WO1994011400A1 - Peptides de icam-2 et icam-1 chez l'homme et leurs analogues s'utilisant en therapie et diagnostic - Google Patents

Peptides de icam-2 et icam-1 chez l'homme et leurs analogues s'utilisant en therapie et diagnostic Download PDF

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Publication number
WO1994011400A1
WO1994011400A1 PCT/FI1993/000480 FI9300480W WO9411400A1 WO 1994011400 A1 WO1994011400 A1 WO 1994011400A1 FI 9300480 W FI9300480 W FI 9300480W WO 9411400 A1 WO9411400 A1 WO 9411400A1
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seq
peptide
icam
cells
amino acid
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PCT/FI1993/000480
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English (en)
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Carl G. Gahmberg
Pekka Nortamo
Rui Li
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Helsinki University Licensing Ltd. Oy
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Priority to AU54667/94A priority Critical patent/AU5466794A/en
Publication of WO1994011400A1 publication Critical patent/WO1994011400A1/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
    • C07K14/70525—ICAM molecules, e.g. CD50, CD54, CD102
    • 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 present invention relates in general to ICAM molecules, and in particular to peptides from human ICAM-2, to peptides from human ICAM-1, and to analogs of both.
  • BACKGROUND Many leukocyte functions depend on the ability of the cells to specifically adhere to other leukocytes, to various target cells, or to homotypic leukocytes. Adherence involves the binding of heterodimeric leukocyte integrins, which consist of one of three specific ⁇ -chains (CDlla, CDllb, or
  • CDllc a common ⁇ -chain
  • CD18 a common ⁇ -chain
  • the CD11/CD18 leukocyte integrins which bind to intercellular adhesion molecules (hereinafter referred to as "ICAM") present on leukocytes and various other cells, are important in mediating diverse cell-cell interactions and leukocyte adhesion.
  • the ICAM polypeptides belong to the immunoglobulin superfamily of adhesion ligands.
  • ICAM-1 CD54
  • ICAM-2 contains two immunoglobulin domains which show a high degree of homology to the two NH 2 - terminal domains of ICAM-1.
  • ICAM-1 is widely distributed, and its expression may be up-regulated by various cytokines. It acts as a receptor for the malaria parasite P . falciparum and for rhinoviruses. Soluble ICAM-1 may be useful in the treatment of asthma and rhinovirus infections. ICAM-1 binds both to CDlla/CD18 and CDllb/CD18.
  • ICAM-2 In contrast to ICAM-1, ICAM-2 has been reported to bind only to CDlla/CD18, and not to CDllb/CD18 or to rhinovirus. ICAM-2 is reported to be cloned by a functional adhesion assay using COS- cells transfected with an endothelial cell expression library. ICAM-2 cDNA may be synthesized by polymerase chain reaction, and may be expressed in E . coli as a fusion protein. ICAM-2 may also be used to generate polyclonal antiserum. A monoclonal antibody raised against expressed ICAM-2, such as the monoclonal antibody designated 6D5, inhibits ICAM-2-dependent cell adhesion. The ICAM-2 protein has an apparent molecular weight of approximately
  • the 6D5 antibody reacts with the first immunoglobulin domain of ICAM-2.
  • ICAM-2 appears to be constitutively expressed in most cells and tissues. However, in vivo, the reactivity of monoclonal antibody 6D5 with endothelium in malignant lymph nodes is higher than in nonmalignant lymph nodes. ICAM-2 has a costimulatory effect during the activation of human T cells.
  • RGD arginine-glycine-aspartic acid
  • CDlla/CD18-ICAM interactions The regulation of CDlla/CD18-ICAM interactions has been extensively studied using phorbol ester-stimulated aggregation as a model.
  • Phorbol esters induce a rapid and sustained increase in lymphocyte aggregation which is mediated by CDlla/CD18 interaction.
  • Patarroyo, et al . , Scand . J . Immunol . , 22 : 171 (1985) is known.
  • ICAM-2 interactions with the leukocyte integrins.
  • the present invention relates generally to purified and isolated ICAM-1 and ICAM-2 peptides and fragments thereof (hereinafter collectively referred to as "ICAM peptides or fragments”) .
