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EP0511302A1 - Procedes et molecules apparentees a une protease specifique aux cellules cytotoxiques - Google Patents

Procedes et molecules apparentees a une protease specifique aux cellules cytotoxiques

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Publication number
EP0511302A1
EP0511302A1 EP91903747A EP91903747A EP0511302A1 EP 0511302 A1 EP0511302 A1 EP 0511302A1 EP 91903747 A EP91903747 A EP 91903747A EP 91903747 A EP91903747 A EP 91903747A EP 0511302 A1 EP0511302 A1 EP 0511302A1
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EP
European Patent Office
Prior art keywords
cells
cytotoxic
cell
cll
protease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP91903747A
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German (de)
English (en)
Other versions
EP0511302A4 (en
Inventor
Robert C. Bleackley
Corrine G. Lobe
Verner H. Paetkau
Michael N. G. James
Michael Murphy
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Seragen Inc
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Seragen Inc
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Publication of EP0511302A1 publication Critical patent/EP0511302A1/fr
Publication of EP0511302A4 publication Critical patent/EP0511302A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6467—Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Thymus derived lymphocytes play a major role in the immune system. Maturation of the T cell lineage involves three distinct stages: (a) generation of a T cell precursor from a pluripotent stem cell, (b) differentiation in the thymus, and (c) migration of mature cells to the peripheral tissues. Maturation of T cells within the thymus is antigen independent. However, once they have left the thymus, upon interaction with an antigen they are driven through the final steps of differentiation to become mature cells. These final steps are complex and involve interactions with other cells and soluble effector molecules.
  • helper T lymphocytes potentiate immune responses (both humoral and cell-mediated) either by cell-cell contact or by synthesis and secretion of factors. These factors, although synthesized in response to an antigen-specific signal, can be either antigen-specific or antigen-nonspecific.
  • Suppressor T lymphocytes inhibit the functions of other lymphocytes, again either directly or via soluble factors. Cytotoxic T lymphocytes are the effector cells in cell mediated immune reactions. They specifically recognize foreign antigens on the surface of cells, bind to them, and cause the target cell to lyse. Cytotoxic T lymphocytes are known to cause or exacerbate autoimmune diseases such as rheumatoid arthritis, and are also involved in allograft rejection and graft-versus-host disease.
  • cytotoxic T lymphocytes A general description of cytotoxic T lymphocytes, natural killer cells, and killer (K) cells is contained in Stites et al., Basic & Clinical Immunology 227-31 (Lange Medical Publications, Los Altos, Ca. , 1984).
  • the invention features a vector containing a DNA sequence encoding the CCP1 protein.
  • the invention features a vector containing a DNA sequence encoding the CCP2 protein.
  • the invention features a vector containing a DNA sequence encoding the hCCPl protein.
  • the invention features a vector containing a DNA sequence encoding the hCCPX protein.
  • the invention features substantially pure CCP1 protein expressed from a vector containing a DNA sequence encoding the CCP1 protein.
  • substantially pure means a preparation with a purity of 95% or greater by weight, and free of the proteins, lipids, and carbohydrates with which the protein is naturally associated.
  • the invention features substantially pure CCP2 protein expressed from a vector containing a DNA sequence encoding the CCP2 protein.
  • the invention features substantially pure hCCPl protein expressed from a vector containing a DNA sequence encoding the hCCPl protein.
  • the invention features substantially pure hCCPX protein expressed from a vector containing a DNA sequence encoding the hCCPX protein.
  • the invention features a peptide of the formula: Asp-Val-Asp-Ala; Ala-Pro-Asp-Ala; Ala-Asn- Pro-Ala; Phe-Pro-Arg-Phe; Ala-Pro-Arg-Phe; Phe-Pro-Asp-Phe; Phe-Pro-Asn-Phe; Phe-Asn-Pro-Phe; or Phe-Asp-Pro-Phe.
  • competitive inhibition refers to inhibition in which the inhibitor combines with the free protease such that it competes with the normal substrate of the protease.
  • Competitive inhibition is described, e.g., in Lehninger, Biochemistry 197-200 (Worth, 2d ed. 1975) .
  • protease refers to an enzyme that hydrolyzes, and thus cleaves, peptide bonds.
  • Cytotoxic lymphocytes e.g. cytotoxic T lymphocytes (sometimes called T killer cells) and natural killer cells are described in Jandl, Blood: Textbook of Hematology (Little, Brown and Co., Boston, 1987) hereby incorporated by reference.
  • serine protease refers to a protease which has a serine residue at the active site of the enzyme.
  • peptide as used herein, includes proteins as well as peptides too short to be characterized as proteins. Generally those peptides having a molecular weight of greater than 5,000 are characterized as proteins.
  • cytotoxic cell protease refers to any protease, preferably a serine protease, that has 30% or greater homology, more preferably 50% or greater ho ology, with the protein encoding sequence of the murine Cll gene, and which cleaves at different sites than does plasmin.
