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WO1997044351A1 - Dimeres immunomodulateurs - Google Patents

Dimeres immunomodulateurs Download PDF

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Publication number
WO1997044351A1
WO1997044351A1 PCT/US1997/008689 US9708689W WO9744351A1 WO 1997044351 A1 WO1997044351 A1 WO 1997044351A1 US 9708689 W US9708689 W US 9708689W WO 9744351 A1 WO9744351 A1 WO 9744351A1
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WIPO (PCT)
Prior art keywords
peptide
compound
peptides
days
amino acids
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PCT/US1997/008689
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English (en)
Inventor
Carol Clayberger
Alan M. Krensky
Roland Beulow
Original Assignee
The Board Of Trustees Of Leland Stanford Junior University
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Publication date
Priority claimed from US08/653,294 external-priority patent/US7011834B1/en
Application filed by The Board Of Trustees Of Leland Stanford Junior University filed Critical The Board Of Trustees Of Leland Stanford Junior University
Priority to AU32098/97A priority Critical patent/AU3209897A/en
Publication of WO1997044351A1 publication Critical patent/WO1997044351A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the immune system plays a crucial role in the defense of mammalian hosts against pathogenic organisms and aberrant indigenous cells, such as in neoplasia
  • cytotoxic T-lymphocytes CTL
  • CTL cytotoxic T-lymphocytes
  • autoimmune diseases a group of diseases referred to as autoimmune diseases
  • transplantation and autoimmune diseases one would wish to be able to inhibit the CTLs from attacking the tissue.
  • immunosuppressants are employed, which generally debilitate the immune system
  • the patient is much more susceptible to adventitious infection, as well as numerous side effects resulting from the drugs, such as cyclosporin A and FK5O6.
  • drugs such as cyclosporin A and FK5O6.
  • compositions for immunomodulation so as to inhibit cytotoxic T-lymphocytes ("CTL") from undesirably attacking cells in a host or in vitro
  • CTL cytotoxic T-lymphocytes
  • the compositions contain peptides or peptide-like compounds comprising amino acid sequences related to a Class I HLA-B ⁇ ,-domain.
  • the compounds are employed in accordance with conventional administration and formulation techniques and find use in vitro and in vivo.
  • the invention is directed to peptide-type compounds or variants thereof having immunomodulating activity including the N-terminal acylated and C-terminal amidated or esterified forms thereof of up to 60 amino acids;
  • the peptide-type compounds comprise the formula: ⁇ - ⁇ wherein: ⁇ and ⁇ are the same or different and are of the formula:
  • the invention is directed to pharmaceutical compositions which contain, as an active ingredient, the above-described peptide-type compounds and to methods of modulating the immune response using the compounds and compositions of the invention.
  • the invention is directed to recombinant materials for the production of those of the peptide-type compounds of the invention which are themselves peptides and which contain amino acid residues encoded by the gene Also included within the scope of the invention are antibodies immunoreactive with the peptide-type compounds of the invention.
  • CTL immunomodulating peptide compositions are provided in which the active ingredient is a peptide or peptide-like compound with sequences based on a sequence present in a Class I HLA-B ⁇ i -domain, wherein the sequence is of at least 6 amino acids.
  • the compounds are dimers (thus containing a minimum of 12 amino acids) optionally having substitution of amino acids which do not adversely affect the activity of the peptides, and may include D-isomers or nongene-encoded amino acids.
  • the peptides will be based on a 6 amino acid sequence found at positions 79-84 in the HLA-B sequence, but may include amino acids related to those at HLA-B positions 60 to 84, as well as additional irrelevant sequences.
  • the compounds of the invention are peptide-type compounds defined as follows and variants thereof, and include the N-terminal acylated and N-terminal amidated or esterified forms thereof, of up to 60 amino acids. All have immunomodulating activity.
  • the peptide-type compounds comprise the formula: ⁇ - ⁇ wherein: ⁇ and ⁇ are the same or different and are of the formula: (a) ⁇ R aa 76 - 77 L ⁇ (aa 79 - 84 ) or (b) (aa 84"79 ) ⁇ L aa 77"76 R ⁇ wherein: aa 76 is E or V; aa 77 is D, S or N; aa 79 is R or G; aa 80 is I or N; aa 81 is a hydrophobic or small amino acid; aa 82 is R or L; aa 83 is G or R, aa 84 is a hydrophobic or small amino acid, wherein the sequence in brackets may be absent or, beginning with L, may be extended by one or more amino acids in accordance with the sequence in the brackets, that is, the sequence may optionally be truncated at any peptide-type bond within the brackets
  • the term "substantially pure” means a preparation of the compound which is usually greater than about 70% free of materials with which the peptide may naturally be associated, and preferably greater than about 80% free of these materials, "these materials,” however, exclude materials with which the peptide may be mixed in the preparation of pharmaceutical compositions
  • peptide-type compound is meant compounds which are derived from amino acid sequences but wherein one or more of the peptide bonds is optionally replaced by an isostere as further described hereinbelow.
