WO1997011959A1

WO1997011959A1 - Biologically active mhc peptides having a repeated amino acid sequence motif

Info

Publication number: WO1997011959A1
Application number: PCT/US1996/015426
Authority: WO
Inventors: Lennart Olsson
Original assignee: Receptron, Inc.
Priority date: 1995-09-29
Filing date: 1996-09-26
Publication date: 1997-04-03

Abstract

Oligopeptides having a tandemly repeated amino acid sequence, where the repeat has the amino acid sequence of an MHC Class I α-1 domain regulate surface membrane receptor responses by modulating interaction between MHC Class I antigen and the surface membrane receptor. These oligopeptides show decreased aggregation and increased bioavailability when compared to a monomeric peptide. The methods and compositions of the subject invention may be used in diagnosis and therapy of diseases which involves inadequate or inappropriate receptor response as well as in the screening of drug candidates that may affect surface expression of the receptors.

Description

BIOLOGICALLY ACTIVE MHC PEPTIDES HAVING A REPEATED AMINO ACID

SEQUENCE MOTIF

INTRODUCTION Technical Field

The field of the subject invention concerns peptides useful in modulating surface membrane receptor responses.

Background The complex regulatory balance between hormones, receptors and responding cells is critical to the correct functioning of multicellular organisms.

Subtle environmental and genetic factors can disrupt this balance, sometimes resulting in disease. The advent of molecular biology has meant that medically important hormones can be made available in therapeutically useful amounts. Among them are human growth hormone, insulin-like growth factor, insulin, epidermal growth factor, and numerous others.

A condition of great economic and medical significance is insulin resistance, which is an essential feature of a great variety of clinical disorders, such as diabetes mellitus, obesity and certain types of hypertension Individuals with non-insulin dependent diabetes present with insulin resistance in peripheral tissues They have a subnormal glucose utilization in skeletal muscle, where glucose transport across the cell membrane of skeletal muscle is the rate limiting step in glucose metabolism It is possible that a defect exists in insulin-dependent glucose transport in skeletal muscle in diabetic states, where decreased levels of the glucose transporter 4 protein (GLUT4) have been observed

Insulin resistance may also be attributed to a defect in insulin action at the cellular level The insulin receptor is activated by binding of insulin to the alpha-subunit of the receptor, which causes autophosphorylation of the intracellular beta- subunit region The activated insulin receptor couples to cytosolic receptor substrates that can affect signaling cascades, resulting in the pleiotropic hormone response Most proteins involved in the signal transduction pathway are not known yet, but each of them might play a role in the various forms of insulin resistance The heterogeneous nature of insulin resistance makes treatments that can act "upstream" of the signal transduction pathways very attractive, because a number of different pathologies could be treated with a single drug Specific peptides derived from the a1 domain of MHC class I antigens, including those having the ammo acid sequence of D^k-(69-85), have been shown to enhance the cellular response to certain hormone This effect has been attributed to inhibition of the intemalization of the corresponding hormone receptors Insulin-stimulated glucose uptake is increased by adding the peptides to responding cells, offering the possibility of improved therapy for insulin dependent and insulin resistant diabetes The enhanced response may also be exploited in therapies involving other hormones Improvements in the stability and bioavailability of agents that enhance the activity of insulin and other hormones are of considerable interest for their therapeutic benefits.

Relevant Literature

U.S. Patent no. 5,385,888, issued January 31 , 1995, describes Class I MHC peptide modulation of surface receptor activity. Data presented in International patent application PCT/US94/09189 suggest that these peptides must be in an ordered conformation to be biologically active. Peptides having a random coil conformation were found to be inactive. The peptide may therefore need to be held in a specific conformation in order to be biologically activity. The sequences of known HLA and H-2 alleles may be found in Kabat et al. M991 Ϊ Sequences of Proteins of Immunological Interest. N.I.H. publication no. 91-3242, vol. I, pp. 738-740, 761 , 770-771 , 779-780, 788-789 and 802-804. The composition and uses of such peptides are further described in International application PCT/US93/01758. The peptides are further disclosed in International application PCT/US89/00876.