  • ICAM peptides or fragments fragments which stimulate aggregation of cells expressing at least one heterodimeric leukocyte integrin.
  • ICAM peptides or fragments stimulate homotypic leukocyte aggregation.
  • ICAM peptides or fragments which stimulate natural killer cell activity are disclosed.
  • ICAM peptides or fragments according to the invention are shown to bind to purified CDlla/CD18 leukocyte integrin.
  • the present invention further provides a peptide having an amino acid sequence selected from the group consisting of: (SEQ ID NO: 4) ; and an amino acid sequence differing from SEQ ID NO: 4 by the addition or substitution of an amino acid which does not destroy integrin binding activity such as preferably, the amino acid sequence of SEQ ID NO: 12.
  • Another peptide according to the present invention has an amino acid sequence selected from the group consisting of: (SEQ ID NO: 5) ; and an amino acid sequence differing from SEQ ID NO: 5 by the addition or substitution of an amino acid which does not destroy integrin binding activity; preferably the amino acid sequence of SEQ ID NO: 13.
  • Yet another peptide according to the present invention has an amino acid sequence selected from the group consisting of: (SEQ ID NO: 6) ; and an amino acid sequence differing from SEQ ID NO: 6 by the addition or substitution of an amino acid which does not destroy integrin binding activity; such as the amino acid sequence of SEQ ID NO: 14.
  • Still another peptide according to the present invention has an amino acid sequence selected from the group consisting of: (SEQ ID NO: 11) ; and an amino acid sequence differing from SEQ ID NO: 11 by the addition or substitution of an amino acid which does not destroy integrin binding activity; such as the amino acid sequence of SEQ ID NO: 17.
  • Methods of treating a patient suspected of being infected by a virus comprising administration of an amount of an ICAM peptide or fragment sufficient to stimulate natural killer cell activity are disclosed.
  • the present invention also provides methods for inhibiting transplant rejection including the steps of identifying a patient as being susceptible to transplant rejection, and administering to the patient a peptide according to the present invention in combination with a pharmaceutically acceptable diluent.
  • the present invention provides substitution analogs of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 9 in which a substituted amino acid is similar to the amino for which it was substituted in at least one of the properties of hydropathicity, charge, size of side groups, hydrogen bonding capacity, salt bridge formation capacity, optical rotation, disulfide bridge formation, and effect on peptide backbone conformation.
  • Substitution analogs of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 according to the present invention minimally retain integrin binding properties as exhibited by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • Addition analogs according to the present invention may be shorter in length than naturally- occurring ICAM-1 and ICAM-2 peptides and may not include integrin binding sites other than those in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 9.
  • addition analogs according to the present invention are shorter than human ICAM-2 (SEQ ID NO: 1) or human ICAM-1 (SEQ ID NO: 2), or have a different amino acid sequence therefrom.
  • a method for activating natural killer cells in a patient includes the steps of identifying a patient as benefitting from activation of natural killer cells, and administering to the patient a peptide according to the present invention in a pharmaceutically acceptable diluent.
  • a method for constructing a peptide having integrin binding activity according to the present invention includes the step of chemically synthesizing according to methods known to those skilled in the art a peptide according to the present invention.
  • a method for constructing a peptide having integrin binding activity includes the step of expressing from DNA, according to methods known to those skilled in the art, a peptide having the amino acid sequence of a peptide or fragment according to the present invention.
  • kits for performing an assay according to the present invention includes a peptide according to the present invention in combination with a signal for identifying integrin binding of the peptide.
  • the present invention also provides alternative forms of peptides according to the present invention, including anti-idiotypic antibodies produced according to methods known to those skilled in the art.
