WO1997036606A1

WO1997036606A1 - Constrained cd4 peptides and methods of using the same

Info

Publication number: WO1997036606A1
Application number: PCT/US1997/004847
Authority: WO
Inventors: Mark I. Greene; Xin Zhang
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 1996-03-29
Filing date: 1997-03-25
Publication date: 1997-10-09
Also published as: AU2589497A

Abstract

Constrained peptides comprising an amino acid sequence that consists of 30 amino acid residues or less are disclosed. The peptides comprise a human CD4 sequence within a cyclic portion and two exocyclic portions, which each comprises an aromatic amino acid residue, are disclosed. Pharmaceutical compositions comprising the peptide are disclosed. Methods of inhibiting the oligomerization of membrane bound CD4 in a CD4+ cell by contacting the cell with a peptide are disclosed. Methods of inhibiting activation of a T cell and methods of treating individuals with diseases, disorders and conditions whose causes and/or symptoms are associated with and/or characterized by T cell activation are disclosed.

Description

CONSTRAINED CD4 PEPTIDES AND METHODS OF USING THE SAME

FIELD OF THE INVENTION

The present invention relates to aromatically modified exocyclic constrained peptides which include an active sequence derived from human CD4 and to methods of using such peptides to modulate CD4-associated protein-protein interactions.

BACKGROUND OF THE INVENTION

The T cell lymphocyte surface protein, CD4, is a transmembrane glycoprotein that acts as an accessory molecule or co-receptor for T cell activation through association with the class II major histocompatibility complex (MHC II) antigens and T cell antigen receptors (TCR) . The immunoglobulin-like extracellular domains of CD4 contacts nonpolymorphic regions of the MHC II molecules, either in an adhesion interaction or as part of a ternary complex with the TCRs. The cytopiasmic domain of CD4 associates with p56, a sarc-like tyrosine kinase, involved in T cell activation. Although soluble monomeric CD4 has been unable to inhibit T cell responses, the oligomerization of CD4 may be relevant to T cell activation and may reflect dimerization of the T cell receptor assembly. A variety of studies have demonstrated that different domains and surfaces of CD4 are required for the formation of higher ordered complexes of the CD4 holoreceptor with MHC II molecules .

SUMMARY OF THE INVENTION Peptide analogs have been developed to analyze precise CD4 substructures involved in MHC class II binding. Forms of the complementarity determining-like regions (CDRs) of the Dl domain of human CD4 were reproduced as synthetic aromatically modified exocyclic (AME) analogs and tested for their ability to block CD4-MHC II interactions and T cell activation. The exocyclic derived from CDR3 (residues 82-89) of human CD4 which specifically associated with CD4 on the T cell surface to create a heteromeric CD4 complex, blocked IL-2 production and antagonized the normal function of the CD4 receptor. The approach of creating novel synthetic antagonistic receptor complexes may represent a new receptor specific pharmaceutical approach to modulate biological function.

The present invention relates to aromatically modified constrained peptides which have an active sequence from human

CD4. Aromatically modified constrained peptides are constrained peptides which have free aromatic amino acid residues linked to the constrained peptide.

The aromatically modified peptides of the invention comprise an amino acid sequence that consists of no more than 30 amino acid residues and has the formula:

R₁ - R₂ - R₃ - R₄ - R₅ - R₆ - R₇ wherein:

R_x is 1-6 amino acid residues, at least one of which is tyrosine or phenylalanine;

R₂ is a linking amino acid residue, preferably cysteine;

R₃ is 0-13 amino acids;

R₄ is an active sequence of 3-26 amino acids including human CD4 sequences ;

R_s is 0-13 amino acids;

R₆ is a linking amino acid residue, preferably cysteine;

R₇ is 1-6 amino acid residues, at least one of which is tyrosine or phenylalanine; and wherein R₂ and R₆ are bound to each other, thereby forming a cyclic portion which includes R₂, R₃, R₄, R₅ and R₆ with R_x and R₇ forming exocyclic portions.

The present invention relates to pharmaceutical compositions which comprise peptides of the invention. The present invention relates to methods of inhibiting the oligomerization of membrane bound CD4 in CD4+ cells.

The present invention relates to methods of inhibiting the T cell activation.