Nisco et al. (1994) J. Immunol. 152:3786 demonstrate the induction of allograft tolerance in rats by an HLA Class I derived peptide and cyclosporin A. Similar tolerance in mice was shown by Beulow ef al. (1995) Transplantation 59:455-460. Prolongation of allogeneic heart graft survival in rats by administration of a peptide from the a1 helix of the first domain of HLA-B7 is described in Cuturi et al. (1995) Tra n s p la n tat io n 59 : 661 -669. Immunomodulation by soluble Class I molecules is reviewed in Beulow ef al. (1995) Transplantation 59:649-654.

For a review of biological functions of MHC Class I antigens see Ohno (1977) Immunol. Rev. 33:59-69; and Simonsen (1985) Proα. Allerαv 36: 151 - 176. For a suggestion that Class I antigens and insulin receptors interact, see Olsson (1984) in Cell Fusion: Gene Transfer and Transformation (eds. Beers & Bassett) 395-403 (Raven Press, New York); Simonsen and Olsson (1983) Ann. Immunol. 134D:85-92; and Stagsted ef al. C_eh (1990) 62:297-307.

SUMMARY OF THE INVENTION Methods and compositions are provided for modulating the activity of cell surface receptors by administration of oligopeptides having a repeated amino acid sequence motif, where the repeated motif has at least substantially the amino acid sequence of a mammalian MHC Class I α-1 domain. Oligopeptides having a repeated sequence show decreased aggregation and increased bioavailability in the enhancement of the cellular response to specific hormones, when compared to the corresponding monomeric peptide. The methods and compositions of the subject invention may be used in diagnosis and therapy of diseases which involve inadequate or inappropriate receptor response as well as in the screening of drug candidates that may affect surface expression of receptors.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Oligopeptides having a repeated amino acid sequence motif, where the motif has at least substantially the sequence of an MHC Class I α-1 domain, are provided. The oligopeptides enhance the cellular response to specific protein hormones and growth factors. Oligopeptides with a repeated amino acid sequence are an improvement over the previously described monomeric peptides due to increased biological activity.

The sequence of the subject oligopeptides is of the formula: (X-Z-X')_n or (X'-Z-X)_n; where X is an amino acid sequence as defined below, Z is a linker sequence, X' is a repeat of X and n is the number of repeats. Usually n will be at least one, and not more than three, preferably n will equal one. X and X' may be identical in sequence, or X' may be a truncated or a modified repeat of the X sequence. X and X' may be direct repeats, i.e. head to tail, or an inverted repeat, i.e. head to head, or tail to tail.

The sequence of X will comprise at least a portion of one of the following sequences, where the oligopeptide comprises, as an active motif sequence, at least 8 amino acids, usually at least about 12 amino acids, and fewer than 40 amino acids, more usually fewer than 24 amino acids. It is understood that up to about three substitutions or deletions may be made in the subject sequences, where the change will not be more than about 20 number %, usually not more than about 10 number %, of the number of amino acids in the active motif. In a preferred embodiment, the sequence of X is at least 12 amino acids, and is contained within the following amino acid sequence (SEQ ID

Where two or more amino acids are indicated at a particular site, they may be employed interchangeably. Most preferably, the sequence motif (SEQ ID NO:2) FRVDLRTLLRYA is included in the oligopeptide sequence. The amino acid sequence of SEQ ID NO:1 is substantially the sequence of an MHC Class I α-1 domain from residues 69 to 85.