  • FIG. 1 is a schematic depiction of the location of certain peptides in the amino acid sequence of human ICAM-2 (SEQ ID NO: 1) , human ICAM- 1 (SEQ ID NO: 2) and mouse ICAM-1 (SEQ ID NO: 3) ;
  • FIG. 2 is a reproduction of a gel exhibiting the results of polyacrylamide gel electrophoresis of purified CDlla/CD18;
  • FIG. 3A is a bar graph depicting the binding of 125 I-labeled PI (SEQ ID NO: 12) peptide to purified CDlla/CD18;
  • FIG. 3B is a bar graph depicting the relative binding of 125 I-labeled PI (SEQ ID NO: 12) peptide to purified CDlla/CD18 in the presence of competing peptides;
  • FIG. 3C is a graph depicting the relative binding of 125 I-P1 (SEQ ID NO: 12) peptide to purified CDlla/CD18 by comparison with the concentration of peptide;
  • FIG. 4A is a bar graph depicting inhibition endothelial cell binding to purified CDlla/CD18 by various peptides and antibodies
  • FIG. 4B is a graph depicting inhibition of endothelial cell binding to purified CDlla/CD18 versus concentration of peptides used
  • FIG. 5 is a bar graph depicting binding of NAD-20 cells to peptide-coated plates
  • FIG. 6 is a three dimensional view of the first domain of ICAM-2
  • FIG. 7 is a graph depicting an aggregation score versus time for various peptides showing the effects on homotypic aggregation of Ramos cells;
  • FIG. 8 is a bar graph of an aggregation score for various monoclonal antibodies in a blocking assay
  • FIG. 9 is a bar graph depicting the PI (SEQ ID NO: 4) and P7 (SEQ ID NO: 10) inhibition of the relative binding of 125 I-P1 (SEQ ID NO: 12);
  • Fig. 10A is a graph of the time-course of phorbol ester (squares)- and PI (circles) -induced aggregation of T-cells;
  • Fig. 10(B) is a graph of percent T-cell aggregation with differing amounts of PI
  • Fig. 10(C) shows the percent T-cell aggregation when cells were pretreated with an inhibitor or control and then treated with PI
  • Fig. 10(D) shows various microscopic fields of cells treated with P(Bu) 2 or PI;
  • Fig. 11 shows the extent of inhibition of aggregation induced by peptides according to the present invention with several compounds
  • Fig. 12A shows the effect of peptides according to the invention and control peptides on natural killer cell binding
  • Fig. 12B shows the effect of peptides according to the invention and control peptides on natural killer cell cytotoxicity
  • Figs. 13A-13D show the kinetics and dose response of Na alwa B lymphoma cells and U-937 myelomonocytic leukemia cells upon treatment with peptides according to the invention
  • Fig. 14 shows the expression of CD11/CD18 integrins and ICAMs on Namalwa and U-937 cells
  • Fig. 15 shows the inhibition of Pl-induced adhesion in Namalwa and U-937 cells with different monoclonal antibodies
  • Figs. 16A and 16B show inhibition of Pl- induced cell adhesion in Namalwa and U-937 cells with various monoclonal antibodies which were the same as those used in Figure 15;
  • Figs. 17A and 17B show the inhibition of Pl-induced aggregation in Namalwa and U-937 cells at 4 °C and with various chemical pre-treatments;
  • Figs. 18A-18F show blockade of Pl-induced aggregation of Namalwa cells with protein kinase, phosphatase, and protein G inhibitors;
  • Fig. 19A and 19B show the inhibitory effects of staurosporine and okadaic acid, respectively, on PI (circles)- and p(Bu) 2 -induced aggregation of T-cells;
  • Fig. 20 is a graph of the kinetics of PI (ICAM-2 peptide) -induced migration of natural killer cells; and Fig. 21 is a graph of the dose-dependent
  • PI IGF-2 peptide promotion of natural killer cell migration in a Boyden chamber.
  • the abbreviations used in the present application include CD, cluster of differentiation antigens; CDlla/CD18, lymphocyte function-associated antigen-1 (also abbreviated "LFA-1") CDllb/CD18, complement type 3 receptor (also abbreviated as Mac- 1) ; ICAM-1, intercellular adhesion molecule-1; ICAM-2, intercellular adhesion molecule-2 ; HAT, hypoxanthine-aminopterin-thymidine; BSA, bovine serum albumin; TNF- , tumor necrosis factor- ⁇ ; PI, peptide 1, etc.; and VLA-4 , very late antigen-4.
  • FIG. 1 shows an alignment of the amino acid sequences from the first domains of human ICAM-2 (SEQ ID NO: 1), ICAM-1 (SEQ ID NO: 2), and mouse ICAM-1 (SEQ ID NO: 3) which show the highest homology.