  • the cytotoxic cell protease is expressed by cytotoxic lymphocytes, more preferably exclusively by cytotoxic lymphocytes.
  • Cytotoxic lymphocytes produce, as part of their cytotoxic activity, proteases, some of which, we have discovered, cleave proteins at sites different from the sites cleaved by proteases such as plasmin produced by other cells of the body. These proteases are members of the cytotoxic cell protease family.
  • the inhibitory molecules of the invention since they mimic the unique cleavage sites recognized by cytotoxic cell proteases, can exclusively inhibit cytotoxic cell proteases e.g., those produced by cytotoxic lymphocytes.
  • a person suffering from an immune disorder, or experiencing allograft rejection can be administered a molecule of the invention to inhibit the cytotoxic lymphocytes involved in the disease or rejection process, and the administered molecule will not interfere with, for example, lysis of blood clots, or other normal protease-dependent functions.
  • Fig. l is a graph showing the correlation of a protease mRNA expression (•) with cell activation in a mixed lymphocyte culture.
  • Fig. 2 is a partial nucleotide sequence comparison of two protease-encoding cDNA's.
  • Fig. 3 is the nucleotide sequence of one of said cDNA's and the predicted protein structure it encodes.
  • Fig. 4 is a partial amino acid sequence comparison of five serine proteases.
  • Fig. 5 is the sequence of CCP2.
  • Fig. 6 is the sequence of hCll, the human analog of the murine Cll gene.
  • Fig. 7 is a restriction map of the hCCPX gene.
  • Fig. 8 is the nucleotide sequence of the hCCPX gene.
  • Fig. 9 is the predicted cDNA sequence encoded by the hCCPX gene.
  • Fig. 10 is the amino acid sequence of proteins encoded by hCCPX and the CCP genes.
  • Fig. 11 is the amino acid sequence of some protease inhibitors of the invention.
  • Table 1 shows the expression of Cll mRNA in infiltrating cells of tissue grafts.
  • Table 2 shows the degree of homology between CCP1 and various proteins.
  • Table 3 shows the effect of peptides of the invention on the cytotoxicity of cells from a cyclosporine- A mixed lymphocyte reaction.
  • Table 4 shows the effect of peptides of the invention on the cytotoxicity of cytotoxic T-cells activated with ConA and interleukin 2.
  • the inhibitory molecules of the invention competitively inhibit the activity of cytotoxic cell proteases produced e.g. by cytotoxic lymphocytes, while not inhibiting the activity of proteases produced by other cell types or any other proteases produced by the cells producing the cytotoxic cell proteases.
  • the inhibitory molecules are peptides.
  • Cytotoxic lymphocytes synthesize a characteristic set of cytotoxicity-related proteases which are expressed at much reduced levels, if at all, in other subsets of lymphocytes.
  • the cytotoxicity- related proteases can be divided into two groups, effector proteases and non-effector proteases. Effector proteases are released by a cytotoxic lymphocyte when it comes in contact with a target cell, and break down proteins in the membrane of the target cell or enter the target cell and hydrolyze intracellular proteins, leading to the cell's destruction.
  • Non-effector proteases are involved in the enzymatic processes that lead to the production and/or release of the effector proteases (or other effector molecules) from the lymphocyte. Inhibiting the action of either an effector protease or a non-effector protease inhibits the ability of cytotoxic lymphocytes to destroy a target cell.
  • the preferred peptides contain the two amino acids that constitute the cleavage site recognized by the protease, and have between 3 and 20 (more preferably between 3 and 5) amino acids residues. Shorter peptides are preferred because they are, in general, readily taken up cells.
  • the peptides should not contain a cleavage site recognized by other proteases, for example, those sites described by Zreighton, Proteins: Structure and Molecular Properties 336-37, 427-38 (W.H. Friedman, N.Y., 1983).
  • Example 1 Described in Example 1 below is the isolation, cloning, and characterization of two genes expressed exclusively in the cytotoxic T lymphocytes of mice.
  • Example 2 describes the sequencing of the two genes, the determination of the amino acid sequence of the protease which one of the genes encodes, and the characterization of the protease.
  • Example 3 describes the identification and isolation of a human gene (hCII) encoding a cytotoxic cell protease (hCCPl) produced exclusively by human cytotoxic T lymphocytes.
  • Example 4 describes the isolation, cloning, and characterization of a gene encoding another human cytotoxic cell protease, human cytotoxic cell protease X (hCCPX) .
  • Example 5 describes the sequencing of the hCCPX gene, the determination of the amino acid sequence of the hCCPX protease, and the characterization of the protease.
  • Example 6 describes the determination of three dimensional structure of a cytotoxic cell protease and the structure of a peptide that can act as a competitive inhibitor of that protease.
  • Example 7 describes several inhibitors of the invention.
  • Example 8 describes the production of substantially pure proteases and their use in the design of inhibitors.
  • Example 1 describes the sequencing of the hCCPX gene, the determination of the amino acid sequence of the hCCPX protease, and the characterization of the protease.