  • peptide-type includes both peptides and these modified forms
  • immunomodulating activity is meant that the compound can be shown to inhibit CTL-mediated lysis in the cytotoxicity assays set forth in Example 1 below and/or inhibits the proliferation of purified T cells in response to anti-CD3 according to the assay described in that example.
  • the invention compounds also include "variants", i.e., peptide-type compounds which differ from those specifically set forth above by conservative substitutions which do not destroy the immunomodulating activity of the compound.
  • conservative substitution is meant that an amino acid which is of the same general group as that for which substitution is made replaces the referent amino acid
  • the replacing amino acids may or may not be gene-encoded They may also include either D or L-isomers where relevant.
  • conservative substitutions are those within groups defined as follows
  • the residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH -
  • the residue has a positive charge due to association with H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium
  • Amino acid residues can be further subclassified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues
  • subclassification according to the foregoing scheme is as follows
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains, and is not, therefore, included in a group
  • either the standard three-letter code or standard one- letter code will be used to denote the gene-encoded amino acids
  • a superscripted dagger C to the right of the one-letter code symbol will be employed, thus, D + represents D-aspartic acid, as does D-asp, L f represents D-leucine as does D-leu
  • Certain commonly encountered amino acids include, for example, beta-alanine (beta-Ala), or other omega-amino acids, such as 3 aminopropionic, 2,3 diaminopropionic (2,3-diaP), 4 aminobutyric and so forth, alpha-aminisobutyric acid (Aib), sarcosine (Sar), ornithine (Orn
  • Sar, beta-Ala, 2,3-diaP and Aib are small, t BuA, t BuG, N Melle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic are hydrophobic,
  • the D-isomers of the nongene-encoded amino acids will be denoted by a prefix D- when the nongene-encoded amino acid appears in a sequence generally using the three-letter code, however, a superscripted dagger ( f ) will be used when the nongene-encoded amino acid appears in a sequence where the gene-encoded amino acids are denoted by a one-letter code.
  • omega-amino acids are classified according to size as small (beta- Ala and 3 aminopropionic) or as large and hydrophobic (all others)
  • Other amino acid substitutions for those encoded in the gene can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme according to their structure.
  • the number of substitutions as compared to the basic sequences set forth above to provide variants that come within the scope of the invention will include relatively few substitutions As previously indicated, even though specific amino acids or a limited number of amino acids have been indicated at each site, individual amino acid substitutions may be made to determine which amino acids are essential Usually, the number of substitutions will not exceed 20% of the total number of amino acids in the sequence, usually not exceed 15%, more usually not exceed 10%, generally being not more than 3 substitutions, more usually not more than 2 substitutions; and still more usually not more than 1 substitution.
  • the amino terminus of the compound may be in the free amino form or may be acylated by a group of the formula RCO-, wherein R represents a hydrocarbyl group of 1-6C.
  • R represents a hydrocarbyl group of 1-6C.
  • the hydrocarbyl group is saturated or unsaturated and is typically, for example, methyl, ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, and the like.
  • the C-terminus of the compounds of the invention may be in the form of the underivatized carboxyl group, either as the free acid or an acceptable salt, such as the potassium, sodium, calcium, magnesium, or other salt of an inorganic ion or of an organic ion such as caffeine.
  • the carboxyl terminus may also be derivatized by formation of an ester with an alcohol of the formula ROH, or may be amidated by an amine of the formula NH 3 , or RNH 2 , or R 2 NH, wherein each R is independently hydrocarbyl of 1-6C as defined above Amidated forms of the peptides wherein the C-terminus has the formula CONH 2 are preferred
  • the peptides of the invention may be supplied in the form of the acid addition salts
  • Typical acid addition salts include those of inorganic ions such as chloride, bromide, iodide, fluoride or the like, sulfate, nitrate, or phosphate, or may be salts of organic anions such as acetate, formate, benzoate and the like The acceptability of each of such salts is dependent on the intended use, as is commonly understood
  • This replacement can be made by methods known in the art
  • the following references describe preparation of peptide analogs which include these alternative-linking moieties Spatola, A F , Vega Data (March 1983), Vol 1 , Issue 3, "Peptide Backbone Modifications” (general review), Spatola, A F , in “Chemistry and Biochemistry of Amino Acids Peptides and Proteins," B Weinstein, eds , Marcel Dekker, New York, p 267 (1983) (general review), Morley, J.S , Trends Pharm Sci (1980) pp 463-468 (general review), Hudson
  • ⁇ and/or ⁇ in the above formula are peptides from HLA-B0701 (B7) or B2702, where one or more amino acids may be substituted while retaining CTL modulating activity
  • the portions of B0701 (B7) and B2702 at positions 75-84 differ at sites 77, 80, 81, 82 and 83, having identity at sites 75, 76, 78, 79, and 84 It is found that the amino acid at sites 77, 81 and 84 may be substituted without loss of immunomodulating activity
  • a consensus sequence is REX'LRX 2 X 3 X 4 X 5 X 6 , where X 1 may be any amino acid, polar or non-polar, preferably polar, either charged or uncharged, X 2 is preferably an amino acid of at least five carbon atoms, which may be polar or non-polar, particularly asparagine and isoleucine, X 3 may be non-polar aliphatic of from two to six carbon atoms (i.
