Classifications

• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• TNT ODTICTTON Technical Field The field of this invention is immunomodulating therapies.
• tolerization which may be associated with cell depletion, suppression, or anergy.
• T cell proliferation and/or activation are of interest. These include autoimmunity, cancer, T cell mediated cytotoxicity, or the like. There would therefore be a substantial value to be able to enlist naturally occurring processes associated with tolerization to provide for acceptance of allogeneic implants.
• Peptides corresponding to at least a portion of the ccj helix of the alpha chain of Class ⁇ major histocompatibility complex antigens are employed for immunomodulation.
• the peptides, mutant derivatives thereof, and peptidomimetric agents are administered to a host where the immune response is to be modulated.
• compounds, particularly oligopeptides having the same sequence, or being able to compete with a sequence, of at least 8 amino acids coming within the t helix of the alpha chain of a Class ⁇ major histocompatibility complex antigen, more particularly a human lymphocyte antigen (HLA).
• HLA human lymphocyte antigen
• the domain of the alpha chain comprising the amino acids from about amino acid 50 to amino acid 85, more usually from about amino acid 53 to 80, and particularly comprising the amino acids in the range from about71 to 80.
• hile substantially longer amino acid sequences may be employed, these additional flanking amino acids groups will serve specific purposes, rather than providing the immunomodulation activity. substituted, particularly with conservative substitutions. Furthermore, the entire c ⁇ helix does not seem to be essential for binding, so that only conserved regions among the oligopeptides are required for the desired effect. Even in the conserved regions, various substitutions may be made, where polarity and size variations may be accommodated by the binding partner. Conveniently substitutions may be made with alanine and valine, or the alternative amino acid, among the acidic, basic and amio acids, i.e. N and Q.
• the peptide compounds of this invention will include an oligopeptide of at least about 8 amino acids, coming within the following sequence:
• Formula 1 aa 53 aa 54 aa 55 aa 56 F aa 58 aa 59 Q aa 61 aa 62 L aa 64 N I A aa 68 aa 69 aa 70 aa 71 N L aa 74 aa 75 aa 76 aa 77 aa 78 R aa 80 aa 81 aa 82 aa 83 wherein: aa 53 , aa 54 and aa 55 may be any amino acid, including neutral amino acids other than proline, both aliphatic and aromatic, acidic amino acids, or basic amino acids, more particularly V, I, L , D, E, K, R or F; aa 56 is K, R, S or T, particularly R or S; aa 58 is D or E; aaa 59 is G, A or P, particularly A or P; aa 61 is A, G or F, particularly G or F; aa 62 is A or
• sequence having at least 8 amino acids coming within the sequence 71-79 where these amino acids in particular will be more subject to reduction in activity by replacement, although even in this region, some replacement will be permissible without total loss of activity.
• the subject peptides may be modified in a wide variety of ways. As already indicated, the subject peptides may be mutagenized, by employing natural or unnatural amino acids, particularly the D-stereoisomer. While for substitutions and deletions, usually the restrictions indicated previously will apply, where the substitution involves the different stereoisomer, all of the amino acids may be substituted with the unnatural stereoisomer. Sites 74and 77permit modification with retention of activity. For example, replacement of I with a neutral amino acid having about the same chain length (including heteroatoms, in the range of about 5 to 8), e.g.
• T leads to only a partial loss of activity, while replacement of neutral N at position 74had substantially no effect on activity, where an acidic amino acid, D, was used as the replacement. By contrast, replacement of N with D abrogated the activity.
• the activity was inhibition of Conconavalin A induced T cell proliferation by the peptide.
• the assays are straight forward and the peptides may be screened for activity, where conservative and non-conservative substitutions may be employed to detrmine whether an amino acid at a particular site is essential. Usually, the number of substitutions will not exceed 3, more usually not exceed 2, and generally not exceed 1.
• the subject peptides may be obtained from any mammalian source: such as domestic or laboratory animals; e.g. murine, bovine, canine, feline, equine, lagomorpha, primate; and particularly human.
• the peptides may be joined by covalent bonds at any convenient site along the peptide to a variety of other compounds for different purposes, particularly at the termini, where the N-amino group may be substituted and/or the C-carboxyl group may be substituted.
• the peptides may be joined to: a) immunogens for administration to a host for immunization for production of antibodiesl; b) a non-adjacent MHC sequence of the particular MHC antigen by means of synthesis, expression of a synthetic gene, or the like; c) a lipid or polyalkyleneoxy group; d) a sugar; or e) a nucleic acid.