  • Example 6 describes the determination of three dimensional structure of a cytotoxic cell protease and the structure of a peptide that can act as a competitive inhibitor of that protease.
  • Example 7 describes several inhibitors of the invention.
  • Example 8 describes the production of substantially pure proteases and their use in the design of inhibitors.
  • cytotoxic T-cell lines MTL2.8.2 and MTL11.1 were generated from CBA/J mice as described by
  • EL4.E1 is an interleukin 2 (IL-2)-producing variant of the EL4 cell line described by Farr et al., (1980) J. Immunol. 125:2555.
  • CHI is a CBA/J a CBA/J X BALB/c antigen- specific helper T-cell line. It was produced from a 2-day mixed lymphocyte culture by continuous restimulation with irradiated F ⁇ spleen cells in RPMl 1640 medium supplemented with 10% fetal bovine serum and 100 ⁇ M 2-mercaptoethanol (RHFM) .
  • peripheral blood lymphocytes were incubated in RHFM and stimulated with irradiated allogeneic cells at days 0 and 7 and harvested at day 10.
  • the fetal-derived cells used are described by Teh et al., (1985) J. Immunol. !35_:1582.
  • spleen cells from CBA/J mice were incubated in RHFM (10 cells per ml) and purified IL-2 (described by Riendeau et al., (1983) J. Biol. Chem.
  • the cDNA was ligated to EcoRI/bacterial alkaline phosphatase-treated pUC13 (P-L Biochemicals) in 66 mM Tris-HCl, pH 7.6/6.6 mM MgCl 2 /10 mM dithiothreitol/1 mM ATP. Reactions were heated to 37°C for 5 min, quick-chilled before the addition of 1 unit of T4 DNA ligase, and incubated at 14°C for 2 hr. Escheri ⁇ hia coli JM83 cells were made competent by using the CaCl_/RbCl procedure described by Maniatis et al.
  • Filters were washed in 5x SET buffer for 15 min at 22°C and then in 2x SET buffer/50% formamide for 20 min at 42°C and were exposed to film (Kodak X-Omat AR) with an intensifying screen for 1 to 3 days at - 70°C. Hybridized probe was removed by boiling the filters for 10 min in distilled water.
  • Results - Triplicate copies of the library were hybridized first with cDNA synthesized from MTL2.8.2 mRNA, then, after autoradiography and washing, with helper T-cell cDNA, and finally with thymocyte cDNA. Colonies that gave a higher hybridization signal with killer cell mRNA in at least two of the three copies of the library were picked. Upon rescreening, again in triplicate, 36 of these 121 colonies appeared to be clearly CTL-specific. Plasmid DNA isolated from these colonies was cut with EcoRI. and a series of cross-hybridizations was performed.
  • clone BIO because it appeared to be the most abundant in the library, cross- hybridizing strongly with eight other inserts, and clone Cll because it weakly cross-hybridized with BIO but not with all BlO-related clones (one other Cll-related sequence was found) .
  • Cytodots prepared from a variety of cells and tissues were hybridized with nick-translated BIO and Cll.
  • the number of cells per dot was 10 .
  • the data with probe Cll are similar and are not discussed.
  • the highest signal was detected in MTL2.8.2—i.e., the killer cell line that was used to generate the cDNA library.
  • MTL-III a variant of MTL2.8.2 that had a low level of cytotoxicity and had become IL-2 and antigen independent.
  • a similar level of expression was observed in a novel T-cell clone derived from murine fetal thymus of Teh, supra. In all over 20 cytotoxic T cell lines and cultures have been tested and all have been positive for BIO and Cll expression.
  • Natural killer (NK) and T killer (TK) cells were purified, cultured, and tested for the expression of Cll mRNA by the methods described in Manyak et al. (1989) J. Immunol. 142:3707-3713. Culturing NK cells in IL-2 induced: i) lytic activity, ii) chymase and tryptase enzymatic activities and iii) the total mRNA levels of the Cll gene in a dose-dependent manner. Cll mRNA reached peak activity on days 5 to 7 of culture. Similar results were seen with TK cells.
  • RNA samples were reprobed with either a lymphocyte-specific probe or oligo(dT) or the T-cell antigen receptor ⁇ -chain gene (Hendrick et al. (1984) Nature 308:153) . Although the level of signal varied, all samples were positive.
  • lymphocytes were separated from adherent cells on Petri dishes and then treated with anti-Thy-1.2 antiserum. The enriched B cells were then incubated with lipopolysaccharide (LPS) or Con A or RHFM medium.
  • LPS lipopolysaccharide
  • Poly(A) RNA was isolated from a variety of cell sources, run on a denaturing agarose gel, and transferred to nitrocellulose. The same filter was probed first with nick- translated B10, then with Cll, and finally with probe 10, a cloned gene that detects mRNA in a variety of cell types
  • Probe B10 detected a single band (approximately 900 bases) in two different murine cytotoxic T cell clones, MTL2.8.2 and MTL11.1. No bands were detected in RNA from thymocytes, an antigen-specific helper cell line, or murine thymoma EL4. When the blot was reprobed with Cll, again only the two cytotoxic T cell clones showed bands. However, in contrast to BIO, this probe hybridized to two bands, one of approximately 900 bases and the other of 1200 bases.