The sequence of X' may be identical to X, or may be a truncated or modified repeat of the X sequence. In a preferred embodiment, X and X' are identical. However, the increased bioavailability found with the repeated sequence, as compared to a monomeric sequence, does not require that X' be an exact repeat of X. The sequence of X' will desirably include the active sequence motif SEQ ID NO:2. The sequence of X' may be truncated after aa¹² or before aa⁶, based on the numbering shown for SEQ ID NO:1 The linker sequence Z will be from 0 to 10 amino acids in length, usually at least 1 amino acid and usually not more than 8 amino acids. The amino acids of Z may be any amino acids that do not interfere with the formation of a biologically active oligopeptide Preferred are small neutral am o acids, defined as having 2 to 5 carbon atoms, and 0 to 1 heteroatoms on the side chain, e g serine, methionine, threonine, glycine, alanine, valine, etc Particularly preferred are glycine and alanine Where X and X' are identical repeats, Z will usually be from 1 to 3 ammo acids in length Where X' is a truncated repeat of X, truncated at the am o terminal portion of the sequence for X-Z-X', or the carboxy terminal portion for X'-Z-X, then Z will usually be from about 5 to 8 ammo acids in length

An exemplary oligopeptide has the following am o acid sequence (SEQ ID NO 3) GNEQSFRVDLRTLLRYAGGGNEQSFRVDLRTLLRYA

The oligopeptides of this invention may be prepared in accordance with conventional techniques, such as synthesis (for example, use of a Beckman Model 990 peptide synthesizer or other commercial synthesizer) Peptides may be produced by directly by recombinant methods (see Sambrook ef al Molecular Cloning A Laboratory Manual, CSHL Press, Cold Spring Harbor, NY, 1989) or as a fusion protein, for example to a protein that is one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at a site engineered to yield the desired peptide (see for example Dπscoll et al M993 J Mol Bio 232 342- 350)

The oligopeptides may be extended to provide convenient linking sites, e g cysteine or lysine, to enhance stability, to bind to particular receptors, to provide for site-directed action, to provide for ease of purification, to alter the physical characteristics (e σ solubility, charge, etc ), to stabilize the conformation, etc The oligopeptides may be joined to non-wild-type flanking regions as fused proteins, joined either by linking groups or covalently linked through cysteine (disulfide) or peptide linkages The oligopeptide may be linked through a variety of bifunctional agents, such as maleimidobenzoic acid, methyldithioacetic acid, mercaptobenzoic acid, S-pyridyl dithiopropionate, etc. The oligopeptides may be joined to a single amino acid at the N-or C-terminus of a chain of amino acids, or may be internally joined. For example, the subject peptides may be covalently linked to an immunogenic protein, such as keyhole limpit hemacyanin, ovalbumin, etc. to facilitate antibody production to the subject oligopeptides.

Alternatively, the subject oligopeptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the chain, either internal, or at the N- or C- terminus. Various post-expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation, such that the subject peptide will be bound to a lipid group at one terminus, and will be able to be inserted into a lipid membrane, such as a liposome.

The subject oligopeptides may be PEGylated, where the polyethyleπeoxy group provides for enhanced lifetime in the blood stream. The subject oligopeptides may also be combined with other proteins, such as the Fc of an IgG isotype to enhance complement binding, or with a toxin, such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain. The oligopeptides may be linked to antibodies for site directed action. For conjugation techniques, see, for example, U.S. Patent Nos. 3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081 ; 4,069,105; and 4,043,989, which are incorporated herein by reference.

The subject oligopeptides act to enhance the cellular response to specific hormones that act through surface membrane receptors. Of particular interest are hormones or growth factors that act through receptors that are either internalized or are recycled, that is, internalized into the cytoplasm and optionally returned to the plasma membrane surface, e.g. insulin, insulin-like growth factor, human growth hormone, glucose transporters, transferrin, epidermal growth factor, low density lipoprotein and epidermal growth factor, herein referred to as "therapeutic hormones".

This enhancement of the cellular response to therapeutic hormones provides a means of improving the response of patients that are unresponsive, e.g. resistant, to the action of such hormones. The subject oligopeptides may be administered to patients requiring enhancement of the response to naturally occurring levels of the therapeutic hormone. Altematively, the oligopeptides may be administered to patients in conjunction with a therapeutic hormone. Of particular interest is the treatment of insulin resistance, which may be associated with defects in glucose transport, or in the cellular response to insulin. Administration of the subject oligopeptides improves the response to insulin therapy.