The present invention relates to methods of treating individuals with diseases, disorders and conditions whose causes and/or symptoms that are associated with and/or characterized by T cell activation.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of U.S. Serial Number 08/257,783 filed June 10, 1994 filed June 10, 1994 is incorporated herein by reference in its entirety. U.S. Serial Number 08/257,783 describes aromatic modified exocyclic constrained peptides generally. As used herein, the disclosure in U.S. Serial Number 08/257,783 is meant to refer to aromatic modified exocyclic constrained peptides which include active regions derived from human CD4. Whenever aromatic modified exocyclic constrained peptides are referred to U.S. Serial Number 08/257,783 for the purposes of this disclosure Applicants includes express description of such peptides having CD4 active regions. Thus, for example, the disclosure of synthesis methodology contained in U.S. Serial Number 08/257,783 is intended to be included in this disclosure and to describe the synthesis of peptides of the present invention. Similarly, the description of the peptides having various sizes in U.S. Serial Number 08/257,783 is intended to specifically describe peptides of the invention having such size limitations.

As used herein, the terms "conformationally restricted peptides", "constrained peptides" and "conformationally constrained peptides" are used interchangeably and are meant to refer to peptides which, for example through intramolecular bonds, are conformationally stable and remain in a sufficiently constant conformation to maintain the peptide' s level of function and activity more consistently. Many conformationally restricted peptides whose structures are modeled upon the active region of a protein have been shown to have biological active similar to that of the protein.

As used herein, the terms "aromatic amino acids" and "aromatic amino acid residues" used interchangeably are meant to refer to phenylalanine and tyrosine.

As used herein, the term "exocyclic amino acid residue" is meant to refer to amino acid residues which are linked to cyclicized peptide but which are not within the portion of the peptide that makes up the circularized structure.

As used herein, the term "exocyclic portions" is meant to refer to an amino acid sequence having one or more amino acid residues which is linked to cyclicized peptide but which are not within the portion of the peptide that makes up the circularized structure.

As used herein, the term "linking amino acid residue" is meant to refer to an amino acid residue in an amino acid sequence which when linked to a non-adjacent amino acid residue results in cyclicizing at least a portion of the peptide.

As used herein, the terms "active sequence of human CD4", and "active region of human CD4" are used interchangeably and are meant to refer to the amino acid sequences of CDR3, specifically about amino acids 82-89 of the human CD4 molecule and may further include some additional amino acids on either side of the region such as about 80-91. The active region of CD4 is directly involved in CD4 dimerization. In some embodiments, the active region of CD4 may refer to amino acids 45-50.

The present invention relates to improved constrained CD4 peptides and methods of using the same. Constrained peptides according to the present invention comprise a cyclic portion which comprises a CD4 active region and which further comprise amino acid residues that have aromatic groups, specifically phenylalanine and tyrosine, linked to, but outside of, the cyclic portion.

The peptides of the present invention have the following features: 1) they consist of between 7 and 30 amino acids;

2) they are conformationally restricted such that they comprise a cyclic portion;

3) the cyclic portion includes a CD4 active sequence, preferably which consists of 3-18 amino acid residues;

4) the cyclic portion is linked to two exocyclic portions; and,

5) each exocyclic portion consists of 1-6 amino acids residues and comprises at least one aromatic amino acid residue.

Constrained peptides are typically produced as linear peptides that are then cyclicized by non-peptide bonds, usually disulfide bonds between distally positioned cysteine residues, often N-terminal and C-terminal cysteines. According to the present invention, aromatic amino acid residues are provided as exocyclic amino acid residues in association with constrained peptides in order to provide increased interactions between the active sequence of the constrained peptide and other molecules. According to the invention, aromatic amino acids are exocyclic; that is, they are linked to the constrained peptides but are not within the cyclicized portion of the molecule.

Peptides may be constrained by any of several well known means. In preferred embodiments, disulfide bonds between two non-adjacent cysteines cyclicize and thereby conformationally restrict the peptide. The cyclization of linear peptides using disulfide bonds between non-adjacent cysteines is well known. Similarly, other non-adjacent amino acid residues may be linked to cyclicize a peptide sequence and the means to do so are similarly well known. Other methods of cyclization include those described by Di Blasio, et al . , (1993) Biopolymers, 33:1037-1049; Wood, et al . , (1992) J. Pep . Prot . Res . , 39:533-539; Saragovi, et al . , (1992) Im unomethods , 1:5-9; Saragovi, et al. , (1991) Science , 253:792-795; Manning, et al . , (1993) Reg. Peptides, 45:279-283; Hruby, (1993) Biopolymers, 33:1073-1082; Bach, et al . , (1994) New Adv. Peptidomimetics Small Mol . Design, 1:1-26; and Matsuyama, et al . , (1992) J. Bacteriol . , 174:1769-1776, each of which are incorporated herein by reference.