  • Probe 10 detected a band in all cell samples.
  • blot- hybridization analysis was performed on poly(A) RNA from a number of murine cells including eleven CTL lines, two helper lymphocyte lines, brain cells, liver cells, three helper T-cell lines, unstimulated and LPS-stimulated B lymphocytes, and one B-cell myeloma. Of these, only the actively cytotoxic T cells expressed mRNAs that hybridized with B10 and Cll. To ensure that all tracks contained hybridizable RNA, the blot was rehybridized with probe 10. A band of the expected size was seen in all tracks.
  • Fig. IB On each of the 6 days after stimulation, the level of cytotoxicity was measured in a chromium-release assay against EL4 (H-2 b ) (Q, S194(Hr2 d ) ( ⁇ ), and RI(H-2 k ) (0) cell lines. Cytodots were also prepared on each of these days, and the blots were hybridized with nick- translated B10 and Cll. Data are presented only for B10, as Cll gave indistinguishable results. Relative B10 mRNA levels (•) were determined by scanning densitometry on an ELISA plate reader. In the allo-specific response (Fig.
  • Fig. 1 The experimental results illustrated in Fig. 1 indicates that the maximum expression of BIO and Cll mRNAs precedes the peak of cytotoxicity in an in vivo allogenic or mitogen-induced cytotoxic response by 24hrs; thus, they both fulfill the primary prerequisite for genes encoding proteins that are important in the lytic process.
  • In situ hybridization experiments indicate that a high proportion of T lymphocytes that infiltrate incompatible heart allografts in vivo express the Cll gene. Complete details of the in situ hybridization procedure, and all related techniques, are found in Mueller et al., (1988) J. Exp. Med. 167:1124-1136,
  • a 1.1-kb fragment of the Cll gene was subcloned into the polylinker of the transcription vector pSPT 672 using standard techniques.
  • This vector ha a SP6 and a T7 promotor at the 5' and 3' end of the multicloning site, respectively.
  • sense and antisense probes were prepared using SP6-polymerase and T7-polymerase (both from New England Biolab, Beverly, MA) reactions and (S-35) UTP No. SJ 1303, Amersham Corp., Arlington Heights, IL) at a final concentration of 12 ⁇ M.
  • the labelled nucleotide was dried down before adding the other reagents of the reaction mixture.
  • a typical reaction 35 ⁇ l contained 7 ⁇ l 5X SP6 buffer (final concentration; 40mM Tris-HCl, pH 7.9; 5mM
  • DNA template was digested with DNase I (2U/ ⁇ g DNA; Worthington) for 15 min at 37°C.
  • the RNA probe was subsequently extracted with phenol/chloroform, separated on a Bio-Gel P-60 spin column, and ethanol precipitated after adding 7.5 ⁇ g of yeast tRNA per 10 cpm-labelled probe.
  • the probe was subsequently resuspended at 2 X 10 cpm/ ⁇ l in Tris-EDTA (TE) , boiled for 2 min. and stored frozen at - 70°C.
  • this probe was mixed with formamide (final concentration 50%) , dextran sulfate (10%) , DTT (lOOmM), NaCl (300mM) , Tris-HCl, pH 7.5 (20mM) , EDTA (5mM) Denhardt's solution (IX) at a concentration of 2 X 10 cpm/ ⁇ l hybridization solution.
  • cytospin preparations were fixed and hybridized as described for cryostat sections.
  • the fixed sections or cytospin preparations were treated with proteinase K (Boehringer Mannheim, Federal Republic of Germany), 1 ⁇ g/ml in 100 mM Tris-HCl, pH 8.0, and 50 mM EDTA at 37 ⁇ C for 30 min.
  • the slides were postfixed again with 4% paraformaldehyde for 20 min. Free amino groups on tissue sections were acetylated by treatment with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min.
  • the first cells with detectable levels of Cll mRNA were found on day 2 after transplantation both in animals with an allogeneic and those with a syngeneic graft. These positive cells, however, were extremely rare at this timepoint and were normally not found on every section of the same animal. On day 4 after transplantation, the experimental animals showed a 5-10-fold higher frequency of
  • Cll specific mRNA per cell increases steadily during the entire observation period in experimental animals, indicating that the gene was expressed over long periods, perhaps after local induction by alloantigen and/or mediators. In control animals, the expression level increased only slightly after more than 4 days after transplantation.
