treatment of systemic lupus erythematosus

United States Patent:  6,613,536

Issued:  September 2, 2003

Inventors:  Mozes; Edna (Rehovot, IL); Waisman; Ari (Tel-Aviv, IL)

Assignee:  Yeda Research and Development Co. Ltd. (Rehovot, IL)

Appl. No.:  913994

Filed:  September 29, 1997

PCT Filed:  March 27, 1996

PCT NO:  PCT/US96/04206

PCT PUB.NO.:  WO96/30057

PCT PUB. Date:  October 3, 1996

Abstract

Synthetic peptides based on a complementarity-determining region (CDR) of the heavy or light chain of a pathogenic anti-DNA monoclonal antibody that induces a systemic lupus erythematosus (SLE)-like disease in mice, and analogs, and salts and chemical derivatives thereof; dual peptides comprising two such peptides or analogs covalently linked to one another either directly or through a short linking chain; peptide polymers comprising a plurality of sequences of said peptide or analog thereof; and peptide polymers attached to a macromolecular carrier, are disclosed, and pharmaceutical compositions comprising them for the treatment of SLE in humans.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means for specific treatment of patients with SLE.

For this purpose, the invention provides peptides and analogs thereof based on the CDR regions of pathogenic monoclonal autoantibodies isolated from mice with experimental SLE.

Thus, in one aspect, the invention relates to a synthetic peptide selected from the group consisting of:

(i) a peptide of at least 12 and at most 30 amino acid residues based on a complementarity-determining region (CDR) of the heavy or light chain of a pathogenic anti-DNA monoclonal antibody that induces a systemic lupus erythematosus (SLE)-like disease in mice (hereinafter CDR-based peptide), a salt or a chemical derivative thereof,

(ii) an analog of a CDR-based peptide defined in (i), a salt or a chemical derivative thereof;

(iii) a dual synthetic peptide comprising two such peptides of (i) or analogs of (ii) covalently linked to one another either directly or through a short linking chain;

(iv) a peptide polymer comprising a plurality of sequences of said peptide (i) or analog thereof (ii); and

(v) a peptide polymer (iv) attached to a macromolecular carrier.

In one embodiment of this aspect, the synthetic peptide is capable of:

(i) inhibiting specifically the proliferative response and cytokine secretion of T lymphocytes of mice that are high responders to SLE-inducing autoantibodies; or

(ii) inhibiting development of SLE in mice that are susceptible to SLE-induction by pathogenic autoantibodies.

The synthetic peptides and analogs thereof according to the invention may be selected from the group consisting of peptides having the sequences I to V herein, wherein:

(i) the peptide of sequence I has the formula:

T G Y Y X1 X2 X3 X4 X5 Q S P E K S L E W I G (SEQ. ID NO:1) [I]

wherein X1 is Met, Ala or Val; X2 is Gln, Asp, Glu or Arg; X3 is Trp or Ala; X4 is Val or Ser; and X5 is Lys, Glu or Ala;

(ii) the peptide of sequence II has the formula:

E I N P S T G G X6 X7 X8 X9 X10 X11 X12 K A K A T (SEQ ID NO:2) [II]

wherein X6 and X7 are each Thr, Val or Ala; X8 is Tyr or Phe; X9 is Asn or Asp; X10 is Gln or Glu; X11 is Lys or Glu, and X12 is Phe or Tyr;

(iii) the peptide of sequence III has the formula:

Y Y C A R X13 X14 X15 X16 P Y A X17 X18 Y W G Q G S (SEQ ID NO:3) [III]

wherein X13 is Phe, Thr or Gly; X14 is Leu, Ala or Ser; X15 is Trp or Ala; X16 is Glu or Lys; X17 is Met or Ala, and X18 is Asp, Lys or Ser;

(iv) the peptide of sequence IV has the formula:

G Y N X19 X20 X21 X22 X23 X24 S H G X25 X26 L E W I G (SEQ ID NO:4) [IV]

wherein X19 is Met or Ala; X20 is Asn, Asp or Arg; X21 is Trp or Ala; X22 is Val or Ser; X23 is Lys or Glu; X24 is Gln or Ala; X25 is Lys or Glu, and X26 is Ser or Ala; and

(v) the peptide of sequence V has the formula:

Y Y C A R X27 X28 X29 Y G X30 X31 X32 G Q G T L (SEQ ID NO:5) [V]

wherein X27 is Ser or Phe; X28 is Gly or Ala; X29 is Arg, Ala or Glu; X30 is Asn or Asp; X31 is Tyr or Phe, and X32 is Trp, His or Ala.