It is contemplated that in preferred embodiments, the cyclized portion consists of 5 to 25 amino acid residues. In some preferred embodiments, the cyclized portion is 9 to 20 amino acid residues. In some preferred embodiments, the cyclized portion is 8 to 12 amino acid residues. In some preferred embodiments, the cyclized portion is 10 to 20 amino acid residues. In some preferred embodiments, the cyclized portion is 12 to 16 amino acid residues. It is contemplated that the active sequence of the cyclized portion consists of at least 3 amino acid residues. In some preferred embodiments, the active sequence of the cyclized portion is at least 4 to 12 amino acid residues. In some preferred embodiments, the active sequence of the active sequence of the cyclized portion is at least 6 to 10 amino acid residues. In some preferred embodiments, the active sequence of the cyclized portion is at least 6 to 8 amino acid residues.

The active sequence of a constrained peptide is derived from human CD4 protein. According to the present invention, the cyclic portion is linked to two exocyclic portions. Essentially, each exocyclic portion is an amino acid sequence consisting of 1-6 aromatic amino acid residues linked to the cyclic portion but not within the cyclicized conformationally restricted peptide. Each exocyclic portion extends out from the cyclic portion and comprises at least one aromatic amino acid residue. In some embodiments, each exocyclic portion consists of one amino acid residue. In some embodiments, one exocyclic portion consists of one amino acid residue and the other exocyclic portion consists of 1-6 amino acid residues. In some embodiments, one exocyclic portion consists of 1-3 amino acid residues and the other exocyclic portion consists of 1-6 amino acid residues. In some embodiments, each exocyclic portion consists of a single aromatic amino acid residue. In some embodiments, each exocyclic portion comprises a single aromatic amino acid residue. It is preferred that the exocyclic residues are linked to the residues furthest from the active sequence. In some embodiments, it is preferred that the exocyclic residues occupy the N- and C-terminal positions and that the bonds are formed between the second and penultimate residues which cyclicized the remainder of the peptide, providing the N- and C-terminal residues as exocyclic residues.

In preferred embodiments the second and penultimate residues are cysteines which are linked by disulfide bonds. In preferred embodiments, one of either the N- and C-terminal residues is phenylalanine and the other is tyrosine.

The bonds which result in cyclization of a portion of the peptide are formed between one of the second, third, fourth, fifth, sixth or seventh residues and one of the penultimate, third to last, fourth to last, fifth to last, sixth to last residues or seventh to least residue. The binding of non adjacent residues forms the cyclized portion of the constrained peptide which has two exocyclic sequences of exocyclic amino acid residues between 1 and 6 residues each, respectively. Peptides can be synthesized by those having ordinary skill in the art using well known techniques and readily available starting materials. According to the invention, references to synthesizing or constructing peptides is herein construed to refer to the production of peptides similar in sequence or structure to the corresponding regions identified by the method of the invention. These peptides may be produced using any method known in the art, including, but not limited to, chemical synthesis as well as biological synthesis in an in vi tro or in vivo in a eukaryotic or prokaryotic expression system. In a preferred method, peptides of the invention are produced by solid phase synthesis techniques as taught by Merryfield, (1963) J. Am. Chem . Soc , 15:2149-2154 and J. Stuart and J.D. Young, Solid Phase Peptide Synthelia , Pierce Chemical Company, Rockford, IL (1984) , each of which is incorporated herein by reference.

The present invention relates to methods of using the aromatically modified exocyclic CD4 (AME-CD4) constrained peptides of the present invention. The AME-CD4 constrained peptides bind to CD4 on cells and prevent the CD4 molecules from interacting with other molecules. The oligomerization of CD4 with other molecules is associated with activating CD4* cells. Accordingly, AME-CD4 constrained peptides may be administered to individuals in order to prevent or inhibit activation of CD4⁺ cells.

The present invention relates to methods of treating individuals with diseases, disorders and conditions whose causes and/or symptoms that are associated with and/or characterized by T cell activation. Diseases, disorders and conditions which can be treated using the pharmaceutical compositions that comprise AME-CD4 constrained peptides of the invention include psoriasis, contact dermatitis, ocular inflammation, allogenic grafts, immunological suppression, and HIV treatment .