  • the phenotype of the Cll transcript positive cells was determined as detailed in Mueller et al. (1988) J. Exp. Med. 167:1124-1136. Briefly, small pieces of the allograft were digested with collagenase, and the resulting suspension of the isolated infiltrating T cells were sorted on a fluorescence activated cell sorter for subsequent in situ hybridization. The infiltrating cells of the allograft and the splenocytes of six animals that received a heart muscle graft 6 days before were pooled and sorted according to their phenotype. The frequency of Cll-positive cells in the CD8 subset was generally 10-20 times higher in the infiltrate of the allograft than in the spleen of the same animals.
  • CD4 cells from the infiltrate were always very low and the frequency of positive cells in this subpopulation was at least 10-fold lower than in the CD8 subset of infiltrating cells; of 84 Cll mRNA + cells analyzed, 82 were CD8 (98%) and 2 CD4 (2%) .
  • CD8 98%)
  • CD4 2 CD4
  • cytospin preparations from sorts cells Cll-transcript positive cells were mainly found among the blast-like CD8 cells.
  • no evidence for a significant contribution of CD4 ⁇ , CD8 ⁇ or CD4 +, CD8+ T cells among the allograft infiltrating cells and the Cll transcript positive cells were found.
  • CTL complementary DNA (cDNA) library was screened, in which inserts greater than 1000 base pairs were cloned in ⁇ gtlO. Forty thousand recombinants were screened and 39 plaques corresponding to Cll were isolated.
  • this sequence may act as a signal to direct secretion or intracellular organelle location.
  • NBRF National Biomedical Research Foundation
  • CCPl and CCP2 The cytotoxic T lymphocyte-specific proteins (CCP's) encoded by Cll and B10 will be referred to as CCPl and CCP2, respectively.
  • CCPl and CCP2 The cytotoxic T lymphocyte-specific proteins (CCP's) encoded by Cll and B10 will be referred to as CCPl and CCP2, respectively.
  • Fig. 4 the optimal protein alignment with CCPl is presented for RMCPII, bovine chymotrypsin, bovine trypsin, and CCP2 (not numbered, as the full sequence is not presented) .
  • the full sequence of CCP2 can be obtained by application of the procedures applied to Cll and CCPl.
  • the full sequence of CCP2 is presented in Fig. 5.
  • RMCPII is an intracellular serine protease found in the granules of atypical mast cells.
  • the high level of homology of CCPl with RMCPII is particularly interesting as RMCPII has a number of structural features that make it exceptional in the serine protease superfamily.
  • Protein CCPl contains cysteines in precisely the same positions as RMCPII which, by analogy with RMCPII, form three disulfide bonds. These occur in the same positions in chymotrypsin, trypsin, and elastase.
  • CCPl and RMCPII lack a disulfide bond that is present in all other known serine proteases, including several from prokaryotes, and that links Cys 191 with Cys220 in chymotrypsin.
  • the first of these two half-cysteines is replaced by a phenylalanine, while the second half-cysteine has been deleted along with other residues.
  • Cys is thought to be important in stabilizing the conformation of the substrate binding site (Woodbury et al. ,
  • CCPl the amino acid six residues before the active-site serine is alanine.
  • chymotrypsin-like proteases it is serine and in trypsin-like proteases, aspartic acid.
  • the residue in this position lies at the bottom of the S. binding site, so the change to a less polar residue would indicate a preference for a hydrophobic amino acid at the
  • the initial step in determining the structure of a protease expressed exclusively by human cytotoxic T lymphocytes and recognizing a unique protein cleavage site is to clone human cytotoxic T lymphocyte specific cDNAs.
  • PolyA RNA from a human cytotoxic T lymphocyte cell line e.g., one of the lines on deposit at the Coriel Institute for Medical Research, Copewood and Davis Street, Camden, NJ, is used as a template for the synthesis, by standard procedures, of double stranded complementary DNA.
  • EsoRI recognition sequences are then ligated onto the ends of the dscDNA by standard methods, and the resultant molecules are size selected on low melt agarose and then inserted into the EcoRI site of ⁇ gtll, all by conventional procedures.
  • recombinant molecules are then packaged into ⁇ phage heads (Gigapack plus, Stragene) and used to infect £. coli Y1088.
  • DNA from plaques harboring recombinant molecules are hybridized with radioactive probes generated from BIO and Cll by standard procedures to identify corresponding human genes. The screening is conducted in duplicate to minimize the possibility of false positives.
  • hCll a human counterpart of Cll, was found using the above procedures.
  • the phage DNA from any positive plaques are isolated and immediately recloned, using conventional procedures, in the plasmid vector pUC13. Large amounts of these recombinant plasmid DNAs are then isolated for further analysis.
  • the human cytotoxic T lymphocyte specific clones can be characterized by restriction enzyme digestions and. ultimately, sequence analysis. In addition, their relationships to one another can be investigated by standard cross-hybridization and heteroduplex mapping.