For therapy, the oligopeptides may be administered topically or parenterally, e.g. by injection at a particular site, for example, subcutaneously, intraperitoneally, intravascularly, intranasally, transdermally or the like. Formulations for injection will comprise a physiologically-acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like. Water soluble preservatives which may be employed include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in individual amounts of from about 0.001 to about 5% by weight and preferably about 0.01 to about 2%. Suitable water soluble buffering agents that may be employed are alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives such as carboxymethylcellulose may be used as carrier in amounts of from about 0.01 to about 5% by weight. The formulation will vary depending upon the purpose of the formulation, the particular mode employed for modulating the receptor activity, the intended treatment, and the like. The formulation may involve patches, capsules, liposomes, time delayed coatings, pills, or may be formulated in pumps for continuous administration. The specific dosage can be determined empirically in accordance with known ways. See, for example Harrison's, Principles of Internal Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987.

Generally, a therapeutically effective dose of the subject oligopeptides will be in the range of about 0.005 - 10, more usually from about 0.01 - 1 mg/kg of host weight. Such a dose will be sufficient to enhance the action of the therapeutic hormone by as much as 100%, more usually by as much as 50%. Administration may be as often as daily; usually not more than one or more times daily, or as infrequent as weekly, depending upon the level of drug which is administered. The oligopeptides may be administered alone, or in combination with the therapeutic hormone. The hormone may be administered at a normally therapeutically effective dose, or the dose may be decreased by as much as 50%, usually by as much as 25%, to compensate for the oligopeptide enhancement. The host may be any mammal including domestic animals, pets, laboratory animals and primates, particularly humans. The amount will generally be adjusted depending upon the half life of the peptide, where dosages in the lower portion of the range may be employed where the peptide has an enhanced half life or is provided as a depot, such as a slow release composition comprising particles, introduced in a matrix which maintains the peptide over an extended period of time, e.g., a collagen matrix, use of a pump which continuously infuses the peptide over an extended period of time over a substantially continuous rate, or the like. Heller, Biodegradable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 1 , CRC Press, Boca Raton, FL, 1987, pp 39-90, describes encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13:850-856 describes controlled drug release from hydrophobic polymers.

The subject oligopeptides may also find use in a drug screening assay. Drug candidates capable of inhibiting surface receptor intemalization may be identified by first screening the drug candidates for the ability to compete with peptide for association with either Class I MHC antigen, receptor, a saturable cell-surface binding site, or an accessory molecule(s) involved in surface receptor intemalization. Drug candidates that affect receptor intemalization may also be identified by screening drugs for the ability to either enhance or reduce the effect of peptides on the intemalization of a selected surface receptor.

In one embodiment of the screening assay, a peptide derived from Class I MHC antigen and having modulatory activity or a substantially purified Class I MHC antigen is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate). The insoluble supports may be made of any composition to which peptide, Class I MHC antigen, or other protein can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, membranes and beads. These are typically made of glass, plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose. Microtiter plates are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the peptide, Class I MHC antigen or other protein is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the peptide and is nondiffusable. Following binding peptide or Class I MHC antigen, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein.

The drug candidate and varying concentrations of the oligopeptide are added to each of the sample receiving areas containing support-bound peptide or Class I MHC antigen. The oligopeptide added is of substantially the same amino acid sequence as the oligopeptide bound to the support and is labeled. The oligopeptides could be labeled, directly or indirectly, with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, particle, chemiluminescer, etc. Positive controls for binding of active peptide and competitive binding of active peptide may include samples containing labeled active peptide alone and a mixture of labeled active peptide and unlabeled active peptide, respectively. Samples containing labeled active peptide and unlabeled inactive peptide which does not aggregate with the bound peptide may serve as a negative control for competitive binding with peptide. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the labeled active peptide to the support-bound peptide. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, labeled peptide determined. For example, where a radiolabel is employed in labeling the peptide, the samples may be counted in a scintillation counter to determine the amount of bound, labeled peptide.