In preferred embodiments, peptides I to V have the sequences Ia-Va herein:

T G Y Y M Q W V K Q S P E K S L E W I G (SEQ ID NO:6) (Ia)

E I N P S T G G T T Y N Q K F K A K A T (SEQ ID NO:7) (IIa)

Y Y Y C A R F L W E P Y A M D Y W G Q G S (SEQ ID NO:8) (IIIa)

G Y N M N W V K Q S H G K S L E W I G (SEQ ID NO:9) (IVa)

Y Y C A R S G R Y G N Y W G Q G T L (SEQ ID NO:10) (Va)

Peptides Ia to IIIa are based on the CDR1, CDR2 and CDR3 regions, respectively, of the VH chain of mAb 5G12, and peptides IVa and Va are based on the CDR1 and CDR3 regions, respectively, of the VH chain of mAb 2C4C2 (Waisman and Mozes, 1993).

In another aspect, the invention relates to pharmaceutical compositions for the treatment of SLE comprising a synthetic peptide or peptide polymer of the invention and a pharmaceutically acceptable carrier.

In still another aspect, the invention relates to a method of treatment of a SLE patient comprising administering to a SLE patient an effective amount of a synthetic peptide or peptide polymer of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to synthetic peptides that are based on the CDR of monoclonal pathogenic autoantibodies isolated from mice with experimental SLE. Such monoclonal antibodies are obtained from supernatants of hybridomas produced by fusion, for example, of spleen cells of C3H.SW mice immunized with an anti-16/6 Id mAb, with X63.653 plasmacytoma cells (Waisman and Mozes, 1993).

Examples of such peptides are those of formulas Ia to Va herein, based on, respectively, the CDR1, CDR2 and CDR3 regions of the heavy chain of mAb 5G12 and the CDR1 and CDR3 regions of the heavy chain of mAb 2C4C2 (Waisman and Mozes, 1993), and analogs thereof.

Analogs of parent peptides Ia-Va contemplated by the invention include substitution, deletion and addition analogs as described herein. Substitution analogs have amino acid substitutions at different positions, these substitutions being made based on the volume, hydrophobic-hydrophilic pattern and charge of the amino acids.

Amino acids may be divided along the lines of volume, hydrophobic-hydrophilic pattern and charge. With respect to volume, those of ordinary skill in the art understand that the amino acids with the largest volume are Trp, Tyr, Phe, Arg, Lys, Ile, Leu , Met and His, while those with the smallest volumes are Gly, Ala, Ser, Asp, Thr and Pro, with others being in between.

With respect to hydrophobic-hydrophilic pattern, it is well known that the amino acids Gly, Ala, Phe, Val, Leu, Ile, Pro, Met and Trp are hydrophobic, whereas all of the remaining amino acids are hydrophilic. Among the hydrophilic amino acids, Ser, Thr, Gln, and Tyr have no charge, while Arg, Lys, His and Asn have a positive charge and Asp and Glu have negative charges.

In selecting peptides to be tested for their potential in inhibiting the proliferative response of T lymphocytes of mice that are high responders to SLE-inducing autoantibodies, it is important that the substitutions be selected from those which cumulatively do not substantially change the volume, hydrophobic-hydrophilic pattern and charge of the corresponding portion of the unsubstituted parent peptide. Thus, a hydrophobic residue may be substituted with a hydrophilic residue, or vice-versa, as long as the total effect does not substantially change the volume, hydrophobic-hydrophilic pattern and charge of the corresponding unsubstituted parent peptide.

It should be understood that other modifications of the peptides and analogs thereof are also contemplated by the present invention. Thus, the peptide or analog of the present invention is intended to include a "chemical derivative" thereof which retains at least a portion of the function of the peptide which permits its utility in preventing or inhibiting T cell proliferative responses and autoimmune disease.

A "chemical derivative" of a peptide or analog of the present invention contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Many such chemical derivatives and methods for making them are well known in the art.

Also included in the scope of the invention are salts of the peptides and analogs of the invention. As used herein, the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptide molecule. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids, such as, for example, acetic acid or oxalic acid. Such chemical derivatives and salts are preferably used to modify the pharmaceutical properties of the peptide insofar as stability, solubility, etc., are concerned.