  • Tissue specific expression and transcript sizes of isolated genes can be established using the same methods as described for BIO and Cll. Using Northern blot analysis, as described above, a number of different cell lines (all obtained from ATCC) were tested for expression of hCll. CEM-CM3 (acute lymphoblastic leukemia) , CCRF-CEM (acute lymphoblastic leukemia) , CCRF-SB (acute lymphoblastic leukemia) , RPMl 7666 (B lymphoblast) , DLD-1 (colon adenocarcinoma) , and CRL-7123 (spleen line) all failed to express hCll. Human thymocytes and peripheral blood lymphocytes were also negative.
  • Cytolytic T cells activated by mitogen, interleukin 2, anti-T cell-receptor antibody, or fucose, were all positive for hCll expression.
  • a human cytotoxic T cell line was also positive.
  • expression of hCll appears to be specific to cytotoxic T cells.
  • the correlation between the level of cytotoxicity and the expression of the human genes also can be examined using the above-described methods.
  • the expression of hCll was found to correlate with the cytolytic activity of the cells in which it was expressed. Expression of hCll was detected in lympho ine activated killer (LAK) cells.
  • LAK lympho ine activated killer
  • hCll was sequenced, as described above, and found to be very similar to murine gene Cll (Fig. 6) .
  • hCCPl appears to have an Arg at S_, imparting the unusual specificity of Asp at P..
  • the only other difference is the substitution of an aromatic amino acid two residues downstream from the Arg. Due to the similarity of the proteins encoded by hCll and Cll inhibitors synthesized to inhibit one should inhibit the other.
  • a partially purified preparation of hCCPl does not cleave at sites recognized by trypsin and chymotrypsin.
  • a human placental genomic library, in ⁇ charon 4A, 5 was screened by hybridization in 20% formamide and 6 x SSC (1 x SSC is 0.15 M sodium chloride, 0.3 M sodium citrate, pH7) at 41°C with a mixture of radioactivity labelled cDNAs corresponding to the murine cytotoxic cell proteases CCP1- 4, Bleackley et al., (1988) FEBS Letters 234: 153-159 and 0 Lobe et al. (1976) Science 122: 858-861.
  • Phage DNA from one of the positive plaques gave a 6.3 kb EcoRI fragment (and ultimately a 1.5 kb Bam fragment) (Fig. 7) that hybridized with the murine probes but failed. under conditions of high stringency, to hybridize with hCCPl.
  • Preliminary sequence analysis revealed that the 1.5 kb fragment encoded a protein which was highly homologous to the murine cytotoxic proteases. Thus this gene is a new member of the human CCP family but is different from hCCPl.
  • hCCPX is expressed in cytotoxic cells.
  • Poly A RNA was purified from resting and activated peripheral blood lymphocytes and subjected to Northern blot analysis using the 1.5 kb genomic fragment as a probe.
  • a transcript is clearly present in the activated cells that is absent in RNA from the unstimulated control. Sometimes a small amount of transcript is seen in the unstimulated cells, perhaps due to cellular contamination, however, the transcript is always induced upon stimulation. Because of the high level of homology between the various CCP family members cross-hybridization can occur. In the case of the murine genes, CCPl can be distinguished from the others because of a difference in transcript size. However, the transcripts detected by hCCPl and HCCPX are very similar in mobility. Therefore, high stringency washing conditions were used to minimize cross- hybridization. With washing at 41°C the 1.5 kb probe detects transcripts in both human and mouse cytotoxic cells.
  • RNA samples from a number of human cell lines obtained from the ATCC including CEM-CM3, CCRF-CAM, CCRF-SB (acute lymphoblastic leukemias) , RPMl 7666 (EBV-transfor ed B lymphoblast) , DLD-1 (colon, adenocarcinoma) , CRL-7020 (thymus) , CRL-7123 (spleen) and freshly isolated human splenocytes and thymocytes.
  • Fig. 8 The nucleotide sequence of the region indicated by the heavy line in Fig. 7 is presented in Fig. 8. A comparison of this sequence with those of the murine CCP genes revealed high levels of homology (-70% identity) in regions which correspond to exons and dissimilarity in regions which correspond to introns. By placing the introns (the underlined regions in Fig. 8) in exactly the same places that they occur in the murine sequences (all four murine genes have introns in precisely the same positions.
  • the sequence of a cDNA could be determined (Fig. 9) . A cDNA corresponding to exons 3, 4 and 5 has been isolated and confirms the positioning of the introns.
  • the amino acid sequence is shown below the nucleotide sequence in Fig. 9.
  • This protein was not found in the GenBank data base. It is however, homologous to a wide variety of serine proteases. The highest level of identity was with the cytotoxic cells proteases (human 70%) , murine (61%) , cathepsin G (human 57%) , and mast cell proteases (40-50%) . In addition, a significant level of identity (-30%) was found with many other trypsin and chymotrypsin like enzymes.
  • This protein is a serine protease and is related to the cytotoxic cell proteins, it will be referred to as human cytotoxic cell protease-X (hCCPX) .