The pharmaceutical compositions of the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. Topical or parenteral administration, i.e., intravenous, subcutaneous, intramuscular, ordinarily are used to optimize absorption. In some preferred embodiments, pharmaceutical compositions which comprise the compounds of the present invention are administered topically or as a lavage for treatment of psoriasis, contact dermatitis or ocular inflammation. In some preferred embodiments, pharmaceutical compositions which comprise the compounds of the present invention are administered systemically by parenteral administration for treatment of allogenic grafts, immunological suppression such as in the case of preventing organ or tissue rejection in transplantation procedures, and HIV infection. Pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice. The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 0.001 to 1 grams per kilogram of body weight, in some embodiments about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily dosages are in the range of 0.5 to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day. In some embodiments, the pharmaceutical compositions are given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95 by weight based on the total weight of the composition.

For parenteral administration, the compound can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) . The formulation is sterilized by commonly used techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington ' s Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.

According to the present invention, the AME-CD4 constrained peptides may be administered to tissue of an individual by topically or by lavage. The AME-CD4 constrained peptides may be formulated as a cream, ointment, salve, douche, suppository or solution for topical administration or irrigation. Formulations for such routes administration of pharmaceutical compositions are well known.

Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are used. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical preparations according to the present invention are preferably provided sterile and pyrogen free.

One of skill in the art of pharmaceutical formulations, e.g., having an advanced degree in Pharmaceutics or Pharmaceutical Sciences, can prepare a variety of appropriate dosage forms and formulations for the compositions of the invention with no more than routine experimentation. A number of texts in the field, a,g., Remington ' s Pharmaceutical Sciences and The U. S. Pharmacopoeia/National Formulary, latest editions, provide considerable guidance in this respect. A pharmaceutically acceptable formulation will provide the active ingredient (s) in proper physical form together with such excipients, diluents, stabilizers, preservatives and other ingredients as are appropriate to the nature and composition of the dosage form and the properties of the drug ingredient (s) in the formulation environment and drug delivery system.

In addition to the usual considerations of stability and bioavailability, in order to achieve adequate mucosal immunity, the dosage form will provide adequate physical and temporal contact with the selected mucosa. The active ingredients) can be formulated as a single phase or two-phase system, and in liquid, solid or semisolid dosage form, for example, cream, gel, emulsion, suspension, ointment, suppository, tablet. The formulation vehicle may be aqueous, oleaginous, or an oil-in-water or water-in-oil emulsion, preferably water/oil. The active ingredients may be formulated in sterile water or saline. EXAMPLE

Summary Immunoglobulin gene superfamily members, including CD4, possess complementarity determining-like regions (CD4) whose conformational properties define many of the biological activities of these polypeptides. In the first domain (Dl) of human CD4, the three CDR loops are juxtaposed along one surface of the molecule. To probe the atomic features of the exposed CD4-D1 CDR-like regions required for interaction with MHC II, a new class of constrained forms of peptides was developed. The forms have been cyclized and constrained with cysteine disulfide bridges in order to preserve the predicted configuration of the adjacent CDR turn reversals, aromatic residues were added to the termini of the cyclic constructs to improve binding efficacy. These aromatically modified exocyclic molecules (AMEs) have been designed to resemble the authentic three dimensional structure of the CDR-like loops of the Dl domain of the CD4 molecule. The structure of the CDR3 synthetic analog, CDR3.AME (82-89) , was modeled and found to structurally mimic the authentic CDR3 of the crystallized CD4 molecule. The CDR3.AME(82-89) analog was also found to inhibit CD4-MHC II binding and antagonize CD4 function.

Results CDR3.AME(82-89) inhibits CD4 binding to MHC II β fragment (134- 148) . To examine the activities of these CD4.AME forms, competition binding assays were performed. Different species of the CD4.AME analogs were tested for their ability to inhibit the binding of the recombinant soluble ¹²⁵I-labeled human MHC II DR4 molecules produced in insect cells to immobilized recombinant soluble CD4 (sCD4) . The constrained exocyclic forms of both CDR3.AME(82-89) and CDR2.AME(45-50) inhibited 50 percent of ¹²⁵I human MHC DR4 binding to immobilized sCD4 at 4 uM and 62 uM, respectively. The analog of the CDRl-like region, CDR1.AME (25-28) , inhibited only 20-30% of the interaction at the highest concentration tested. The linear peptides, CDR3LIN (82-89) and CDR3LIN (85-91) , as well as other cyclized AME forms derived from other CDR-like regions, did not inhibit the interaction between the CD4 and purified DR4 molecules .

