WO2001089555A1 - Compositions antiproliferatives et modes d'utilisation - Google Patents

Compositions antiproliferatives et modes d'utilisation Download PDF

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Publication number
WO2001089555A1
WO2001089555A1 PCT/US2001/016868 US0116868W WO0189555A1 WO 2001089555 A1 WO2001089555 A1 WO 2001089555A1 US 0116868 W US0116868 W US 0116868W WO 0189555 A1 WO0189555 A1 WO 0189555A1
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WIPO (PCT)
Prior art keywords
cells
peptides
composition
cell
activity
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PCT/US2001/016868
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English (en)
Inventor
Oleh G. Pankewycz
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Philadelphia, Health And Education Corporation
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Priority to AU2001266606A priority Critical patent/AU2001266606A1/en
Publication of WO2001089555A1 publication Critical patent/WO2001089555A1/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
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Diabetes mellitus is the most commonly occurring endocrine disease. Although an accurate frequency of the disease is not available because of differing standards for diagnosis, millions of Americans suffer from some form of the disease. There are several distinct forms or classes of diabetes, including both primary (no associated disease is present) and secondary (some other identifiable cause allows for development of diabetes) forms. Primary diabetes is classified into Type I and Type II, while secondary forms are classified by their proximal cause such as pancreatic disease- induced, hormonal abnormalities-induced, drug-induced, etc.
  • IDDM Human Type I diabetes mellitus
  • insulin-dependent diabetes is a disorder of immunity characterized by the selective destruction of insulin-producing pancreatic beta cells ( ⁇ -cells) .
  • ⁇ -cells are the targets of autoreactive T lymphocytes which are the primary mediators of the disorder.
  • Typical IDDM is characterized by destruction of more than 90% of the ⁇ -cells.
  • the precise cellular mechanisms and immunological mediators involved in IDDM have not been elucidated.
  • NTD non-obese diabetic mouse
  • This model is currently accepted as the principal animal model and the optimal tool for examining mechanisms underlying the disease, as well as for preclinical screening of potential treatments for IDDM.
  • therapeutic strategies that target T cells have been successful in preventing IDDM (Makino et al. 1980. Exp . Anim . 29:1-13).
  • T cell clones have been isolated from NOD mice that proliferate in response to syngeneic islets and induce insulitis but not IDDM, whereas other T cell clones can severely damage mouse islets when co-mixed with islet cell transplants derived from NOD mice (Reich et al . 1989. Nature 341:326-328; Haskins et al . 1989. Proc . Natl . Acad. Sci . USA 86:8000-8004) .
  • T cell clone IS-2.15 CD8 + T cell clone
  • the T cell clone IS-2.15 was isolated directly from the islets of disease-resistant NOD male mice. Although both male and female NOD mice develop a strong autoimmune reaction against pancreatic ⁇ -cells, in males the process aborts by an unknown mechanism and does not lead to total destruction of insulin-producing ⁇ -cells.
  • IS-2.15 cells exhibited an anergic phenotype as they failed to kill any tested target or produce any known cytokine when stimulated with anti-CD3 monoclonal antibodies.
  • IS-2.15 cells prevented the expression of overt diabetes. Spleen cells harvested from older diabetic mice were transferred into young, female, irradiated NOD mice which led to diabetes within 28 days. In contrast to a 100% incidence of diabetes in control mice, only 60% of those that received IS-2.15 cells became diabetic. Young male NOD were also tested by administering cyclophosphamide, which leads to diabetes within 10 days. In control animals, 75% developed diabetes while only 18% of mice that received IS-2.15 cells became diabetic.
  • IS-2.15 cells regulated the immune response and were able to prevent the autoimmune process. Later studies with IS-2.15 cells demonstrated that these cells produced a soluble immunoregulatory factor that was able to block T cell proliferation in vi tro (Pankewycz et al . 1992. Eur. J. Immunol . 22:2017-2023). This unidentified T cell- derived substance was not cytotoxic and did not prevent T cell activation.
  • T cell clone of NOD mice produces novel lymphocyte-derived peptides with anti- proliferative activity.
  • these peptides or active fragments or variants thereof and molecular mimics of these peptides can be used to treat or prevent autoimmune disorders, transplant rejection and immunoproliferative disorders.
  • compounds which inhibit the activity of these peptides can be used in the treatment of immunodeficient conditions.
  • An object of the present invention is to provide peptides derived from a T cell clone which are useful in preventing or treating autoimmune disorders including diabetes and transplant rejection.
  • these peptides comprise PI, MCACVCPSACASVSLKNNLLCDFLWSFCSGYSAAPQ