Examples of peptides and analogs thereof are as follows:

(i) Peptide Ia of the Formula:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 T G Y Y M Q W V K Q S P E K S L E W I G (SEQ ID NO:6) (Ia)

and substitution analogs thereof in which Met at position 5 is substituted by either Ala or Val; Gln at position 6 is substituted by either Asp, Glu or Arg; Trp at position 7 is substituted by Ala; Val at position 8 by Ser; and Lys at position 9 is substituted by either Glu or Ala; and deletion analogs thereof in which up to 5 amino acid residues are deleted from the C-terminal of peptide Ia.

(ii) Peptide IIa of the Formula:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 E I N P S T G G T T Y N Q K F K A K A T (SEQ ID NO:7) (IIa)

and substitution analogs thereof in which Thr in positions 9 and 10 are each substituted by either Val or Ala; Tyr at position 11 is substituted by Phe; Asn at position 12 is substituted by Asp; Gln at position 13 by Glu; Lys at position 14 by Glu; and Phe at position 15 by Tyr, and deletion analogs thereof in which up to 5 amino acid residues are deleted from the C-terminal of peptide IIa.

(iii) Peptide IIIa of the Formula:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Y Y C A R F L W E P Y A A M D Y W G Q G S (SEQ ID NO:8) (IIIa)

and substitution analogs thereof in which Phe at position 6 is substituted by either Thr or Gly; Leu at position 7 is substituted by either Ala or Ser; Trp at position 8 is substituted by Ala; Glu at position 9 is substituted by Lys; Met at position 13 by Ala; and Asp at position 14 by either Lys or Ser; and deletion analogs thereof in which up to 5 amino acid residues are deleted from the C-terminal of peptide IIIa.

(iv) Peptide IVa of the Formula:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 G Y N M N W V K Q S H G K S L E W I G (SEQ ID NO:9) (IVa)

and substitution analogs thereof in which Met at position 4 is substituted by Ala; Asn at position 5 is substituted by either Asp or Arg; Trp at position 6 is substituted by Ala; Val at position 7 by Ser; Lys at position 8 by Glu; Gin at position 9 by Ala; Lys at position 13 by Glu; and Ser at position 14 by Ala; and deletion analogs thereof in which up to 5 amino acid residues are deleted from the C-terminal of peptide IVa.

(v) Peptide Va of the Formula:

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Y Y C A R S G R Y G N Y W G Q G T L (SEQ ID NO:10) (V)

and substitution analogs thereof in which Ser at position 6 is substituted by Phe; Gly at position 7 is substituted by Ala; Arg at position 8 is substituted by either Ala or Glu; Asn at position 1 is substituted by Asp; Tyr at position 12 by Phe; and Trp at position 13 by either His or Ala; and deletion analogs thereof in which up to 5 amino acid residues are deleted from the C-terminal of peptide Va.

Once an analog in accordance with the present invention is produced, its ability to inhibit the proliferative response of T lymphocytes of mice that are high responders to SLE-inducing autoantibodies may be readily determined by those of ordinary skill in the art without undue experimentation using tests such as those described herein. One test which may be readily conducted is for the ability of substituted peptides to inhibit in vitro the proliferative responses of certain T cell lines and clones specific to SLE-inducing autoantibodies. The T cell lines and clones may, for example, be the T cell lines and clones specific to the 16/6 Id mAb (Fricke et al., 1991) established from immunized lymph node cells of mice by previously described methodology (Axelrod and Mozes, 1986). Cells are exposed to the stimulating antibody presented on irradiated syngeneic spleen cells in the presence of enriched medium every two weeks. The T cell lines are cloned by the standard limiting dilution technique.The proliferative responses of these T cell lines and clones are tested, for example, by the method described in Materials and Methods, section (g), herein.

Another test which can be conducted in order to select analogs having the desired activity is to test for the ability of the substituted peptides to inhibit the ability of the T cell lines and clones to provide help to peptide-specific B cells in the presence of the parent peptide. The substituted peptides may also be tested for their ability to bind directly, following biotinylation, to MHC Class II products on antigen-presenting cells of the relevant strains. For this purpose, N-terminal biotinylation of the relevant peptides is performed at 0oC. with an excess of biotin-N-hydroxysuccinimide in aqueous solution (Mozes et al., 1989). Mouse splenic adherent cells or human peripheral blood lymphocyte (PBL)-adherent cells (1x106 /sample) are incubated with biotinylated peptides in PBS containing 0.1% bovine serum albumin (PBS/BSA) at 37oC. for 20 hr, followed by incubation with phycoerythrin-streptavidin for 30 min at 4oC. After each incubation, the cells are washed twice with the above solution. Thereafter, the cells are analyzed by flow cytometry using FACScan. In each analysis, a minimum of 5000 cells are examined (for above procedures, see, for example, Mozes et al., 1989; Zisman et al., 1991).