  • SEQ ID NO: 1 shows an alignment of the amino acid sequences from the first domains of human ICAM-2 (SEQ ID NO: 1), ICAM-1 (SEQ ID NO: 2), and mouse ICAM-1 (SEQ ID NO: 3) which show the highest homology.
  • a peptide has been defined from this part of the first domain of ICAM-2. This peptide specifically binds to CDlla/CD18. Numerous leukocyte functions depend on adhesive intercellular interactions.
  • the present invention provides a defined peptide region from the first immunoglobulin domain of human ICAM-2 (SEQ ID NO: 4) and a peptide region from human ICAM-1 (P6, SEQ ID NO: 9) , which peptide regions are specifically involved in binding to CDlla/CD18.
  • Synthetic peptides from these portions of ICAM-2 and ICAM-1 bind to purified CDlla/CD18 and inhibit the adhesion of endothelial cells to the CDlla/CD18 integrin.
  • Leukocytes also bind to plastic coated with the aforementioned peptides. Furthermore, such peptides induce leukocyte adherence to target cells.
  • Controlling leukocyte functions by inhibiting leukocyte target cell binding with peptides such as those disclosed in SEQ ID NO: 4 and SEQ ID NO: 9 or their analogs, or by inducing leukocyte activation with peptides according to SEQ ID NO: 4 and SEQ ID NO: 9 or their analogs, is useful for therapeutic application in conditions of undesired leukocyte target cell binding (e.g., to inhibit transplant rejection) , and is also useful in the treatment of infection and cancer or in cases where activation of leukocytes is desired (e . g . , activation of natural killer cells) .
  • Peptides according to the present invention may be administered to patients in order to interfere with leukocyte binding to endothelial cells. In this way, inflammation and leukocyte migration into tissues are prevented.
  • ICAM-2 The external portion of ICAM-2 is relatively simple, containing only two immunoglobulin domains. Therefore, ICAM-2 offers some advantages over ICAM-1 as a model protein. In both ICAM-1 and ICAM-2, domain 1 is most important in binding to CDlla/CD18. Because not only human ICAM-1 and ICAM-2, but also murine ICAM-1, binds to human integrin, the binding regions of the first immunoglobulin domains in each of those proteins may be closely related.
  • Synthetic peptides were prepared by solid- phase synthesis on an Applied Biosystems model 430A peptide synthesizer (Applied Biosystems, Foster City, California) , using t-BOC-chemistry. Peptides were reduced with dithiothreitol for two hours at room temperature before purification by HPLC to greater than 98% purity using a C18 ⁇ Bondapak column with a linear gradient of 0-70% acetonitrile in 0.1% trifluoroacetic acid. The structures of the synthetic peptides so made were confirmed by amino acid analysis and plasma desorption mass spectrometry.
  • the synthesized peptides comprised: a 22- amino acid peptide (residues 21 to 42) from the first domain of ICAM-2 , as shown in FIG. 1 and designated PI (SEQ ID NO: 4) ; sub-peptides of PI (SEQ ID NO: 4) which represent sequential deletions of the NH 2 -terminal amino acid(s) of PI to form P2 through P4 [P2 (SEQ ID NO: 5), P3 (SEQ ID NO: 6), and P4 (SEQ ID NO: 7)]; P5 (SEQ ID NO: 8)]; P5a (SEQ ID NO: 11); P5b (SEQ ID NO:19) ; the peptide corresponding to PI from human ICAM-1 (P6, SEQ ID NO: 9) ; and a control peptide, P7 (SEQ ID NO: 10) , which contains the same amino acids as PI (SEQ ID NO: 4) in random order with the exception of two conserved cysteine residues
  • a COOH-terminal tyrosine was added to all peptides to facilitate 125 I-labeling as follows: Pl-Tyr (SEQ ID NO: 12) ; P2-Tyr (SEQ ID NO: 13); P3-Tyr (SEQ ID NO: 14); P4- Tyr (SEQ ID NO: 15); P5-Tyr (SEQ ID NO: 16); P6-Tyr (SEQ ID NO: 17); and P7-Tyr (SEQ ID NO: 18) .
  • FIG. 1 shows an alignment of homologous sequences from the first immunoglobulin domains in human and mouse ICAMs.