Previous studies have identified the 134-148 region of the β chain of the human MHC II molecule as a primary determinant for MHC II/CD4 interaction. It was therefore determined whether any of the CD4 analogs could inhibit CD4 binding to this region of the MHC II molecule. An assay was devised in which CD4 CDR analogs competed with the specific binding of ¹²⁵I-labeled human CD4 to human MHC β2 134-148 peptides immobilized on plates. Our results showed that the

CDR3-like compound CDR3.AME (82-89) inhibited CD4 binding in a dose dependent manner. Other compounds, including the CDR2.AME

(30-55) and CDR3.AME (85-91) could not inhibit CD4 interaction with the MHC II peptide. Therefore our studies have shown that the CDR3 region of CD4 plays an important role in the interaction with the 134-148 fragment of the MHC II molecule. The CDR3.AME (82-89) was also found to inhibit rosette formation between CD4 expressing COS cells and MHC II positive B lymphocytes inducing a half-maximum at 25μM while the other AME forms, CDR2.AME (45-50) and CDR3.AME (85-91) , were ineffective at the same concentration. CDR3.AME (82-89) inhibits T cell activation.

Despite reports that soluble CD4 was unable to affect T cell activation, the effects of these CD4 analogs were studied on antigen induced T cell activation. The human CD4⁺ DO.11.10 T-cell hybridoma is activated by OVA peptide presented by RT.2.3.3 H6 cells (restricted by murine 1-A^d) . The activation of DOU.IO is CD4 dependent and can be detected and quantitated by measuring IL-2 produced by the hybridoma cells. In these studies, linear forms of each compound, OKTE (anti- CD8) as well as GK1.5 (anti-murine CD4) were used as controls. The cyclized CDR3.AME (82-89) inhibited 85% of the IL-2 production by CD4⁺ DOU.IO cells at lOuM. At a similar concentration, CDR1.AME(23-28) , CDR2.AME (45-50) and CDR3.AME (85-91) inhibited 56%, 52% and 33% of the IL-2 production, respectively. It is noteworthy that the three CDRs 82-89, 45- 50 and 23-28 all align on one surface of the CD4 molecule (Figs. IA and IB) . These studies indicate that several CD4 surfaces may be involved in T cell activation and CD4-MHC II interaction. Importantly, in both binding and functional studies, the CDR3-like analog was the most effective inhibitor. CDR3.AME (82-89) specifically binds to the CD4 receptor.

It has been suggested that the oligomerization of CD4 may be required to form a stable MHC II binding site. This issue was addressed with the CD4 forms we have generated. To examine whether the most efficient constrained CDR3 analog could physically associate with cell surface CD4, CDR3.AME (82- 89) was labeled with fluorescein. We assayed the CDR3.AME (82- 89) -PITC molecule for its ability to bind to a variety of transformed lymphoid or non-lymphoid cells by FACS analysis (Table 1) . Anti-human CD4 (OKT4) , anti-human CD8 (OKT8) , anti- I-A^d (MKD-6) and anti-mouse L3T4 (GK1.5) were used as antibody controls. Our results indicated that CDR3.AME (82-89) -PITC is able to bind to both CD4⁺ DOU.IO cells and CD4⁺ Jurkat cells. The CDR3.AME (82-89) form also binds slightly to mouse L3T4⁺ DOU.IO cells, but not to CD4^" or L3T4^" lymphocytes

(DOll.10.Agδ.dul, the parental cell from which DOU.IO was derived) or CD8^* 3887.27 cells. Furthermore, CDR3.AME (82-89) does not bind to non-lymphoid L-cells. Independently, it was verified that none of these cell lines expresses MHC II molecules excluding the possibility of promiscuous binding of the CD4 AMEs. These data indicate that CDR3.AME (82-89) binds specifically to the CD4 molecules on the CD4⁺ T cells, and O 97/36606 PC17US97/04847

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suggests that the synthetic CDR3.AME (82-89) forms heteromers with the cell surface CD4 molecule. The heteromers appear to be responsible for interference with IL-2 production and T cell activation. It is likely that the CDR3.AME (82-89) /CD4 heteromer prevents ordered and specific binding to MHC class II polypeptides.