In test samples containing the drug candidate, if the amount of labeled active peptide bound to the support-bound peptide or Class I MHC antigen is in the range of values of the positive control samples for competitive binding and is significantly less than binding of labeled peptide in the active peptide alone and negative control samples for competitive binding, then the drug candidate in the test sample is able to successfully competitively bind the support-bound peptide or Class I MHC antigen. Drug candidates capable of such competitive binding may mediate modulation of cell surface expression of a receptor with a peptide-like activity. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1. Analysis of Peptide Structure

The structure of inactive and active forms of the monomeric peptide were determined by X-ray diffraction. Materials and Methods

Peptides: The synthesis and characterization of peptides have been described previously (Stagsted et al. (1983) supra). The active peptide (SEQ ID NO:4) GNEQSFRVDLRTLLRYY and inactive peptide (SEQ ID NO:5) GNEQSARVDLRTLLRYY were synthesized. Briefly, peptides were assembled on a phenylacetamidomethyl (PAM) resin using the t-boc NMP/HOBt protocol of an Applied Biosystems 403A peptide synthesizer. The peptides were purified to greater than 99% homogeneity by preparative high performance liquid chromatography using Vydac C18 (2.2x25 cm) column and a gradient of acetonitrile in 0.1 % aqueous TFA. The desired peptides were confirmed by sequence analysis, amino acid composition, and fast atom bombardment mass spectrometry. X-ray diffraction: A suspension of the fibers was lightly centrifuged to concentrate the material toward the bottom of the centrifuge tube. The liquid containing the fibers was then drawn up into a 1 mm diameter thin-walled glass capillary tube. The flow of the solution along the tube appeared to preferentially align the fibers parallel to the tube.

An X-ray diffraction pattern of this solution was taken with graphite- monochromatized CuK_a radiation using a 0.3 mm diameter collimator and a standard precision camera. The capillary tube was then left with the end open under reduced pressure to permit slow evaporation and concentration of the mateπal. Overnight the sample was concentrated against the side of the tube with little if any surplus liquid apparent. A diffraction pattem of this material was again taken with the monochromatic CuK_a radiation.

Electron Microscopy (EM): the peptides were prepared in a 1 mM solution in water and studied in the electron microscope by 3 different methods.

(a) Sections. Aggregated peptide was sedimented at 12,000g for 10 min at room temperature, the supernatant discarded, leaving a gel. To the top of the gel was added a solution of 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, and the gel fixed for 48 hours. The fixed gel was then removed from the tube, and postfixed for 2 hours in 1 % OsO₄ in cacodylate buffer. To enhance contrast, the gel was incubated with 1 % tannic acid in water for 2 hours, rinsed briefly in water and immersed in 0.5% uranyl acetate in water overnight. Specimens were dehydrated in graded ethanols and finally embedded in epon. Sections were contrasted with lead citrate.

(b) Negatively stained preparations. Solutions were applied to formvar/carbon coated, glow-discharged electron microscope grids and negatively stained with 1 % Uranyl acetate in water. Some specimens were fixed for 5 min by floating the grids on a drop of 1% glutaraldehyde in 0.1 M sodium cacodylate buffer before being negatively stained.

(c) Shadowed preparations. Solutions were applied to pieces of freshly split mica. The pieces of mica were then rinsed by a few drops of distilled water and the peptides fixed on the mica by floating the mica pieces on glutaraldehyde, as above, for 5 min, followed by a rinse in distilled water and air dried. Shadowing with platinum was done in a Balzer freeze-etch apparatus at a shadowing angle of 18°. After shadowing with Pt, a reinforcing layer of carbon was evaporated onto the specimen at 90^°. The resulting replicas were floated off the mica onto water and the replicas picked up on electron microscope grids. Some specimens were rotation shadowed at 15°. Specimens were studied in a Philips 300 electron microscope and photographed at 10-80,000 primary magnification.