  • hCCPX is very basic (14% basic, 6% acidic amino acids) and contains a hydrophobic leader sequence of 18 residues followed by a putative zymogen dipeptide which precedes the mature protease amino terminal lie residue. It is believed that the basic nature of the proteins may play a role in sequestering them within granules bound to proteoglycans, Stevens et al.
  • CCPl and 2 both contain unusual residues in regions that are believed to be important in defining substrate specificity.
  • the protease described here also has unusual residues in these same sites and lacks the disulfide bond.
  • the use of comparative molecular modeling to predict the structure of a protease and its characteristic substrate is particularly reliable when the protein of unknown structure is relatively homologous with a protein of known three dimensional structure.
  • the existence of a large database of known three dimensional structures of related proteins and their substrate is also very helpful. In the case of the cytotoxic cell proteases both of these criteria are met.
  • the model building procedure (as applied to CCPl and another unrelated serine protease) is described in detail in Murphy et al. (1988) Proteins: Structure, Function, and Genetics 4.:190-204 which is included herein as an appendix.
  • the computer program MUTATE referred to in the appendix is available from Dr. R. Read, Department of Medical Microbiology, University of Alberta, Canada) . Briefly, the process begins with aligning the sequence of the protein of unknown structure with the sequence of a template protein, a protein of known three-dimensional structure. In the case of highly homologous proteins the alignment is straightforward: the sequences are aligned and a computer generated model of the template protein is modified to yield a model of the structure of the unknown protein.
  • the side chain of each amino acid of the template is then replaced with the side chain of the corresponding amino acid of the protein of unknown structure.
  • the replacement side chain conformations are adjusted to follow the conformation of the replaced, i.e., template, side chain conformations when possible. When this is not possible preferred side chain angles are selected from a dictionary of preferred side chain conformations.
  • Subsequent refinements include adjusting the model to remove unacceptably close non-bonded intramolecular contacts and adjusting the placement of deletion and insertion loops.
  • the deduced structure is adjusted to relieve any remaining unacceptably close non- bonded contents.
  • the prediction of substrate structure is drawn from several types of information. This procedure begins with an examination of the deduced three dimensional structure of the protease and an analysis of the identity of amino acid residues in key positions on the catalytic site of the protease. This information is compared to the reactive site on the substrate of a closely related protease. The sequence of that substrate can then be altered to achieve a sequence complementary to the catalytic site of the modeled protein.
  • amino acid residues of a substrate are designated p 4 P 3 P 2 p ⁇ p ⁇ ,p 2' P 3' P 4' with cleavage by the protease occurring between P. and P. ' .
  • the corresponding interacting amino acids of the binding pocket of an enzyme are designated S.S-S j S ⁇ 'S 'S 'S ' with S 1 for e.g., example interacting with P..
  • the computer generated three-dimensional structure of CCPl indicates that the residues of the binding pocket which might interact with a substrate are: Pro 28-Cys 42; His 57-Asn 65; Leu 32; lie 41; lie 73; Tyr 151; Gly 153; Phe 99; Ser 214-Asp 219; Phe 191-Ser 195; Arg 226; and Asn 174- Arg 175. (See pages 198-200 of Murphy et al.. Appendix). The most important prediction is that S. equals Arg 226. This predicts an acid substrate specificity (probably Asp) at P., the site of cleavage. This specificity is unique among eukaryotic serine proteases.
  • S 2 appears from the computer analysis to be Phe 99, indicating a small amino acid e.g., Val, at P 2 .
  • the presence of basic residues in S_ and S. predict acidic residues at P_ and P.. Guided by these considerations the inhibiting peptides, corresponding to residues P -P • of the substrate, were synthesized. These peptides are shown in Fig. 11.
  • the effect of the inhibitors on the cytotoxic properties of cytotoxic T lymphocytes is shown in Tables 3 and 4.
  • Cytotoxicity was measured with cells from a cyclosporine-A induced mixed lymphocyte reaction mixed lymphocyte reaction (CsA-MLR) .
  • Spleen cells were obtained aseptically by pressing the spleen through a wire mesh into a medium of RPMl 1640 (GIBCO Laboratories, Grand Island, .NY), 10% (v/v) fetal bovine serum (GIBCO Laboratories) , 10- M 2- mercaptoethanol, and 10 mM HEPES buffer (Sigma, St. Louis, MO) (RHFM) .
  • Responder cells (1-2x10 /ml) were cocultured with eq al numbers of allogenic stimulator cells (1500 rad from a Cs source) in RHFM plus 300mg/ml CsA and 200 units/ml interleukin 2 in a final volume of 4 ml (Costar 6 well cluster) or 25 ml (Costar 75 cm tissue culture flask). The cultures were incubated at 37°C in 5% CO. and 90% relative humidity. Cells from the primary MLR cultures were harvested, washed in RHFM and then 5 re ⁇ ultured with cytokines at a cell density of 2-5 x 10 cells/ml for 24 or 48 hours. In some experiments, viable cells were isolated by gradient density centrif gation.