  • X 1"6 will be S, N, L, R, G, Y or N, I, A, L, R, Y or D, I, L, L, R, Y, respectively, where one of the amino acids in one group may be substituted at the same site for the amino acid in the other group
  • Particularly preferred embodiments of ⁇ and ⁇ are
  • RIALRY and RJLLRY and these sequences read in reverse, e.g., YRLLIR and YRLAIR, and extensions thereof, e.g., RENLRIALRY and YRLAIRLNER, RENLRILLRY and YRLLIRLNER, REDLRIALRY and YRLAIRLDER, and REDLRJLLRY and YRLLIRLDER.
  • the sequences may be extended at either or both the N- or C-terminus by additional amino acids in the HLA sequence or by other amino acids, which would not be interfering with the activity of the defined peptides.
  • the dimers of the invention are of the formulas, (a)-(a); (b)-(b), (a)-(b), or (b)- (a), wherein (a) and (b) are defined as set forth above.
  • Particularly preferred are compounds of the formula (a)-(b) and (b)-(a), especially (b)-(a).
  • examples of compounds within the scope of the invention are. YRLLIRRILLRY, YRLLIRRIALRY, YRLAIRRILLRY, YRLAIR + RIALRY,
  • YRLAIRI lAlLLRY AYRLLIKVIRIVLKY
  • SYKLVIKINNIRIWKF including those wherein one or more peptide bonds has been replaced by an isostere and the extended or acylated or amidated/esterified forms thereof
  • the subject peptides may be modified in a wide variety of ways
  • the peptides may be joined by covalent bonds at any convenient site along the peptide to a variety of other compounds for different purposes.
  • the peptides may be joined to immunogens for administration to a host for immunization for production of antibodies, or may be joined to a non-adjacent MHC sequence of the particular MHC antigen by means of synthesis, expression of a synthetic gene, or the like; joined to a lipid or polyalkyleneoxy group; joined to a sugar; or joined to a nucleic acid.
  • the subject peptides may be joined to another peptide by synthesis or expression of a synthetic gene where the other peptide provides for extended stability of the subject peptides when administered to a host
  • Various peptides may be used, such as the immunoglobulin constant region, e.g., IgG Fc.
  • the subject peptides may be joined to a toxin, such as diphtheria toxin, ricin, abrin, and the like, particularly where the binding chain has been removed or inactivated, so as to prevent non-specific binding of the binding chain to cells.
  • sequences may be modified in a variety of ways depending upon their ultimate purpose. Different N- or C- terminal groups may be introduced which allow for linking of the peptide to solid substrates or other molecules. In a synthetic procedure, any molecule may be introduced at the N- or C-terminus which would allow for subsequent reaction, depending upon the purpose for which the peptide is prepared.
  • labels may be linked to the terminus, which may provide, directly or indirectly, a detectable signal.
  • fluorescers may be introduced at the terminus or other molecules which provide a linkage to labels such as fluorescers, enzymes, particles, or the like.
  • linkage may be introduced at the terminus, e.g. , biotin, which will bind to an avidin conjugate with enzymes or fluorescers.
  • various reactive sites may be introduced at the terminus for linking to particles, solid substrates, macromolecules, or the like.
  • an internal amino moiety of a growing chain bound to a solid substrate with the intermediate side groups protected may be conjugated with methyldithiobenzoic acid (MDTB).
  • MDTB methyldithiobenzoic acid
  • the free mercaptan group may then be used for conjugating with activated olefins.
  • proteins such as serum albumin, keyhole limpet hemocyanin, bovine ⁇ -globulin, or the like, may be conjugated to the peptide to provide for an immunogen to produce antibodies to the peptide for use in immunoassays, for affinity chromatography, or the like.