  • the sequences corresponding to ICAM-2 peptides P1-P5 (SEQ ID NO; 4 - SEQ ID NO: 8) and the ICAM-1 peptide P6 are indicated by lines beneath the sequences.
  • the boundary between the two immunoglobulin domains (DI and D2) is also shown.
  • CDlla/CD18 and CDllb/CD18 were purified from human buffy coat cell lysates by immunoaffinity chromatography on TS2/4-monoclonal antibody 60.1-
  • Sepharose® Sepharose®, respectively, and eluted at pH 11.5 in the presence of 2 mM MgCl 2 and 1% octylglucoside
  • 125 I-labeled PI (SEQ ID NO: 12), labeled by the chloramine T method [Greenwood et al., Biochem . J . , 89, 114-123 (1963)] was added to each well in 50 ⁇ l of medium (0.15 M NaCl, 0.01 M sodium phosphate, pH 7.4, PBS, 2 mM MgCl 2; 0.5% BSA, 0.02% NaN 3 ) with or without non-radioactive peptides, and incubated at 37°C for one hour. After washing, the attached 125 I-P1 was solubilized with 1% SDS and counted.
  • the purified CD11/CD18 preparation was analyzed by polyacrylamide gel electrophoresis in the presence of SDS.
  • the preparation contained the expected CDlla and CD18 polypeptides, and no impurities were observed.
  • FIG. 2 illustrates the results obtained from polyacrylamide gel electrophoresis of purified CDlla/CD18.
  • Purified CDlla/DC18 was run on an 8 percent polyacrylamide gel in the presence of SDS and stained with Coomassie blue.
  • Peptide PI (SEQ ID NO: 4; derived from ICAM-2) bound to plastic which had been coated with purified CDlla/CD18 almost completely inhibited the binding of 125 I-P1 (SEQ ID NO: 12) , as illustrated in (FIG. 3A) .
  • FIG. 3 illustrates binding of 125 I-labeled PI (SEQ ID NO: 12) peptide to purified CDlla/CD18 in the absence or presence of competing peptides.
  • 125 ⁇ - labeled PI (SEQ ID NO: 12) was incubated in
  • SEQ ID NO: 4 shown by X's
  • P6 SEQ ID NO: 9; shown by closed circles
  • P7 SEQ ID NO: 10; shown by closed squares
  • the ability of various Pi subpeptides disclosed above to bind to CDlla/CD18 was determined using 125 I-labeled PI.
  • the peptide P2 (SEQ ID NO: 5) , lacking the NH 2 -terminal glycine of PI (SEQ ID NO: 4) , was about 25 times less efficient than PI at blocking 125 I-labeled PI binding to CDlla/CD18; and P3 (SEQ ID NO: 6) , lacking the next serine was even less active.
  • the ICAM-1 peptide, P6 (SEQ ID NO: 9) , partially blocked binding.
  • Peptides P4 (SEQ ID NO: 7), P5 (SEQ ID NO: 8), and the control peptide P7 (SEQ ID NO: 10) were not inhibitory (FIG. 3B) .
  • the binding of 125 I-P1 (SEQ ID NO: 12) was inhibited in a concentration-dependent manner by nonradioactive PI (SEQ ID NO: 4) , less so by P6 (SEQ ID NO: 9) , and not at all by the control peptide P7 (SEQ ID NO: 10) (FIG. 3C) .
  • a 50% inhibition was obtained with about 5 ⁇ M of PI (SEQ ID NO: 4) peptide.
  • ICAM-2 peptide P5 (SEQ ID NO: 8) , which ended at Pro-34, had no CDlla/CD18 binding activity.
  • Peptide P2 (SEQ ID NO: 5) , lacking the NH 2 -terminal glycine of PI (SEQ ID NO: 4) , was about 25 times less active than PI (SEQ ID NO: 4) in binding to CDlla/CD18, and further deletion of the NH 2 -terminal serine from P2 further lowered activity. Therefore, it is presumed that those NH 2 -terminal amino acids are important for activity.
  • the PI peptide SEQ ID NO: 4
  • the lysine may change the conformation of the peptide by forming a salt bridge to one of the three glutamic acids in the peptide.