Results

The diffraction pattern of the suspended material was diffuse, but showed distinct arcs corresponding to a repeat distance of 4.8 A. On drying, the pattern became much more distinct. The arc corresponding to the d- spacing of 4.8A becomes much more obvious and is the dominant feature in the meridional direction. There is also a weaker, but sharp reflection at 2.4 A that could correspond to a second order of diffraction. In the equatorial direction, a strong arc appears at a spacing corresponding to o^*=11.3A. It might be noted that although the sample was concentrated by evaporation, it was not rigorously dried and dehydrated. Therefore, we assume that the sample retains a limited amount of water.

The 4.8 A spacing on the meridian indicates that the individual polypeptide chains are aligned perpendicular to the fiber axis, with successive β-sheets stack to form altemating glycine-rich and alanine-rich layers with respective interplanar separations of 3.5A and 5.7A. The overall repeat distance is 3.5A + 5.7A = 9.2A. In the present case, the amino acids are, on average, more bulky than both glycine and alanine, which would therefore result in a larger repeat distance, corresponding to the observed spacing of 23.1 A. If the peptide is aligned in an extended conformation, as in a β-sheet, the amino acids on one side of the sheet are predominantly polar whereas they are predominantly non-polar on the other. In particular, all the charged residues are segregated on one side of the sheet. This is consistent with the individual β-sheets assembling to form alternate hydrophobic and hydrophilic layers. Pairs of sheets would tend to form back-to-back dimers with their hydrophobic surfaces away from the solvent. The association of the polar surfaces would be expected to be weak in dilute solution, but should be promoted by concentration of dehydration, as is suggested by the development of the 11.6A reflection on drying the sample. We assume that the overall distance of 11.6A is made up by the combination of separations between the hydrophobic and hydrophilic layers of 4.7A and 6.9A, although the choice of these values is somewhat arbitrary.

Electron Microscopy - The active peptide presents itself in 3 polymeric configurations: as filaments, as aggregates of filaments, either as ribbons or as thicker filaments, or as globules. The most striking structures are the ribbons. These are essentially straight and can curve slightly over their flat side. The longer ribbons are twisted regularly along their length. The period of twist (from cross-over to cross-over) may vary from 150 to 300 nm in different ribbons, but is constant in an individual ribbon. The ribbons are somewhat pliable as seen when they adapt to underlying structures, but they may break when bent over an edge. Some ribbons seemed to fray at their ends, revealing the basic filamentous unit (BFU) of the polymer. The BFU measures approximately 4 nm in width and may show a faintly demarcated midline groove, indicating that it consists of two still thinner filaments. The BFUs also show a periodicity of about 8 nm, in some cases giving the appearance of "pearls on a string". The BFUs may aggregate laterally, forming ribbons with a lateral periodicity of approximately 7 nm, and a thickness of approximately 5 nm. Ribbons vary in width depending upon the number of participating BFUs. Besides ribbons, BFUs may aggregate in bundles to form yet thicker filaments. These thicker filaments show no discernible substructure. There appears to be a limitation on the number of BFUs that pack into thicker filaments, because a diameter of 15 nm seems to be the upper limit.

Finally, globules are observed in some preparations. They are roughly spherical and vary in diameter from 5-20 nm. When oriented favorably to the optical axis they appear layered. Globules are often present among and along ribbons and filaments.

Inactive peptides do not produce ribbons, rather one finds short rods of approximately 10 nm in width, which in shadowed preparations exhibit a filamentous substructure skin to the ribbons.

The data suggest that the monomeric peptides interact through a beta- pleated sheet structure to form large aggregates. The ability to form such large aggregates may be dependent on the "twist" seen between the individual monomers. Therefore, a dimeric peptide capable of self-interaction between the two active motifs, but incapable of twisting, may have a stable structure that is incapable of forming large aggregates.

Example 2.