  • the peptide can be bonded to another polypeptide by preparing a DNA sequence which has the peptide at the N-terminus, C-terminus or internal to the protein, so as to provide a fused protein which includes the binding peptide of interest
  • fused proteins may be produced which have enzymatic activity, which enzymatic activity may be modulated by macromolecules, e.g., antibodies, binding to the peptide of interest
  • macromolecules e.g., antibodies
  • the peptides of the subject invention may be modified in a wide variety of ways for a variety of end pu ⁇ oses while still retaining biological activity (By "biological activity" is intended the ability to bind with specific affinity (>10 6 M "1 ) to an antibody to the same epitope of the native protein )
  • the subject peptides may also be used in combination with antigenic peptides or proteins of interest to activate CTL's
  • the subject peptides may be bound to a protein, either directly or indirectly, so as to
  • lipid particularly a phospholipid to provide for the presence of the peptide or protein on the liposome surface
  • Phosphatidyl choline, phosphatidyl ethanolamine, or other lipid may be used with a bifunctional linking agent, such as MBSE, glutaraldehyde, methyldithiobenzoic acid, or the like
  • MBSE glutaraldehyde
  • methyldithiobenzoic acid or the like
  • the formation of Iiposomes with conjugated proteins finds ample support in the literature, see, for example, U S Patent Nos 3,887,698, 4,261,975 and 4,193,983
  • the modified peptide or protein is combined with the lipids in an aqueous medium and sonicated to provide the desired Iiposomes
  • the Iiposomes may then be harvested and used in the ways indicated
  • the immunomodulating activity of the compounds of the invention makes them useful in preventing rejection of transplants and in the treatment of autoimmune diseases While for the most part allogeneic implants will be involved, there is substantial interest in using xenogeneic sources of organs, such as pig, primate other than human, e.g., baboon, etc Organs of interest include heart, lung, kidney, vascular vessels, eye, gut, bone marrow, liver, etc - - -
  • the organ may be initially bathed in one or more compounds according to the subject invention, which may be used individually or in combination with other drugs
  • concentration for the subject compound will depend upon its particular activity, generally being in the range of about 0 1 ⁇ g/ml to 1 mg/ml
  • the subject compositions may be administered to the recipient host directly
  • Particular peptides act on CTLs having a broad range of Class I MHC antigens
  • These compounds find particular application in protecting against transplantation rejection by providing for a regimen, where the peptides are administered at various times and in various periods, depending upon whether a bolus, slow release, a depot, continuous infusion or other form of dosage is employed, the manner of administration, whether oral, parenteral, inhalation, or the like; the particular times chosen for the administration, the degree of difference between the transplantation antigens of the donor and recipient, and the like
  • the administration of the peptide may be prior to, at and subsequent to the day of transplantation, or combination thereof It is found that various regimens may be employed effectively, so that no particular regimen can be specifically defined
  • administration should begin at least three days prior to the transplantation, preferably at least about five days, and more preferably at least about 7-20 days prior to the transplantation, while if the peptide is administered beginning at or after the transplantation, preferably administration is initiated within one day of the transplantation, preferably on the day of the transplantation, and may be administered during the grafting process
  • administrations usually not more than about 10, more usually not more than about 6, generally at least about 2, frequently ranging from about 2 to 6 administrations, where the administrations may be daily, alternating days, usually at not more than about 3 days, preferably not more than 2 day, intervals
  • an immunosuppressant drug can also be administered, generally at or subsequent to the transplant, either by itself, or in conjunction with the peptide, particularly where the peptide is administered after the transplantation
  • a subtherapeutic dose of the immunosuppressant compound is employed, where the immunosuppressant may be a single agent or a combination of agents, where the
  • the subtherapeutic dose will be not less than about 5% of the therapeutic dosage, usually not less than about 10%, more usually not less than about 25%, and usually not greater than about 75%, more usually not greater than about 60% Where combinations are used, the subtherapeutic dosage is primarily directed to the drug(s) which have significant side effects, although there is a substantial interest in minimizing the effect on the immune system
  • a subtherapeutic dosage is intended a bolus amount, since a direct comparison is difficult, where the subject regimen is terminated within a short period of the transplantation
  • the subject regimen may be daily or less than daily, and other regimens may involve repetitive daily administrations Suffice it to say, that the subject regimen may be terminated within about 20 days, usually within about 10 days of the transplantation, as contrasted with other immunosuppressant regimens, which are for the life of the patient
  • the amount of peptide administered will be in from about 0 1-50, more usually from about 1-25 mg/kg of host This amount will be used for a peptide compound where the half life of the peptide compound is fewer than six hours, more particularly fewer than four hours and greater than about one minute Dosages in the lower portion of the range and even lower dosages 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 at a substantially continuous rate, or the like.
  • 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 at a substantially continuous rate, or the like.
  • the immunosuppressant regimen may vary. For example, where the peptide is given at -7 and -1 day, a single subtherapeutic dosage of cyclosporin A may have insignificant protective capability, while administering cyclosporin A daily, 0-4 days, can be protective. A regimen of administration of peptide on days -14, -12, -10 and -7 followed by cyclosporin A on days 0-4 after transplantation can also have substantial protective effect. Alternatively, by using a combined dosage of the peptide and a subtherapeutic dosage of cyclosporin A, on days 0-4 after transplantation, retention of the grafts can be greatly enhanced.