  • CDllb/DCl ⁇ was purified from human buffy coat cells by immunoaffinity chromatography using anti-CDllb mAb.
  • An adhesion assay was performed in a way similar to that described in Example 3.
  • FIG. 9 illustrates a comparison of the relative binding percent of 125 I-P1 (SEQ ID NO: 12) with the background % binding of BSA and with the degree of inhibition of binding by unlabeled PI (SEQ ID NO: 4) or unlabeled P7 (SEQ ID NO: 10) . Degree of inhibition is shown as lower 125 I-P1 (SEQ ID NO: 12) binding.
  • the antibody 60.1 which reacts with CDllb/CD18, also blocks binding of labeled PI (SEQ ID NO : 12) .
  • PI SEQ ID NO: 4
  • ICAM-2 may also bind to CDllb/CDl ⁇ and not only to CDlla/CDl ⁇ .
  • PI SEQ ID NO: 4
  • the ICAM-1 peptide P6 showed less activity in all test systems, and the binding capacity of 125 I-labeled P6 (SEQ ID NO: 17) to CDlla/CD18 was less than 10% as compared to that of labeled PI (SEQ ID NO: 12; data not shown).
  • P6 (SEQ ID NO: 9) may bind to the same region in CDlla/CDl ⁇ as does PI (SEQ ID NO: 4) , but the affinity is lower. Small adjustments in the peptide sequence may greatly influence its activity as is the case for PI (SEQ ID NO: 4) .
  • Leukocyte functions are modified by peptides according to the present invention. These function may be used to determine the binding sites on CDlla/CDl ⁇ and CDllb/CDl ⁇ , as is reported for the platelet IIB/IIIa integrin [D'Souza et al., J . Biol . Chem . , 263, 3440-3446 (1990); D'Souza et al. , Nature, 350, 66-6 ⁇ (1991)].
  • the cell line Eahy926 is a hybrid between vascular endothelial and carcinoma cells and expresses endothelial cell markers [Eahy 926 cells were obtained from Dr. C. Edgell and are described in Edgell et al., Proc . Natl . Acad . Sci . USA, 80, 3734-3737 (1983)]. Cells of this cell line were cultured in Dulbecco's HAT medium containing 10% fetal calf serum. Eahy926 was induced by incubating the cells with 10 ng/ml of TNF- ⁇ (Boehringer Mannheim, Mannheim, Germany) overnight [Nortamo et al., Eur . J . Immunol .
  • Epstein-Barr virus-transformed B cell line, NAD-20, and Burkitt's lymphoma cell line, Ramos were maintained in RPMI 1640 medium containing 20% fetal calf serum.
  • Cells (7 x 10 5 ) in 1 ml were added to each plate in the presence of peptides as indicated in FIG. 4A and FIG. 4B, and were incubated for one hour at room temperature. Unbound cells were removed by washing three times with the same medium. The binding was quantitated by counting bound cells/microscopic field, with 15 fields for each plate.
  • Patarroyo 60.1 (obtained from Dr. P. Beatty) , IC2/2 [obtained from Dr. T.A. Springer and described in deFougerolles et al., J . Exp . Med . , 174 , 253-267 (1991)], and the irrelevant antibody 84-3C1 (anti- CD43) [obtained from D. J. Vives and described in Borche et al., Eur . J . Immunol . , 17, 1523-1526 (19 ⁇ 7) ] were prepared using precipitation with l ⁇ l mg/ml of Na 2 S0_,.
  • FIG. 4A and FIG. 4B illustrate inhibition of endothelial cell binding to purified CDlla/CDl ⁇ by peptides according to the present invention.
  • Peptide PI (SEQ ID NO: 4) efficiently inhibited the binding of TNF- ⁇ -induced Eahy926 cells to purified CDlla/CDl ⁇ (FIG. 4A) .
  • the ICAM-1 derived peptide P6 (SEQ ID NO: 9) showed a smaller inhibition.
  • the binding was efficiently blocked by the CDl ⁇ antibody 7E4.
  • the inhibition was concentration-dependent as shown in FIG. 4B.
  • a 50% inhibition was obtained with about 18 ⁇ M (43 ⁇ g/ml) of peptide PI (SEQ ID NO 4) .