  • Another object of the present invention is to provide methods for inhibiting T cell proliferation and for treating or preventing autoimmune disorders via these peptides or active fragments or variants thereof or via molecular mimics of these peptides.
  • Yet another object of the present invention is to provide methods for identifying small molecule inhibitors of these peptides for use in treatment of immunodeficient disorders .
  • PI SEQ ID N0:1
  • P2 SEQ ID N0:2
  • the nucleotide sequence of the DNA segment 722415 encodes for PI (SEQ ID N0:1) and P2 (SEQ ID NO:2).
  • PI (SEQ ID NO:l) is encoded by an open reading frame at positions 172 through 295 of SEQ ID NO: 3.
  • P2 (SEQ ID NO: 2) is encoded by an open reading frame at positions 404 through 511 of SEQ ID N0:3.
  • a variant of the PI peptide, namely Plb (SEQ ID NO:5), has also been identified, and is believed to share the immunological properties of PI due to their significant amino acid homology.
  • the nucleotide sequence encoding for Plb (SEQ ID NO:5)
  • TGF ⁇ transforming growth factor beta
  • IS-2.15 cells were cultured alone in either serum-free medium or in the presence of 10% fetal calf serum at a concentration of 10 5 cells/200 ml.
  • a soluble factor was isolated using siliconized tubes and non-tissue culture treated well plates. This soluble factor was added to mixed lymphocyte cells in culture and found to inhibit T cell proliferation by more than 50%.
  • the IS-2.15 cell supernatant was then fractionated into >30 kDa and ⁇ 30 kDa fractions using size selective centrifugation techniques.
  • the entire immunoregulatory activity was found in the ⁇ 30 kDa fraction, with no activity at all noted in the >30 kDa fraction. This activity was confirmed in the mixed lymphocyte cell culture assay. Further size selection fractionation of the ⁇ 30 kDa fraction and testing in vi tro demonstrated that activity was in the ⁇ 10 kDa fraction. Passage of the supernatant through a 3 kDa size exclusion membrane led to a decrease in activity.
  • the IS-2.15-derived factor was a small molecule with a molecular weight close to, but greater than 3 kDa, and less than 10 kDa.
  • the isolated factor supernatant was digested with trypsin. Following digestion, the supernatant was size fractionated into the ⁇ 30 kDa portion which removed the trypsin. The digested ⁇ 30 kDa fraction contained an equal or even greater immunoregulatory activity in terms of T cell proliferation when compared to the original supernatant . When digested with Protease K, all immunoregulatory activity was lost .
  • the IS-2.15-derived factor was a small protein, a peptide or peptides.
  • IS-2.15 Genes in IS-2.15 encoding the immunoregulatory peptide or peptides were then isolated.
  • the mRNA content from IS-2.15 cells was first compared with that of control CD4 + and CD8 + T cell clones derived from NOD mice. Unlike IS-2.15 T cells, control T cell clones were not anergic and proliferated in response to syngeneic or allogenic stimulator cells .
  • the CD8 + T cell clone showed potent cytotoxic activity.
  • Differential Display was carried out using a commercial kit (Display Systems, Vista, CA) . DNA segments unique to IS-2.15 cells were sequenced and compared to the established DNA database, GenBank.
  • PI comprises the amino acid sequence MCACVCPSACASVSLKNNLLCDFLWSFCSGYSAAPQ (SEQ ID NO: 1) .
  • P2 comprises the amino acid sequence MTSLIKAFLDNRCFLSAPVSYSLVGFFSDCLPSYSPGGRGA (SEQ ID NO: 2) .
  • PI is an unusual peptide in that it contains 6 cysteines out of a total of 36 amino acids.
  • the protein structure of PI was shown to be similar to the structure of beta-defensin proteins. The spacing of the six cysteine amino acids and the intervening residues are markedly different from the known canonical sequence of beta-defensin molecules.
  • PI shares no genetic or protein sequence similarity to beta-defensins, when this peptide assumes its normal protein folding structure, it strongly resembles this group of strong biologically active proteins. Since beta-defensins are potent mediators of anti-proliferative activity for immune cells, the striking similarity of PI with these molecules is indicative of PI also being a mediator of the immunoregulatory activity of IS-2.15 T cells.
  • the specific PI sequence was also cloned into an appropriate vector for in vi tro translation using the rabbit reticulocyte lysate method.
  • the presence of PI in vector containing lysates was confirmed by SDS-PAGE gel analysis.
  • PI containing lysate was added to mixed lymphocyte cultures .
  • PI containing lysates were inhibitory to lymphocyte proliferation in mixed lymphocyte reticulocytes .
  • Recombinant PI was also synthesized using a bacterial expression system.
  • the specific sequence coding for PI was cloned into a GST tagged bacterial expression vector.
  • the GST-PI fusion protein was isolated using sepharose beads.
  • the peptide was cleaved from the GST protein with thrombin.
  • the purified PI containing solution was added to mixed lymphocyte reticulocytes at varying dilutions.
  • the inhibitory activity of the PI containing solutions was compared to a control solution containing an equal concentration of GST and thrombin alone.
  • the recombinant peptide containing solution was strongly anti-proliferative for T cells at all dilutions tested.