  • compositions can be prepared as formulations for direct administration, where the compositions may be in a physiologically acceptable medium Where other drugs are present, specialized formulations may be employed to ensure the stable dispersion of the other drug.
  • Media which may be employed include water, saline, phosphate buffered saline, ethanol, vegetable oil, etc Where cyclosporin is involved, the formulation may also include Cremaphor®. Other conventional media may also find use.
  • other components may also be included, such as stabilizers, antibiotics, detergents, dispersants, emulsifying agents, Iiposomes, etc
  • Similar formulations can be used and optimized for use in protocols for treatment of autoimmune diseases.
  • Such treatments can be routinely designed and optimized based on standard medical practice and based initially, at least, on animal models.
  • animal models for a number of autoimmune diseases for example, NOD mice are widely used as murine models for the investigation of autoimmune diabetes, as they spontaneously develop diabetes by about eight weeks of age.
  • Various peptides derived from B2702.75-84 are currently being tested in protocols involving three ip injections per week starting at three weeks of age. The results of these models will be helpful in the design of appropriate therapeutic protocols for diabetes Similar models are useful in the development of protocols for other autoimmune diseases, such as rheumatoid arthritis and lupus erythematosis
  • compositions can be produced in a variety of ways Where the natural amino acids are employed and only peptide linkages are contained in the peptide-type compounds of the invention, the peptides can be prepared using standard recombinant techniques In such methods, expression systems are constructed using conventional methods wherein a nucleotide sequence encoding the desired peptide is operably linked to control sequences capable of effecting its expression in suitable host cells
  • suitable host cells can be used, including prokaryotes, yeast, mammalian cells, avian cells, insect cells and plant cells
  • the choice of appropriate control sequences will, of course, be governed by the choice of recombinant host
  • the recombinant cells modified to contain a DNA molecule which comprises the desired expression system are cultured under conditions wherein the desired peptide is produced and the peptide is recovered from the culture Standard purification processes can be used for recovering the peptide, or cell supernatants or lysates may be used in crude form in some circumstances
  • Antibodies that are "specifically immunoreactive” or “immunospecific” for with the peptide-type compounds of the invention may also be prepared by “specifically immunoreactive” is meant antibodies which react with the dimers of the invention, but fail to react with the native peptides represented by the sequences in the HLA alleles -
  • the dimers are based upon which the dimers are based.
  • standard immunization techniques may be employed whereby a suitable animal is administered the dimer to which an antibody is desired, and administration is continued under appropriate protocols until suitable titers of the desired antibodies are found in the serum or plasma. It may be necessary to associate the immunogen with an appropriate carrier in order to provide sufficient titers, as is understood in the art.
  • the antisera may be used directly; for other uses, it may be desirable to purify the immunoglobulin fraction or to prepare monoclonal forms of these antibodies with the appropriate specificities.
  • antibody-producing cells such as splenocytes or peripheral blood lymphocytes are harvested and immortalized, typically by polyethylene glycol fusion with tumor cells, and the resulting immortalized cells are cultured individually and screened for the production of the desired antibodies.
  • the antibodies prepared as described above can be used intact, or fragments thereof may be used which retain their immunospecificity. Typical such fragments are F ab , F»b , or F (a b )2 fragments.
  • the antibodies may be prepared using standard recombinant techniques by recovering the relevant genes from the immortalized cells.
  • the genes may also be manipulated to provide the above-mentioned fragments, or to provide the antibodies in polyvalent form by coupling a heavy chain/light chain combination immunospecific for one of the peptides of the invention with a similar combination immunospecific for another entity
  • the antibodies may be prepared in single-chain forms such as Fy forms.
  • Various additional modifications, such as humanization of murine-derived antibodies may also be made
  • Antibodies with immunospecificity for the peptide-type compounds of the invention are useful in purifying these peptides, as diagnostic tools in quantitating the peptides of the invention, and in diagnostic assays tracing the activity of the peptide- type compounds of the invention tn vivo
  • Example 1 Assay for Immunomodulating Activity
  • the compounds of the invention can be assessed for their ability to modulate the immune system by convenient in vitro assays
  • the following example illustrates these assays and provides results for compounds of the invention and for control peptides
  • the various peptides were tested for their ability to inhibit lysis by established CTL, for the ability inhibit proliferation of purified T cells in response to anti-CD3, and for their ability affect intracellular calcium levels
  • Nonadherent cells were passed over a nylon wool column and the resulting population was >90% CD3+ by FACS analysis CD8+ CTL lines specific for HLA-A2, B7, B27, B48, or Cw4 were generated and maintained in long-term culture as previously described [Buxton et al , J. Exp. Med. (1992) 775 809, Wesley et al , Hum. Immunol.