A further test which can be conducted is to test for the ability of the analogs to inhibit cytokine secretion by the T cell line or by T lymphocytes oh lymph nodes of mice that are high responders to SLE-inducing autoantibodies. The cytokines are detected as follows: IL-1 activity is assessed either by ELISA using a pair of capture and detecting antibodies (as described below for IL-4, IL-6, IL-10) or using the LBRM-33(1A5) assay (Conlon, 1983) in which 1A5 cells are stimulated in the presence of phytohemagglutinin (PHA), with either supernatants or recombinant IL-1 at various concentrations to secrete IL-2. Following an overnight incubation, supernatants of 1A5 cells are transferred to the IL-2 dependent cytotoxic T lymphocyte (CTLL) line. Stimulation of the CTLL line by IL-2 is measured after 24 hr by incorporation of 3 [H]-thymidine. IL-2 is directly detected using the IL-2 dependent CTLL line or by ELISA. Levels of IL-4, IL-6, IL-10, INF.gamma. and TNF.alpha. in the supernatants are determined by ELISA using antibodies to the various cytokines (Phamingen, San Diego, Calif., USA) according to the manufacturer's instructions.

Peptides which test positive in one or more of these in vitro tests will provide a reasonable expectation of in vivo activity. However, in vivo tests can also be conducted without undue experimentation. Thus, for example, adult mice may be injected with the candidate peptide at either day -3 or day 0. The mice are then immunized with the disease-inducing autoantibody or with the peptide. Ten days later, lymph node cells of the mice are tested for their ability to proliferate to the immunogen in order to find out the inhibitory capacity of the candidate peptide.

Another such it7 vivo animal test consists in measuring the therapeutic activity directly in the murine model in7 vivo for the production of SLE as described above. The peptides can be injected into the mice in which experimental SLE is induced by different routes at different dosages and at different time schedules. In order to determine the pharmacokinetic parameters of the analogs, including volume of distribution, uptake into antigen-presenting cells and clearance, one can use biotinylated derivatives of the analogs. The concentration of the soluble fraction of the analogs in the various body fluids can be determined by ELISA, using avidin-coated plates and specific anti-peptide antibodies. Cell bound analogs can be analyzed by FACS, using fluorochromo-conjugated avidin or streptavidin. Furthermore, the treated mice can be tested periodically in order to determine the effect of the peptides on the autoantibody responses and on disease manifestations elicited in the mice by the SLE-inducing autoantibody.

Another in vivo procedure consists in tolerizing newborn mice with the candidate peptide followed by immunization of the mice with the pathogenic autoantibody, such as 16/6 Id+, or with the same peptide, and following the disease manifestations, such as serological findings associated with leukopenia, elevated erythrocyte sedimentation rate, proteinuria, abundance of immune complexes in the kidneys and sclerosis of the glomeruli.

It can thus be seen that, besides the preferred embodiments which have been shown to be operable in the examples herein, those of ordinary skill in the art will be able to determine additional analogs which will also be operable following the guidelines presented herein without undue experimentation.

A relatively simple iii vitro test can also be conducted in order to assay for the expected therapeutic efficacy of any given substituted peptide on any given SLE patient. In order to assess the ultimate goal of producing peptides that will bind with high affinity to the appropriate MHC Class II molecules but will not lead to further activation of T cells and will therefore have a therapeutic effect on SLE patients, the peptides may be assayed, following biotinylation, for their ability to bind directly to HLA Class II products on antigen-presenting cells in the peripheral blood lymphocytes of the SLE patients. Healthy control donors and control peptides may be used in such assays to verify their specificity.

A preferred form of the therapeutic agent of the invention is a peptide selected from the group of peptides of formulas I to V herein, including peptides Ia to Va and substitution and/or deletion analogs thereof.