Discussion Residues 86-96 of the CDR3 surface as well as other distal residues of CD4 have been shown to be permissive for binding of a CD4 IgG chimeric immunoadhesin to CD4 linear peptides. In addition, a constrained CDR3 peptide derived from murine CD4 was found to inhibit some T cell activities in vitro and in vivo at high concentrations. The murine peptide' s binding pattern could not be established and its mode of action was thought to affect T cell signal transduction. Interestingly, the murine peptide could not affect CD4-MHCII interactions. Using a more extensively modified molecule, the present invention conclusively demonstrate that the CDR3 region of CD4 is relevant to binding MCHII molecules. Furthermore, as a consequence of aromatic modification of the cyclic forms, a dramatic inhibition of T cell function at low micromolar levels was demonstrated. In contrast to the other studies using linear or non-modified peptides, we were able to visualize directly the interaction of the CDR3 exocyclic with CD4 holoreceptor on the cell surface. This suggests that the CDR3-CD4 association is a necessary component in the mechanism of inhibiting T cell activation. Our conclusion is that CD4 must operate as a homomer to mediate binding to MHC II surfaces, and that one of the MHC II surfaces recognized includes the β chain residues 134-148. The CDR3.AME (82-89) is able to associate with the CD4 holoreceptor and effectively create a disabled heteromeric receptor species.

Although it is likely that CDR3.AME (82-89) can directly bind to the 134-148 MHC β chain residues, it is apparent that other CDR-like regions of CD4 are also involved in MHC II binding. While the intact CD4 holoreceptor may be able to make contact with different parts of MHC class II, clearly interference of the CDR3 site of attachment appears sufficient for inhibiting T cell activation.

We have created a form of receptor-specific antagonist that prevents the assembly and function of the normal dimeric form of the CD4 holoreceptor. Since many receptors are arranged as ensembles, it is likely that this general approach may also be applied to create molecules that can specifically antagonize the pharmacological activity of other receptor systems, irrespective of the ligands involved. Experimental Protocol

Peptide synthesis and cyclization.

The following peptides were synthesized: SEQ ID N0:1 CDR1.AME (23-28) FCSIQFHWCY SEQ ID NO:2 CDR2.AME (39-44) YCNQGSFLCY SEQ ID NO:3 CDR2.AME (45-50) FCTKGPSKCY SEQ ID NO:4 CDR2.AME (50-55) FCKLNDRACY SEQ ID NO:5 CDR3.AME (82-89) FCYLCEVEDQCY SEQ ID NO: 6 CDR3.AME (85-91) FCEVECQKECY SEQ ID NO:7 CDR3.LIN (82-89) FCYICEVEDQCY SEQ ID NO: 8 CDR3.LIN (85-91) FCEVEDQKECY

SEQ ID NO: 9 MHC II (134-148) NGQEEKAGWSTGLI

SEQ ID NO:10 OVA (323-339) ISQAVHAAHARTNEINEAGR

All peptides were synthesized by solid-phase methods at the Protein Chemistry Laboratory at the Department of Pathology and Laboratory Medicine of the University of Pennsylvania, deprotected, and released from the resin utilizing anhydrous HE. Peptides were lyophilized and further purified by High Performance Liquid Chromatography (HPLC) utilizing Delta-Park C16 column and then lyophilized. Peptide was more than 95% pure by HPLC analysis and mass spectrometry. The peptides containing internal cysteine residues were refolded and oxidized by dissolving them at 0.1-0.3 mg/ml in distilled water pH 8.0 and stirring them for 3 days exposed to the air at 4°C. The efficiency of the oxidation was tested by determination of free sulfhydryls in peptides and compared with the linear unoxidized peptide. These peptides showed greater than 95% intramolecular disulfide bending at the end of this procedure as determined by HPLC analysis. Molecular modeling.

Computer modelling was performed using Quanta 4.0 (Molecular Simulation Inc., MA) . The model peptides were constructed from their sequences and folded using CHARMM minimization. The side chain of amino acid residues were first positioned to statistically allowed conformation using Pondera rotamer^" database provided in QUANTA. Then the folded peptides were minimized to convergence with dielectric constant set at 60. The Root Mean Square (r.m.s.) deviation for the C atoms was 1.4A. Cells

DOll .10.Ag8.dul and 3887.27 cell lines, provided by Dr. Philipps Marrack and CD4 DOU.IO and L3T4⁺DO11.10 cell lines, provided by Dr. Ronald Germain from NIH, were grown and maintained in RPMI 1640 containing 10% fetal calf serum, 2mM L- glutamine (GIBCO, Grand Island, NY) , 0. ImM nonessential amino acids, ImM sodium pyruvate, lOmM Hepes, 50 uM 2-Me (Sigma Chemical Co., St. Louis, MO) 100 ug/ml atroptomycin, 100 U/ml penicillin (GIBCO, Grand Island, NY) at 37°C and 5% C0₂.