  • Jurkat E6-1 and the T cell receptor negative Jurkat, JRT3-T3 5 were obtained from the American Type Culture Collection (ATCC, Rockville, MD)
  • Other cell lines used included the EBV transformed B cell lines JY, MS, C1R, and 721 221, K562 and HEL, erythroleukemia cell lines, the T cell tumors Peer, Hut-78 and HSB, the Burkitt's lymphoma cell lines Daudi and SUP B17 [Wright et al , J. Exp. Med.
  • NK-like cell line YT2C2 All transformed cells were grown in culture medium [RPMI-1640 supplemented with 10% fetal bovine serum (Hyclone, Ine , Logan, UT) 2mM L-glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin]
  • CTL assays were performed as described using 51 Cr- labeled EBV transformed B cells expressing the appropriate HLA allele as targets [Krensky et al , J. Immunol (1982) 729 2001 ] - -
  • Peptides Peptides were synthesized with an Applied Biosystems Miligen/Biosearch 9050 automated peptide synthesizer using Fmoc chemistry in the Protein and Nucleic Acid Facility at Stanford Medical Center. Peptides were purified by HPLC and the homogenicity of each was confirmed by analytical reverse-phase HPLC. Amino acid content was confirmed by amino acid analysis. Peptides were dissolved at 40 mg/ml in DMSO and then further diluted into culture medium for assay.
  • HLA-B2702 60-84 inhibits lysis by established CTL (Clayberger et al. Transplant Proc (1993) 25 All, whereas HLA- B2705 60-84 does not
  • HLA-B2702 75-84 inhibited lysis by established CTL in this assay
  • B2702 60-84 was approximately 5 fold more potent than B2702 75-84, based on the concentration required to achieve 50% inhibition of cytotoxicity Peptides were therefore prepared to evaluate the effect of peptide length
  • the 15-mer, HLA- B2702.70-84 was comparably effective in inhibiting cytolysis to B2072 75-84
  • an inverted repeat dimer, B2702 84-75/75-84 (YRLAIRLNERRENLRIALRY) was as potent as B2702 60-84 in terms of concentration required to achieve 50% inhibition of cytolysis (approximately 3 ⁇ M) and was superior to this peptide in that it completely inhibited cytolysis B2702 75- 84/75-84 showed similar activity.
  • dimers B2702 84-75/75-84 and B2702 84-79/79-84 were "scanned" by substituting serine for the native residue on both sides of the dimer in each position in turn
  • serine scan only those dimers wherein serine was substituted for the isoleucine at position 80 and/or the leucine at position 78 and/or 82 no longer had activity
  • Substitution of serine at other locations in the molecule was without significant effect
  • the technique of systematically replacing corresponding amino acids in the dimer with serine residues then testing for activity is referred to as a "serine scan"
  • inhibition of cytolysis employed the CTL cell line AJY, a long term CD8+ CTL line specific for HLA-A2 and the target cell was the B-lymphoblastoid cell line JY (HLA-A2, B7)
  • the cytotoxicity assay was performed as described by Clayberger et al , J. Exp. Med.
  • transfectants expressing both HLA- A2 and HLA-B58, an allele that is Bw4a+ were used as targets for HLA-A2 specific CTL. No inhibition of HLA-A2 specific lysis was observed in either case, indicating that the Bw4a sequence is inhibitory only in soluble form.
  • PBL from normal donors were cultured at 5x10 5 cells/round bottom microtiter well in RPMI-1640 supplemented with 10% fetal bovine serum and L-glutamine Cultures were supplemented with Conagglutinin A Cells were incubated at 37°C for three days at which point 3 H-thymidine ( 1 ⁇ Ci/well) was added After 24 hours, wells were harvested and 3 H-thymidine incorporation determined by scintillation counter The following table indicates the results
  • B2702 84-75/75-84 peptide was not due to T cell death or apoptosis T cell lines and freshly isolated pe ⁇ pheral blood lymphocytes were cultured with 1-50 ⁇ M peptide - -
  • BN (RTT), and Lewis (Lew) (RT1 ') rats, weighing 200-250 grams, were used in these studies
  • the rats were purchased from Bantin and Kingman, Fremont, CA (PVG) or Charles River, Boston, MA (ACI, BN and Lew) ACI rats served as recipients of heart or skin allografts from BN or Lew donors Animals were maintained in the Falk Cardiovascular Research Building under standard conditions according to institutional guidelines
  • Peptides were synthesized at the Protein and Nucleic Acid Facility, Beckman Center, Stanford University School of Medicine, or by Multiple Peptide Systems (San Diego, CA) by an automated peptide synthesizer using Fmoc chemistry Peptides were purified by preparative reverse phase HPLC and shown to be >98% homogeneous by analytical reverse phase HPLC. Amino acid content was confirmed by amino acid analysis
  • spleens were removed from naive animals or from animals that had received an allograft a minimum of 60 days earlier and then teased into a single cell suspension
  • Responder cells were plated from 1000 to 40,000 cells per well (24 replicates per - - concentration) into round bottom microliter wells in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, 5 x 10 "5 M ⁇ - mercaptoethanol, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 20% supernatant from Concanavalin A activated rat spleen cells, and 50 ⁇ M ⁇ -methyl mannoside
  • ACI or PVG rats (200 g) were injected intravenously with peptides dissolved in saline on day 0.