Glucose Uptake Assay

The uptake of glucose in rat adipocytes was performed. Adipocytes were prepared from non-starved male rat epididymal fat pads (1.2-1.6 g fat per rat) by collagenase digestion. The digest was filtered (25 μl), washed and resuspended in approximately 4 times the cell volume (estimated by lipocrit) in

Krebs-Ringer's/HEPES (KRH) with 5% BSA. Only plastic tubes were used. An aliquot was removed for Coulter counting after staining with 2% osmium tetroxide, filtration and dilution in saline. 50 μl of adipocyte suspension was added to the pre-incubation mix; 300 μl buffer, 50 μl insulin (8 nM final concentration) or buffer; 50 μl test solution (containing the peptide at a concentration of 10 μM for monomer, 20-50 nM for dimer) or buffer, as a control, and incubated for 30 min at 37°C in a shaking water bath. A blank without cells was included for background counting. D-[¹⁴C]-glucose was subsequently added (about 10⁵ dpm/sample) and incubation continued for 60 min. The incubation was terminated by layering the 400 μl sample on top of silicone oil, followed by a 30 sec. microcentrifuge spin, and cutting the adipocytes (thin layer of cells on top of the oil, buffer under oil) into LS vials with scintillation fluid. Glucose concentration was about 300 nM (specific activity = 295 mCi/mmol).

The dimer enhances insulin-stimulated glucose uptake. At maximum insulin stimulation, the effect of the peptide increases total glucose uptake to about 60% above that of maximal insulin. On the part of the insulin dose response curve where glucose uptake increases with increasing insulin concentrations, the peptide enhances the glucose uptake about 70-90% (i.e. about 1.7 to 1.9 fold) above that of insulin only.

The EC5₀ value for the peptide effect using the dimer (SEQ ID NO:3) is ~3 nM at a low concentration (200 nM) insulin. At maximal insulin the effect is obtain with an ECso~40 nM, which corresponds to the effect on the glucose transporter. The monomeric peptide (SEQ ID NO:4) has an EC5₀ of approximately 1 μM.

Example 3.

Effect on in vivo Glucose Uptake

The effect of the HT dimer (SEQ ID NO:3) and glucose on the blood glucose and insulin levels in rats (100-300 g) was determined. All animals were starved 16-20 hr prior to experimentation. The rats were given an i.v. bolus injection of glucose (1 g/kg) and 20 nmol peptide or saline at time 0 after the animals had been anesthetized with pentobarbital. Blood glucose and plasma insulin was measured for the next 120 min. Saline treated controls have an increase in blood glucose from 6 mM to about 24 mM within 10 minutes, whereafter the blood glucose declines again and is normalized in about 60 minutes. Correspondingly, plasma insulin increases from about 150 pM to 1400 pM within 10 minutes, then declines. Injection of the peptide reduced the intial blood glucose increase to about 14 mM and the corresponding plasma insulin to about 600 pM. Blood glucose and plasma insulin were normalized within 30-40 minutes.

It is evident from the above results that oligopeptides having a repeated sequence motif, where the active amino acid sequence motif is derived from a conserved region of the Class I MHC antigens, is effective in inhibiting the intemalization of receptors to specific hormones. The subject oligopeptides have increased activity when compared to monomeric peptides with the corresponding active amino acid sequence motif. The subject methods are therapeutically useful in enhancing the cellular response to hormones.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT is CLAIMED IS:

1. An oligopeptide having the formula X-Z-X' or X'-Z-X, wherein X is at least 12 amino acids and not more than 24 amino acids, comprising the amino acid sequence of (SEQ ID NO:1 ),

wherein the amino acid sequence of X includes the amino acid sequence motif FRVDLRTLLRYA; X' is a direct repeat of X; and Z is a linker of from 1 to 3 amino acids.

2. An oligopeptide consisting of the amino acid sequence (SEQ ID

NO:3) GNEQSFRVDLRTLLRYAGGGNEQSFRVDLRTLLRYA.

3. A therapeutic formulation comprising a therapeutically effective dose of a hormone selected from the group consisting of insulin, insulin-like growth factor, human growth hormone, glucose transporter, transferrin, epidermal growth factor, low density lipoprotein and epidermal growth factor; and a therapeutically effective dose of an oligopeptide according to Claim 1.