  • P2 proteins was isolated from both 2.15 T cells and mouse thymus .
  • a variant version of the 722415 est species was isolated in which a GG sequence is replaced with a TT nucleotide pair.
  • This variant nucleic acid sequence is depicted in SEQ ID NO : 4 .
  • This naturally occurring substitution yields a variant of PI.
  • This novel 67 amino acid peptide variant of PI is referred to herein as Plb.
  • Plb comprises the amino acid sequence MCACVCPSACASVSLKNNLLCDFLWSFCSFTVLLHNSTDPCPPTPGIKCIIWSQVGFLG TVSFCAVY(SEQ ID NO : 5) . Since PI and Plb share significant amino acid homology including critical Cysteine bonds, Plb is expected to have immunological properties similar to that of PI.
  • the second peptide, P2 contained within the suppressive cDNA clone was also found to have in vi tro biological activity. Unlike PI, P2 has 46 amino acids with only two cysteines forming one potential disulfide bridge. Thus, P2 can be synthesized in pure form for further testing and investigation of the functions of these unique peptides. Based upon the ability of PI and P2 to inhibit T cell proliferation, it is believed that peptides comprising PI or P2 or an active fragment or variant thereof such as Plb will be useful in preventing and treating autoimmune disorders, transplant rejection and abnormal immunoproliferative disorders including cancer.
  • active fragments or variants thereof it is meant amino acid sequences shorter or longer than PI or P2 with similar activity or amino acid sequences with conservative substitutions as compared to PI or P2, such as Plb, which have similar activity to PI or P2.
  • Recombinant PI was also tested for anti-proliferative activity in human mixed lymphocyte reactions. Human T cells were incubated with genetically disparate irradiated antigen presenting cells for 3 days. Cultures were then pulsed with tritiated thymidine and harvested according to standard methods. Similar to murine cultures, PI strongly inhibits the proliferation of human T cells in response to a strong antigenic stimulus.
  • autoimmune disorders linked to T cell function which can be treated with these peptides include, but are not limited to, IDDM, rheumatoid arthritis, asthma, and colitis. It is believed that these peptides, fragments or variants thereof would also be useful in prevention of rejection in human solid organ transplantation and aid in engraftment of the transplants as well as in the prevention or treatment of immunoproliferative disorders such as cancer.
  • the peptides, fragments or variants thereof can be prepared recombinantly via cloning of gene fragments encoding amino acid sequences of PI, P2, or active fragments or variants thereof into mammalian expression vectors to establish the immunosuppressive properties in transfected human cell lines.
  • compositions comprising isolated peptides or active fragments or variants thereof can then be administered to animals, including humans, to prevent or treat an autoimmune disorder, transplant rejection or an immunoproliferative disorder.
  • peptides may be prepared synthetically and incorporated into compositions for such treatment or prevention.
  • one of skill in the art could design chemicals which mimic these peptides, referred to herein as molecular mimics, for use in treating and preventing these disorders.
  • small molecular inhibitors of PI or P2 activity can be designed and synthesized for use in reversing T cell mediated immunosuppression. Such chemicals would be useful in the treatment of patients with immunodeficiencies such as those caused by HIV.
  • the peptides, molecular mimics and small molecular inhibitors of the present invention are preferably administered in a pharmaceutically acceptable vehicle.
  • Examples of such vehicles include, but are not limited to, aqueous solutions of salts or buffers, topical creams or lotions, and solid dosage forms such as pills, gelatin capsules, or liquid-filled gelatin tablets.
  • Peptides are also often administered in the form of nontoxic salts such as hydrochloride, hydrobromide, sulphate, phosphate, maleate, ascorbate, acetate, citrate, benzoate, succinate, and tartrate salts.
  • Design of therapeutic regimens for the peptides, peptide mimics and small molecular inhibitors can be determined routinely by one of skill based upon the determined activity of the peptide, peptide mimic or small molecular inhibitor.
  • the peptide PI encoding cDNA was cloned into a GST fusion protein plasmid containing a trypsin enzyme cut site between GST and PI. After bacterial expression, the fusion protein was purified using sepharose beads and cut using trypsin. The PI containing solution was added to a human mixed lymphocyte reaction using disparate donors for T cells and stimulating antigen presenting cells. Stimulating cells were irradiated (3,000R) prior to incubation with purified T cells in 1% AB human serum media for 3 days, pulsed with 3H- thymidine and harvested according to standard methods. Assays were done in triplicate. Experimental wells contained a 1/100 dilution of GST fusion protein alone. Peptide 1 was found to strongly inhibit human mixed lymphocyte reaction (MLR) .
  • MLR human mixed lymphocyte reaction