  • % lysis experimental - spontaneous release x 100 total release - spontaneous release
  • Spontaneous release was obtained by incubating 5"'1 " ' " C,r-labelled targets alone, and total release from target cells incubated with 1% Zap-Isoton lytic agent (Coulter Electronics of Canada, Ltd., Mississauga, Ontario).
  • Cytotoxicity in cytotoxic T-cells activated with ConA and interleukin 2 (IL2) Cytotoxic T Cells were activated using 10 ⁇ g/ml ConA and 10 U/ml IL2. The cytolytic activity was measured in a standard chromium release assay. Targets were pretreated for 3 hr with 100 ⁇ g/ml peptide and were then mixed with either pretreated 100 ⁇ g/ml peptide and were then mixed with either pretreated 100 ⁇ g/ml peptide) or untreated effectors. All results are at an effector to target ratio of 5:1. Results are calculated as described in the legend of Table 1.
  • the cDNA clones of the invention can be used to generate copious quantities of purified cytotoxic cell proteases by inserting the coding sequence of a cytotoxic cell protease gene into an expression vector and expressing the desired proteins in an expression system. These procedures are well known to those skilled in the art.
  • the possession of purified protease allows for a greatly simplified alternative approach to the design of inhibitor molecules. Rather than the extremely cumbersome and complex immunologically based assays used to produce the results in Tables 3 and 4, the enzymatic action of the purified protease on a given substrate can be followed directly, by cleavage of the substrate, when the purified protease is available.
  • protease cleavage can be followed with standard thioester-based assays such as that described in Harper et al. (1984) Biochem. 23:2995- 3002) . This allows a large number of potential inhibitors to be tested with relative ease.
  • the purified protease based assay can be used alone, or in conjunction with the rational design factors obtained by computer analysis, to screen large numbers of potential inhibitors. Positive compounds could then be tested for their immunosuppressive properties.
  • the inhibitory peptides of the invention can be prepared by standard solid phase synthesis, for example, a method in which a tert-butyloxycarbonylamino acid is attached to either chloromethyl resin containing 0.75 mM Cl ⁇ g , or the p-methylbenzhydrylamine resin containing 0.35 mM NH 2 g ⁇ , followed by the sequential addition of desired amino acid residues to produce the desired peptide.
  • Amino acid analyses of the synthetic peptides are, if desired, performed using a Durrum D-500 analyzer. Cysteinyl residues in the peptides are quantitated as cysteic acid using a modification of the method of Moore (1968) in which 100 mM peptide is oxidized with 2.0 ml performic acid (1 ml 30% H 2 0 2 + 9 ml 88% HCOOH) for 2 hrs. at 0°. Performic acid is removed in a Reacti-Therm at 40° using N 2 , and 0.5 ml distilled water is then added to the residue and re-evaporated. The product is then hydrolyzed using 6 N HCl. Free sulfhydryl groups are determined using the method of Ellman et al. (1959) . Use
  • the inhibitory molecules are effective inhibitors of cytotoxic cells, e.g., cytotoxic lymphocytes.
  • the inhibition of the target cell destroying activity of such cells can be used to treat patients suffering of autoimmune diseases such as Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison's disease, myasthenia gravis, juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis HB g -ve, cryptogenic cirrhosis (some cases), ulcerative colitis, Sj ⁇ gren's syndrome, systemic lupus erythematos
  • the peptides can be administered to a mammal in a dosage of 25 to 500 mg/kg/day, preferably 50 to 100 mg/kg/day.
  • mammals e.g., orally, intravenously, parenterally, nasally, or by suppository
  • the peptides inhibit the ability of cytotoxic T lymphocytes to destroy cells, thus inhibiting the cell-mediated immune response to provide an effective treatment for the above listed disorders.
  • Nucleic acid probes capable of hybridizing to a gene encoding a protease expressed only by cytotoxic lymphocytes can be used in a variety of useful hybridization assays.
  • such probes can be used to monitor cytotoxic T lymphocytes in transplanted tissue, e.g., by the in situ hybridization methods of Cox et al. (1984) Dev. Biol. 101:485.
  • the presence of the lymphocytes in the transplanted tissue is an indication that the tissue is being rejected by the host organism and that appropriate immunotherapy should be undertaken.
  • the probes can also be used to assess the potential cytotoxicity of lymphokine activated killer cells.
  • the generation and use of such cells to treat tumor patients is described by Rosenberg et al. (1985) N.E.J. Med. 313:1485. Rosenberg describe how human peripheral-blood lymphocytes are treated with interleukin-2 (a lymphokine) to generate killer cells that will attack tumor cells when reintroduced into the host.
  • the probes can be used in a hybridization assay with the nucleic acid of the treated lymphocytes by standard methods; the assay monitors the degree to which the activated killer cells have been generated by determining the level of expression of the protease-encoding gene in the cells.

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Abstract

Un peptide est capable d'inhiber, chez un mammifère auquel la molécule a été administrée, l'activité biologique d'une protéase de cellules cytotoxiques.
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