  • the left rear footpads of the rats were injected with 5 x 10 6 splenocytes from a syngeneic donor and the right rear footpads with 5 x 10 6 splenocytes from an allogeneic Lew donor (Moeller et al Transplantation (1993) 55 650) See also Twist and Barnes Transplantation ( 1973) 75 182, Fanslow et al. Science ( 1990) 248 739)
  • the animals were sacrificed and the popliteal lymph nodes removed A single cell suspension was prepared, and the cell number determined using a hemocytometer
  • Vascularized cardiac allografts were heterotypically transplanted into the abdomen of recipient rats using a modification of the technique of Ono and Lindsay (Ono and LindseyJ. Thorac. Cardiovasc. Surg. (1969) 57.225) Abdominal allografts were palpated on a daily basis to assess graft function, and rejection was deemed complete when palpable ventricular contractions ceased
  • Full thickness skin grafts were performed using a modification of the technique described by Billingham and Medawar (J. Exp. Biol (1951) 28 385) Both donor and recipient were shaved and the donor skin was cut in standard 2 x 2 cm pieces and subdermal fat was surgically removed Multiple grafts were obtained from a single donor, preserved in cold saline and transplanted on the same day A piece of skin the - - same size as the donor graft was removed from the flank of the recipient and any loose connective tissue was surgically removed from the fascia. The allograft was then fashioned to the recipient fascia with a 4-0 vicryl suture.
  • Cyclosporin A (CsA, Sandoz Pharmaceuticals Corporation, Base, Switzerland) dissolved in olive oil was given orally through a gavage tube at the indicated dose Peptides were dissolved in water or saline and given intravenously or by gavage as indicated.
  • RTT PVG
  • ACI RTl a
  • rats were treated with a single intravenous injection of saline or 2 mg of A2 75-84, B7 75-84, B2702 75-84, or RTl a 75-84 peptide.
  • splenocytes were removed on different days after peptide treatment, cultured under limiting dilution conditions for 5 days with irradiated (2000R) splenocytes from Lewis rats, i e , Lew (RTl 1 ) stimulator cells, and assayed for lysis of 51 Cr-labeled Lew blasts CTL precursor frequency was determined by linear regression analysis As shown in Table 4, the precursor frequency of Lew specific cells in splenocytes isolated from PVG rats (2/group) treated with either saline, the A2 75-84 peptide, or the RTT 75-84 peptide was approximately 1 in 55,000, independent of the day on which the spleen was removed The same frequency was found in splenocytes from animals that had been treated with either the B7.75-84 or B2702 75-84 peptide on the day of splenectomy or 24 hours earlier However, splenocytes obtained from rats treated with the B7.75-84 or B2702 75-84 peptide
  • the ratio of the number of cells recovered from the side injected with allogeneic versus syngeneic cells was approximately 3 1 in rats treated with saline, the A2 75-84 peptide, or the RTl a 75-84 peptide
  • a similar ratio was observed in lymph nodes obtained from rats treated with the B7.75-84 or B2702 75-84 peptide within 24 hours of footpad injection
  • the ratio fell to 1 1 in rats treated with the B7 75-84 peptide 7 or 10 days prior to footpad challenge
  • the ratio in rats treated with the B2702 75-84 peptide 7 or 10 days prior to footpad injection was approximately 2: 1.
  • 8/12 animals given the peptide orally on days -14, -12, -10, and -7 prior to transplantation and 9/12 animals treated on days 0-4 after surgery in combination with CsA on days 0-4 maintained their grafts for >200 days (p .005 and 0007 respectively, compared with CsA alone)
  • rats that had retained their cardiac allografts were given subsequent skin allografts from donor and third party, they rejected the third party grafts but not the donor skin grafts
  • the immunomodulatory effects of the B7 75-84 peptide could be achieved by either oral or intravenous administration
  • RTl A the only sequence available for a rat MHC Class I molecule is the RTl A molecule, which is identical with the B7.75-84 in seven often amino acids, in contrast to the B2702.75-84 peptide which is identical to the RTl A sequence in only five often residues (RTl A.75-84 RVDLRTLRGY).
  • KLH keyhole limpet hemocyanin
  • Example 3 Effect of the Peptides on Allograft Rejection in Mice According to the protocol in Example 3C, rats treated with B2702.75-84 peptide in combination with CsA did not show prevention of allograft rejection.