• a) immunogens for administration to a host for immunization for production of antibodiesl b) a non-adjacent MHC sequence of the particular MHC antigen by means of synthesis, expression of a synthetic gene, or the like
• c) a lipid or polyalkyleneoxy group d
• a sugar or e) a nucleic acid.
• Various peptides may be used, such as the immunoglobulin constant region IgG Fc.
• the peptides may be linked to various labels, either directly or indirectly, for detection of binding of the peptides to a target molecule.
• Labels include enzymes, fluorescers, chemiluminescers, radioisotopes, or the like.
• the subject peptides may be bound to a solid substrate, such as particles, container walls, or the like, to serve for affinity separation of cells or cell fragments comprising molecules which have a specific affinity for the subject molecules.
• mechansisms associated with T cell activation and anergy. The subvject peptides may be used toprevent activation of T cells to a particular stimulation, e.g. Concanavalin A, whereby the block in the pathway may be investigated.
• the subject peptides may be used in screening compounds as to their ability to override the effects of the subject peptides, to augment the effect of the subject peptides or to direct the T cell into an alternative pathway.
• Subsets of T cells can be classified by their binding affinity to the subject peptides, as well as their other phenotypic characteristics. For example, tumor infiltrating lymphocytes, natural killer cells, cells that home to synovia, mucosal tissue, subcutaneous tissue, lymphoid tissue, or the like, can be classified as to their binding to the subject peptides. In this way, the subject peptides may be used for specific applications associated with specific indications.
• the subject peptides may be joined together to provide a single polypeptide, where the polypeptide is a multimer of the single peptide, or combination of different subject peptides.
• the number of sequences will be at least 2 and not more than 20, usually not more than about 10 sequences or repeats.
• the subject peptides may be combined in a mixture to provide for a broader spectrum of activities or synergistic activity.
• the subject peptides may be prepared in a variety of ways. Conveniently they can be synthesized by conventional techniques employing automatic synthesizers, such as the Beckman, Applied Biosystem Inc., or other automatic apparatus, or may be synthesized manually. Alternatively, DNA sequences may be prepared which encode the particular peptide and may be cloned and expressed to provide the desired peptide. In this instance, methionine may be the first amino acid, or a signal sequence may be provided, whereby the expressed peptide is secreted with processing and removal of the signal peptide. Techniques for preparing synthesized DNA sequences are extensively described in the literature and Sambrook et al. , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 1989.
• the peptides may be isolated from natural sources and purified by known techniques, including, for example, chromatography on ion exchange materials, separation by size, immunoaffinity chromatography and electrophoresis.
• a substantially pure preparation of peptide compound means a preparation of the peptide which is usually greater than about 70% free of materials with which the polypeptide is naturally associated, normally the natural or production source of the peptide, preferably greater than about 80% free of these materials, more preferably greater than about 95% free of these materials. These materials, however, exclude materials with which the peptide may be mixed in the preparation of pharmaceutical compositions.
• sequences may be modified in accordance with their intended purpose. Different N- or C-terminal groups may be introduced which allow for linking of the peptide to a solid substrate or other molecule.
• any molecule may be introduced at an internal site or at a terminus, which may 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, where the other molecules are haptenic or antigenic members of a specific binding pair or receptors, e.g. antibodies.
• 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 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 purposes while still retaining biological activity.
• lipid particularly a phospholipid
• Phosphatidyl choline, phosphatidyl ethanolamine, or other lipid may be used with a bifunctional linking agent, such as MBSE,. glutaraldehyde, methyldithiobenzoic acid, or the like.
• a coding sequence which joins the peptide to phosphatidyl inositol, farnesyl, geranylgeranyl, or myristoyl.
• the modified peptide or protein is combined with the lipids in an aqueous medium and sonicated to provide the desired liposomes.
• the liposomes may then be harvested and used in the ways indicated.
• the subject peptides by themselves, or in combination with other peptides or proteins, may be used for diagnosing the presence of CTLs which bind to a subject peptide or the combination of a subject peptide and other peptide or protein.
• Conjugates of the subject peptide and the other peptide or protein can be prepared by employing linking agents as described previously, where the other peptide or protein may be an enzyme to provide a detectable signal, an antigen for binding to an antibody for separation of binding CTLs.