The IL-2 dependent T cell line, HT-2, was maintained in RPMI 1640 medium containing 10 units/ml (Sigma) mouse recombinant IL-2. The antigen presenting cell line, RT3.3H6 cell lines (provided by Dr. Ronald Germain from NIH) is a transfected line of mouse L cells expressing cell surface murine MHC class II (I-Ad) and was grown in DMEM (GIBCO) at lx HAT (Sigma) . IL-2 assay RT2.3H6 cells were bred in 0.025% glutaraldehyde

(Sigma) , and after 30 s the cells were washed three times and resuspended in RFMI 1640 with 1% FCS in the presence of protease inhibitors. Fixed cells were incubated with ovalbumin peptide 323-339 (1 μg/ml) for two hours or at 37°C. After the incubation period, the cells were washed three times in RPMI 1640 (1096 PCS) , and DO.11.10 T hybrodoma cells and different peptide analogs (50 μg/ml) or antibodies (1 μg/ml) were added. 24 hours later, lOOμl of supernatant was removed to test for IL-2 production using the IL-2 sensitive cell line HT-2. Serial dilutions of supernatant were incubated overnight with 4 X IO⁶ HT-2 cells, and the viability of the HT-2 cells were assessed by a 3- (4, 5-dimethiazol) -2, 5-diphenyltetracollum bromide (MTT) assay. IL-2 titers were measured in U/ml and expressed as the percentages of the untreated control cells. Soluble proteins

DR4 molecules were purified by affinity chromatography from insect cells coinfected with HLA-DR2-and β-DAF recombinant baculoviruses (a gift of P. Sinigaglia and J. Guardiola, Roche Milano Ricerche, Milan, Italy) . Purified recombinant soluble CD4 (sCD4), kindly provided by A. Traunecker (Base) Institute For Immunology, Basel, Switzerland), was a chimeric molecule comprising the CD4 domains 1, domain 2 and part of the human IgG3 heavy chain sCD4 was obtained from R. Sweet (SmithKline Beecham Pharmaceuticals, Philadelphia) . Protein labeling and solid phase radioimmunoassays

DR4 was iodinated with lmCi Na¹⁵I (Amersham) according to the choramine T method. Specific activities was 10 μCi/μg. Purified CD4 molecule (1 to 3 μg/ml) diluted in 50 μl PBS was immobilized onto immulon removestrips (Dynatech, Billingshurst, U.K.) by an incubation for 2 hours at 37°C. After 3 washes with PBS containing 0.1% Tween 20 (PBS-Tw) , coated wells were saturated overnight at 4°C with PBS containing 1% bovine serum albumin. For competition experiments ¹²⁵I-labeled DR4 was pre- incubated in tubes with increasing amounts of CD4 peptide analogs for 1 hour at 37°C. The mixture (50 μl) was then added in triplicate to CD4 coated wells for 1 hour incubation at 37°C. All reagents were diluted in PBS-Tw and the amount of ¹²⁵I-labeled DR4 was 150,000 cpm = 5 ng/well. After 5 washes with PBS-Tw bound radioactivity was measured in Cobra gamma counter (Packard Instruments, Meriden, CT) . Results were expressed as the mean of 3 independent experiments. Background was determined by binding of labeled proteins to uncoated wells . CD4 binding to MHC class II peptide. HPLC purified human MHC II β2 134-148 synthetic peptide (0.1 M ammonium bicarbonate pH 7.8) was immobilized onto 96 wells microtiter plates in quadruplicate and dried by overnight evaporation at 37°C. After washing and blocking in binding buffer (2% BSA in HBSS, pH 7.6) . For two hours at room temperature, 100 μl of each of the oxidized CD4 peptide was added to the appropriate well. 25 μl 2% BSA in PBS was also added to each well as carrier protein. Then 10 μg of ¹²⁵I sCD4 (specific activity = 21,281 cpm/μg) was added and incubated at room temperature for one hour. The final reaction volume was 150 μl . The wells were washed five times with 2% BSA in PBS buffer. Finally each sample was resuspended in 10% SDS for 10 minutes and transferred to small test tubes. The cpm value for each well was read directly in the gamma counter (LKB Instruments) and the radioactivity remaining was counted. Immunofluorescence and flow cytometry.