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  • Chemical & Material Sciences (AREA)
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  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne des peptides dérivés d'un clone de lymphocyte T convenant à la prévention ou au traitement de troubles auto-immunes, y-compris les diabètes et le rejet de greffon. L'invention concerne également un procédé permettant de bloquer la prolifération des lymphocytes T et de traiter ou prévenir des troubles auto-immunes par utilisation de ces peptides, certains de leurs fragments actifs ou certaines de leurs variantes, mais aussi par mimétiques moléculaires de ces peptides. L'invention concerne enfin des procédés permettant de concevoir des inhibiteurs à petites molécules de ces peptides, et des utilisations de ces inhibiteurs pour le traitement ou la prévention de troubles d'immunodéficience.
PCT/US2001/016868 2000-05-25 2001-05-24 Compositions antiproliferatives et modes d'utilisation WO2001089555A1 (fr)

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Application Number Priority Date Filing Date Title
AU2001266606A AU2001266606A1 (en) 2000-05-25 2001-05-24 Compositions with anti-proliferative activities and methods for use thereof

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US20696300P 2000-05-25 2000-05-25
US60/206,963 2000-05-25

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006046957A1 (fr) * 2004-10-22 2006-05-04 The Research Foundation Of State University Of New York Nouveau gene humain aux proprietes immunoregulatrices et antiproliferantes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958403A (en) * 1992-02-28 1999-09-28 Beth Israel Hospital Association Methods and compounds for prevention of graft rejection

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958403A (en) * 1992-02-28 1999-09-28 Beth Israel Hospital Association Methods and compounds for prevention of graft rejection

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7253148B2 (en) 2003-03-07 2007-08-07 The Research Foundation Of State University Of New York Human gene with immunoregulatory and anti-proliferative properties
WO2006046957A1 (fr) * 2004-10-22 2006-05-04 The Research Foundation Of State University Of New York Nouveau gene humain aux proprietes immunoregulatrices et antiproliferantes

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