• the subject peptide and the or the like covalently or through antibody/antigen binding.
• the subject peptide and antigenic peptide or protein may be conjugated to a particle or protein which is fluorescent. The binding of the particle or protein will allow for sorting and counting in a fluorescence activated cell sorter.
• the subject peptides may also be used for modulating CTL activity in the mammalian host.
• the modulation may be by inhibiting CTL activity or by sensitizing target cells. This can be achieved by employing apheresis, ex vivo, where the patient's blood is withdrawn from the patient and circulated through a device in which the peptide is present, either bound to the surface to remove CTLs active with the subject peptide or by adding the peptide in a physiologically acceptable medium to bind to the CTLs and inhibit their activity.
• the subject peptides may be administered to a host intravascularly, in either an artery or vein, or at the site of the implant to provide for inhibition or stimulation of the CTL.
• the subject peptide may be combined with a population of cells taken from a patient, where the cells are only a portion of the total cells in the periphery, or where the population is enriched for a particular cell type, e.g. CTLs, Class I T-cells, Class ⁇ T-cells, NK-cells and the like. Where the population is removed from the patient, the cells may be incubated for sufficient time for the cellular activity to be modulated. The cells may then be washed free of the peptide and restored to the host.
• a population of cells taken from a patient, where the cells are only a portion of the total cells in the periphery, or where the population is enriched for a particular cell type, e.g. CTLs, Class I T-cells, Class ⁇ T-cells, NK-cells and the like.
• the cells may be incubated for sufficient time for the cellular activity to be modulated. The cells may then be washed free of the peptide and restored to the
• the subject peptides may be administered prior to the implantation, usually not exceeding about two weeks prior and repetitive treatment may be employed. At or about the time of implantation the subject compound may be administered during or immediately prior to the operation.
• the subject peptides may be used to treat the organ, by bathing the organ in a medium containing the subject peptides. In this way CTLs present with the organ may be deactivated to prevent graft vs. host disease.
• the peptide may be present at a concentration of 10 "3 to 10 " °M.
• the organ may be washed free of the storage medium or the organ used with the peptide bound to the cells of the organ.
• the subject peptides or compounds comprising the subject peptides can inhibit CTL activity, thus preventing the lysis of target cells.
• the differentiation of inactive precursors into active mature CTLs may be used to reduce the mitogenic response of CTLs to mitogens or other activating compounds, e.g. Concanavalin A or anti-CD3 antibody.
• the subject peptides are found to be active with CTLs having a wide variety of MHC antigens, rather than being specific for the particular HLA antigen from which the subject peptides are derived.
• the subject peptides may be formulated in a variety of ways for administration to a host.
• the subject peptides may be used by themselves or in combination with other immunosuppressant agents, such as cyclosporine A, FK506, immunotoxins, anti-OKT3 or the like.
• immunosuppressant agents such as cyclosporine A, FK506, immunotoxins, anti-OKT3 or the like.
• substantially reduced concentrations of the immunosuppressant may be used in the formulations, usually less than about 60%, more usually less than about 40% of the normal dosage employed in a particular situation, to provide sufficient inhibition of CTL activity. In this way, the substantial side effects of the immunosuppressant compositions may be ameliorated by virtue of the lower concentration.
• the subject agents may be used with peptides derived from the a helix of the a chain of Class I MHC antigens. See particularly, PCT ⁇ US93.01758 and references cited therein. Of particular interest is the peptide B2702.75-84, which may be present in one or more copies, particularly where there is an inverted repeat, e.g. 84-75/75-84.
• peptides may be used in the formulations comprising the subject peptides, by themselves or in conjunction with other drugs, such as deionized water, saline, phosphate buffered saline, aqueous ethanol, etc.
• concentration of the peptide may vary widely, since for the most part, the peptides will be highly soluble in the media. Usually, the peptide will be not more than about 75 weight-percent, more usually not more than about 50 weight-percent, and usually greater than about 1 weight-percent.
• the dosage will vary depending upon the purpose of the administration, the frequency of administration, the efficacy of the peptide, and the like.
• the dosage will range from about 0.01 to 10 mg/kg of host, based on active sequence.
• encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or other techniques may be employed which extend the lifetime of the peptides.
• the subject peptides may be used in assays to evaluate the activity of agents in mimicking the activity of the subject peptides.
• compounds may be screened for their effectiveness in comparison with the subject peptides and related to the mode of action provided by the subject peptides.