  • the control peptide P7 (SEQ ID NO: 10) had no apparent effect on binding.
  • NAD-20 cells ( ⁇ x 10 A ) in 100 ⁇ l (RPMI1640 40 mM HEPES, pH 7.2 2 mM MgCl 2 , 5% fetal calf serum) were incubated for one hour at 22°C in the presence of 5 ⁇ g LB-2 antibody to avoid spontaneous intercellular aggregation. After washing three times, the bound cells in each microscopic field were counted. Four fields were evaluated for each well.
  • FIG. 5 illustrates binding of NAD-20 cells to peptide-coated plates.
  • the binding of NAD-20 cells to PI was significantly (p ⁇ 0.01) higher than binding to the control peptide P7 (SEQ ID NO: 10) .
  • the monoclonal antibody 73R had no effect on the adhesion of cells to PI (SEQ ID NO: 4) .
  • the number of cells bound/microscopic field is given.
  • P7 SEQ ID NO: 10
  • amino-surface plates COSTAR Europe Ltd. Badhoevedorp, the Netherlands
  • 10 ⁇ g of peptide with 100 ⁇ l Qf 100 mM l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride, 5 mM N-hydroxysulphosuccimnimide (Pierce Europe BV, Oud-Beijerland, the Netherlands) in H 2 0 overnight at 4°C.
  • the wells were then saturated with 1% nonfat dry milk solution by incubating for one hour at room temperature.
  • the amount of peptide attached was about 5 ng/well for each peptide as determined with 125 I-labeled peptides.
  • Ramos cells (1 x 10 6 ) in medium 100 ⁇ l RPMI 1640, 40 mM HEPES, pH 7.2-2 mM MgCl 2 , 5% fetal calf serum were incubated at 37°C in the absence or presence of specific antibodies.
  • the stage of aggregation was evaluated by counting the percentage of aggregated cells and showing the data by using aggregation scores as follows: 0, no aggregated cells; 1, less than 5% of cells aggregated; 2, 5-10% of cells aggregated; 3, less than 30% of cells aggregated; 4, 30-50% of cells aggregated; 50-70% of cells aggregated.
  • PI SEQ ID NO: 4
  • P6 SEQ ID NO: 9
  • P7 SEQ ID NO: 10
  • the aggregation induced by the peptide PI is totally blocked with monoclonal antibody 7E4 and partially with monoclonal antibodies H12, 60.1, LB-2 and IC2/2 as illustrated in FIG. ⁇ .
  • PI (SEQ ID NO: 4) coated on plastic efficiently bound leukocytes, but the CDl ⁇ antibody, 7E4, which efficiently blocks CDll/CDl ⁇ -dependent adhesion [Nortamo et al., Scand . J . Immunol . , 28, 537-546 (1988)], had no effect.
  • the 7E4 antibody also did not prevent the binding of 125 I-P1 (SEQ ID NO: 12) to purified CDlla/CDl ⁇ (not shown) .
  • the results show that peptides according to the present invention are able to bind to CDlla/CDl ⁇ in the presence of antibody in situations wherein ICAMs cannot do so. This may be due to less steric hindrance in the case of the smaller peptide fragments as compared to the natural ligands.
  • Peptides PI (SEQ ID NO: 4) and P6 (SEQ ID NO: 9) induced homotypic aggregation of Ramos cells.
  • the control peptide P7 (SEQ ID NO: 10) had no effect, demonstrating that the inducing effect by peptides PI (SEQ ID NO: 4) and P6 (SEQ ID NO: 9) is specific. Since the peptides were attached to solid phase and washed extensively, there was no free peptide present. Thus, the aggregation of cells is due to cellular activation rather than the cross- linking capabilities of the peptides.
  • the monoclonal antibody 7E4 completely blocked the peptide Pl-induced aggregation, showing that the aggregation is CDl ⁇ dependent.
  • peptides according to the invention to activate leukocyte adhesion was determined in an assay involving human T cells.