Another preferred form of the therapeutic agent in accordance with the present invention is the form of a multi-epitope single peptide. Thus, in a preferred embodiment, dual petides consisting of two different peptides selected from the group of peptides of formula I-V herein, are covalently linked to one another, such as by a short stretch of alanine residues or by a putative site for proteolysis by cathepsin. See, for example, U.S. Pat. No. 5,126,249 and European Patent 495,049 with respect to such sites. This will induce site-specific proteolysis of the preferred form into the two desired analogs. Alternatively, a number of the same or different peptides of the present invention may be formed into a peptide polymer, such as, for example, polymerization of the peptides with a suitable polymerization agent, such as 0.1% glutaraldehyde (Audibert et al. (1981), Nature 289:593). The polymer will preferably contain from 5 to 20 peptide residues. Such peptide polymers may also be formed by crosslinking the peptides or attaching multiple peptides to macromolecular carriers. Suitable macromolecular carriers are, for example, proteins, such as tetanus toxoid, and linear or branched copolymers of amino acids, such as a linear copolymer of L-alanine, L-glutamic acid and L-lysine and a branched copolymer of L-tyrosine, L-glutamic acid, L-alanine and L-lysine (T,G)-A-L-, or multichain poly-DL-alanine (M. Sela et al. 1955, J. Am. Chem. Soc. 77:6175). The conjugates are obtained, for example, by first coupling the peptide with a water-soluble carbodiimide, such as 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride, and then performing the conjugation with the macromolecular carrier as described by Muller, G. M. et al. (1982) Proc. Natl. Acad. Sci. USA 79:569. The contents of the coupled peptide in each conjugate are determined by amino acid analysis, in comparison to the composition of the carrier alone.

According to one embodiment of the present invention, one or more active peptides may be attached to a suitable macromolecular carrier or may be polymerized in the presence of glutaraldehyde.

The peptides, polymers thereof or their conjugates with suitable macromolecular carriers, will be given to patients in a form that insures their bioavailability, making them suitable for treatment. If more than one peptide analog is found to have significant inhibitory activity, these analogs will be given to patients in a formulation containing a mixture of the peptides.

The invention further includes pharmaceutical compositions comprising at least one synthetic peptide according to the invention, a conjugate thereof with a suitable macromolecular carrier or a polymer thereof optionally with a pharmaceutically acceptable carrier.

Any suitable route of administration is encompassed by the invention, including oral, intravenous, subcutaneous, intraarticular, intramuscular, inhalation, intranasal, intrathecal, intraperitoneal, intradermal, transdermal or other known routes, including the enteral route.

The dose ranges for the administration of the compositions of the present invention should be large enough to produce the desired effect, whereby, for example, an immune response to the SLE-inducing autoantibody, as measured by T cell proliferation in vitro, is substantially prevented or inhibited, and further, where the disease is significantly treated. The doses should not be so large as to cause adverse side effects, such as unwanted cross reactions, generalized immunosuppression, anaphylactic reactions and the like.

Effective doses of the peptides of this invention for use in treating SLE are in the range of about 1 .mu.g to 100 mg/kg body weight. The dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The synthetic peptides and analogs of the invention, particularly those of sequences I to V herein, are aimed at inhibiting or suppressing specific antigen responses of SLE patients, without affecting all other immune responses. This approach is of the utmost importance since most diagnosed patients are young women that have to be treated for many years and the currently accepted treatment for SLE involves administration of immuno-suppressive agents, such as corticosteroids and/or cytotoxic drugs, that are both non-specific and have multiple adverse side effects.

Claim 1 of 28 Claims

What is claimed is:

1. A synthetic peptide selected from the group consisting of:

(i) a peptide of at least 12 and at most 30 amino acid residues consisting of a sequence including a complementarity-determining region found in the heavy or light chain of a pathogenic anti-DNA monoclonal antibody that induces a systemic lupus erythematosus (SLE)-like disease in mice, or a salt thereof or the reaction product thereof with an organic derivatizing agent capable of reacting with selected side chains or terminal residues, which reaction product retains at least a portion of the function of the peptide to inhibit specifically the proliferative response and cytokine secretion of T lymphocytes of mice that are high responders to SLE-inducing autoantibodies;

(ii) a dual synthetic peptide comprising two different ones of said peptides of (i) covalently linked to one another either directly or through a short linking chain;

(iii) a peptide polymer comprising a plurality of sequences of said peptide (i); and

(iv) a peptide polymer of (iii) attached to a macromolecular carrier.




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