Antibodies (IgG) or peptides which were fluorescene- labeled by using FITC kit from Boehringer Mannheim were incubated with cells on ice in the dark in 150 μl binding buffer (Ca⁺⁺ and Mg⁺⁺ free Hanks' balanced salt solution 0.5% bovine serum albumin 0.05% sodium ozide, pH 7.4) for 30 minutes. The cells were washed twice and then resuspended in 0.5 ml binding buffer. FITC-conjugated anti-mlgG antibody was used to detect the primary antibody. The mean channel fluorescene of different cells were determined by FACScan IV analyzer (Becton Dickinson) . Data was acquired while gating on the live cell population.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Greene, Mark I.

(ii) TITLE OF INVENTION: CONSTRAINED CD4 PEPTIDES AND METHODS OF USING

THE SAME

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz & Norris

(B) STREET: One Liberty Place - 46th floor

(C) CITY: Philadelphia

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: Windows 3.0

(D) SOFTWARE: WordPerfect 6.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/014,294

(B) FILING DATE: 29-MAR-1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: DeLuca, Mark

(B) REGISTRATION NUMBER: 33,229

(C) REFERENCE/DOCKET NUMBER: UPN-3078

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-568-3100

(B) TELEFAX: 215-568-3439

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Phe Cys Ser Ile Gin Phe His Trp Cys Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Tyr Cys Asn Gin Gly Ser Phe Leu Cys Tyr 10

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3

Phe Cys Thr Lys Gly Pro Ser Lys Cys Tyr

1 5 10

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

Phe Cys Lys Leu Asn Asp Arg Ala Cys Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Phe Cys Tyr Leu Cys Glu Val Glu Asp Gin Cys Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

Phe Cys Glu Val Glu Cys Gin Lys Glu Cys Tyr

1 5 10

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Phe Cys Tyr Ile Cys Glu Val Glu Asp Gin Cys Tyr 1 5 10

(2) INFORMATION FOR SEQ ID NO:8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

Phe Cys Glu Val Glu Asp Gin Lys Glu Cys Tyr

1 5 10

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

Asn Gly Gin Glu Glu Lys Ala Gly Val Val Ser Thr Gly Leu Ile 1 5 10

(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ile Ser Gin Ala Val His Ala Ala His Ala Arg Thr Asn Glu Ile Asn 1 5 10 15

Glu Ala Gly Arg 20

Claims

1. A constrained peptide comprising an amino acid sequence that consists of no more than 30 amino acid residues and has the formula:

wherein:

R-_L is 1-6 amino acid residues including at least one of tyrosine or phenylalanine;

R₂ is a linking amino acid residue;

R₃ is 0-13 amino acids;

R₄ is an active sequence of 3-26 amino acids including human CD4 sequence 82-89 or at least a 3 amino acid fragment thereof;

R₅ is 0-13 amino acids;

R₆ is a linking amino acid residue;

R₇ is 1-6 amino acid residues including at least one of tyrosine or phenylalanine; wherein R₂ and R₆ are bound to each other, forming a cyclic portion which includes R₂, R₃, R₄, R_s and R₆ with R and R₇ forming exocyclic portions.

2. The peptide of claim 1 wherein R₂ is cysteine.

3. The peptide of claim 1 wherein R₆ is cysteine.

4. The peptide of claim 1 wherein R₂ is cysteine and R₆ is cysteine.

5. The peptide of claim 1 wherein R₃ is 0 amino acids.

6. The peptide of claim 1 wherein R₅ is 0 amino acids.

7. The peptide of claim 1 wherein R₃ is 0 amino acids and R₅ is 0 amino acids.

8. The peptide of claim 1 wherein said peptide is SEQ ID NO:5 or SEQ ID NO:6.

9. A pharmaceutical composition comprising a peptide of claim 1 and a pharmaceutically acceptable carrier or diluent.

10. A method of inhibiting the oligomerization of membrane bound CD4 in a CD4+ cell comprising contacting said cell with a peptide of claim 1.

11. A method of inhibiting activation of a T cell comprising contacting said T cell with a peptide of claim 1.

12. A method of treating an individual with a disease, disorder or condition whose causes and/or symptoms are associated with and/or characterized by T cell activation comprising administering to said individual a therapeutically effective amount of a peptide of claim 1.