  • mice Similar protocols conducted in mice show that the B2702.75-84 peptide is capable of conferring such tolerance.
  • the subject peptide In combination with a subtherapeutic dose of cyclosporin A (2.7 mg/kg/day; ip; day 0-4) the subject peptide prolonged graft survival to at least 45 days (3 grafts out of 4 still beating) compared to a median graft survival of 14 days in animals that received cyclosporin A monotherapy.
  • a subtherapeutic dose of cyclosporin A 2.7 mg/kg/day; ip; day 0-4
  • the subject peptide prolonged graft survival to at least 45 days (3 grafts out of 4 still beating) compared to a median graft survival of 14 days in animals that received cyclosporin A monotherapy.
  • B2702.75-84 is highly effective in mice.
  • B2702.84-75/75-84 was tested in a murine model of Graft Versus Host Disease (GVHD).
  • Donor/recipient mice were matched at their major histocompatibility complex loci, but mismatched at minor loci, a situation which closely approximates that of 90% of bone marrow transplant recipients.
  • Recipient mice were lethally irradiated (900R) and given a combination of bone marrow and spleen cells from donor animals.
  • all animals showed evidence of GVHD in 30 days.
  • mice that were treated with this peptide at 100 ⁇ g/mouse/ ip daily for 35 days did not evidence GVHD at that time. In the following 20 days, some of these mice, however, developed GVHD. This outcome is expected to improve by combining administration of the peptide with subtherapeutic cyclosporin A.
  • the compounds of the invention can be used to greatly enhance the acceptance of transplants, either by themselves or in conjunction with other drugs, particularly immunosuppressant drugs.
  • the ability to modulate activation of CTL provides many opportunities for inhibiting lysis in vitro and in vivo.

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Abstract

La présente invention se rapporte à des composés de type peptidique et leurs variantes dotés d'une activité immunomodulatrice et comprenant les formes à N terminal acylées et/ou à C terminal amidées ou estérifiées renfermant jusqu'à 60 acides aminés, dans lesquels le composé de type peptidique répond à la formule: α-β où: α et β sont les mêmes ou différents et répondent à la formule: (a) {R aa?76-77 L} (aa79-84¿) ou (b) (aa?84-79) {L aa77-76¿ R} où: aa76 est E ou V; aa77 est D, S ou N; aa79 est R ou G; aa80 est I ou N; aa81 est un acide aminé hydrophobe ou de petite taille; aa82 est R ou L; aa83 est G ou R; aa84 est un acide aminé hydrophobe ou de petite taille; où la séquence entre parenthèses peut éventuellement être absente ou tronquée à n'importe quelle liaison de type peptidique à l'intérieur des parenthèses. Lesdits composés peuvent être utilisés seuls ou combinés avec des médicaments immunosuppresseurs afin de réduire l'activation des lymphocytes T cytotoxiques, en particulier lors de transplantations.
PCT/US1997/008689 1996-05-24 1997-05-22 Dimeres immunomodulateurs WO1997044351A1 (fr)

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US08/653,294 US7011834B1 (en) 1987-01-30 1996-05-24 Immunomodulating dimers
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696545B1 (en) 1997-04-11 2004-02-24 Sangstat Medical Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US7094413B2 (en) 2002-01-24 2006-08-22 Sangstat Medical Corporation Combined therapy for treatment of HIV infection
US7267822B2 (en) 1997-04-11 2007-09-11 Genzyme Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005784A1 (fr) * 1987-01-30 1988-08-11 The Board Of Trustees Of The Leland Stanford Junio Inhibition de lymphocytes par des peptides hla
US5073540A (en) * 1989-05-15 1991-12-17 Receptron Composite binding site drugs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988005784A1 (fr) * 1987-01-30 1988-08-11 The Board Of Trustees Of The Leland Stanford Junio Inhibition de lymphocytes par des peptides hla
US5073540A (en) * 1989-05-15 1991-12-17 Receptron Composite binding site drugs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF IMMUNOLOGY, 15 March 1995, Vol. 59, No. 5, CUTURI et al., "Prolongation of Allogeneic Heart Graft Survival in Rats by Administration of a Peptide (a.a. 75-84) from the Alpha-1 Helix of the First Domain of HLA-B7 01", pages 661-669. *
JOURNAL OF IMMUNOLOGY, 1994, Vol. 152, No. 8, NISCO et al., "Induction of Allograft Tolerance in Rats by an HLA Class-I-Derived Peptide and Cyclosporine A", pages 3786-3792. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696545B1 (en) 1997-04-11 2004-02-24 Sangstat Medical Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US7267822B2 (en) 1997-04-11 2007-09-11 Genzyme Corporation Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation
US7094413B2 (en) 2002-01-24 2006-08-22 Sangstat Medical Corporation Combined therapy for treatment of HIV infection
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection

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