• the subject peptides may be used to standardize assay protocols for use in screening agents.
• the subject peptides may also be used for detecting complementary compounds which bind to the subject peptides.
• proteins which bind to the subject peptides may be isolated and purified, e.g. affinity chromatography.
• Example 1 Preparation of peptides from Class H alpha chain.
• peptides were prepared by conventional synthetic methods using standard solid-phase techniques. See Erickson and Merrifield in: The Proteins, Vol. 2, 3rd ed., eds. Neurath, H. and Hill, R.L., p. 255-527, Academic Press, N.Y. (1970), which is incorporated herein by reference. Three of the peptides had amino acids from the ⁇ x domain of amino acids 53-77, one had amino acids 56-80 of the ocj
• Example 2 Peptides corresponding to residues 50-80 of the ⁇ chain of class II HLA block the differentiation of cytotoxic T lymphocytes (CTL) precursors into effector CTL.
• CTL cytotoxic T lymphocytes
• PBL from normal donors were isolated over Ficoll and cultured in 24 replicates at the indicated numbers in microtiter wells in RPMI 1640 supplemented with 10% fetal calf serum and L-glutamine.
• the EBV transformed cell line JY was used as an allogeneic stimulator cell. To prevent cell division of the JY cells, they were irradiated at 10,000 rad prior to culture.
• Peptides were dissolved at 50 mg/ml in DMSO and added where indicated at a final concentration of 100 ⁇ g/ ml. After 6 days, cultures were tested for lysis of 51 Cr-labeled HLA-A2 transfected C1R cells. Wells were considered positive if lysis was > 10% above spontaneous release.
• Example 3 Effect of peptides on proliferation of human PBL to the mitogen Concanavalin A (Con A.. 4 x 10 5 PBL from normal donors were cultured in microtiter wells with 1 /ig/ml of the indicated peptide. After 24 h, wells were pulsed with 1 ⁇ Ci of 3 H- thymidine and harvested after an additional 24 h of culture. It was found that DQ.56-80 and DR.53-77 peptides strongly block proliferation of PBL to Con A. The DQ.71-80 peptide and the DP.53-77 did not block proliferation as strongly. Inhibition was not allele specific.
• Peptides prepared as in Example 1 were preincubated for 30 min with CTLs before addition of 10 3 cpm of 51 Cr-labelled A2+ target cells at effecto ⁇ target ratios of 1:1, 2.5:1, 5:1, and 10:1.
• the cytotoxicity assay was then performed as described by Clayberger et al. , J. Exp. Med. (1984) 162: 1709-1714; Rice et al. , Proc Natl. Acad. Sci. USA (1980) 77:5432-5436.
• Peptides were added at 100 ⁇ g/ml and the plates were harvested after 4 h. Lysis was blocked by the DQ.56-80 peptide, but not by peptides DQ.68-77, DR.53-77 and DP.53-77.
• Example 4 Determination of critical residues in DQ.56-80 for blocking lysis by CD8+ CTL.
• a series of overlapping 15 amino acid peptides were prepared and tested for effects on lysis as described above.
• the peptides employed were DQ.53-67, 56-80, 65-79, and 71-85.
• the DQ.65-79 and 71-85 were most effective in blocking lysis. Similar results were obtained with CTL specific for other alleles.
• the DQ.71-85 was difficult to synthesize, so the 65-79 was chosen for further testing.
• Example 5 Effect of single amino acid changes in inhibitory effects of DO.65-79 f__I__i___. Single amino acid substitutions were introduced into the DQ.65-79 peptide.
• Blocks Reporter Gene Assay ND + (Rantes Promoter) It is evident from the above results, that the subject peptides provide for therapeutic opportunities to inhibit CTL attack, particularly in association with implantation of allogeneic cells or tissue, particularly solid organs. The peptides do not kill the cells, so that the immune response is readily renewed, once the administration of the peptides is terminated.
• the subject peptides may be used in conjunction with other immunosuppressive agents, which have undesirable deleterious effects, so that lower concentrations of the agents may be employed to reduce their undesired side effects.

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• 1995
  • 1995-06-16 JP JP8502506A patent/JPH09503003A/ja active Pending
  • 1995-06-16 CA CA002169762A patent/CA2169762A1/en not_active Abandoned
  • 1995-06-16 WO PCT/US1995/007673 patent/WO1995034321A1/en not_active Application Discontinuation
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