  • Peptides according to the invention were prepared as above by solid-phase synthesis on an Applied Biosystems model 430A peptide synthesizer, using t-BOC-chemistry. Peptide structures were confirmed by amino acid analysis and plasma desorption mass spectrometry. Monoclonal antibodies were constructed for use as competitive inhibitors in the assays. The monoclonal antibodies used were 7E4, an anti-CD18 antibody reported in Nortamo, et al . , Scand . J . Immunol . , 28 : 537-546 (198 ⁇ ) ; TS1/22, an anti-CDlla antibody reported in Sanchez-Madrid, et al . Proc . Nat . Acad . Sci .
  • Burkitt's lymphoma cell lines Namalwa, Ramos, and
  • BL-41 the T-cell leukemia cell line Jurkat; myelo- monocytic cell line U-937; and the K562 erythroleukemia cell line.
  • Each group of cells was cultured in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Flow Laboratories, Irvine Scotland) , penicillin (50 IU/ml) , streptomycin (50 ⁇ g/ml), and glutamine (0.29 mg/ml) .
  • the EBV-transformed B-cell line, NAD-20, and the promyelocytic cell line, HL-60 were grown in RPMI 1640 medium containing 20% fetal calf serum.
  • T lymphocytes and granulocytes were isolated from blood buffy coat samples obtained from the Finnish Red Cross Blood Transfusion Center, Helsinki using the Ficoll-Paque technique (Pharmacia, Uppsala, Sweden) . T-cells were further purified using a nylon wool column. After isolation, the cells were incubated at 37° C in RPMI 1640 medium with 10% fetal calf serum overnight before the assays.
  • Peptides for use in the assays were prepared as above.
  • the structure of the peptides was confirmed by amino acid analysis and plasma desorption mass spectrometry according to the procedure of Li, et al . , J .Biol . Chem . , 268:17513 (1993) .
  • Peptide PI was confirmed to be the structure indicated in SEQ ID NO: 4, corresponding to amino acids 21-42 of ICAM-2.
  • Monoclonal antibodies identical to those above were also prepared.
  • A. Aggregation Assay In the aggregation assay, cells (as described immediately above) were washed with RPMI 1640 medium containing 40 mM HEPES, 2 mM MgCl 2 , and 5% fetal calf serum. The cells were then resuspended to a concentration of 2 x 10 6 cells/ml for T-cells and granulocytes and 10 6 cells/ml for all other cells. Aliquots of 100 ⁇ l of each cell suspension were added to wells of flat-bottom 96- well microtiter plates either in the presence or absence of P(Bu) 2 or peptide PI or P7 (control) and incubated at 37°C for appropriate times (1,2,4,7, or 24 hours) . For inhibition of Pl-induced aggregation, cells were pre-incubated with inhibitor for 15 minutes at room temperature. Quantitative measurement of cell aggregation was accomplished by counting free cells from four randomly-selected areas (2.5 mm 2 ) per well.
  • Peptide PI induced aggregation of T-cells, granulocytes, NAD-20 cells, Namalwa cells, Ramos cells, and U-937 cells, but had no effect on cell lines which do not express both the CDlla/CDl ⁇ integrin and ICAM-1, such as BL-41, HL-60, and K562.
  • the control peptide, P7 did not induce any aggregation and P(Bu 2 ) produced aggregation similar to PI.
  • Results are shown in Table 2 below, wherein the amount of aggregated cells was express as percent aggregation (100 x [1 - (number of free cells) / (total number of cells)] and "None" indicates the negative control (i.e, nothing was added to the cells) .
  • PI induction of aggregation was stronger than P(Bu) 2 aggregation in U-937 cells as shown in Figure 13B. Further, PI induced aggregation in a concentration-dependent manner ( Figures 13C and 13D) . For example, a 50% aggregation was achieved with 5 ⁇ g/ml of PI for
  • Natural killer cells were preincubated with 100 ⁇ g/ml of either PI or P7 in RPMI 1640 medium complemented with 0.5% bovine serum albumin in standard cell culture conditions (albumin at 37 °C in a humidified 5% C) 2 atmosphere) for various periods (See, Figure 19B) before being tested for cytotoxicity against K562 target cells in a 4-hour 51 Cr assay as described in Timonen, et al . , J. Exp . Med . , 153 : 569-5 ⁇ 2 (19 ⁇ l), at a 10:1 effector/target cell ratio.
  • the ability of PI to induce migration of natural killer cells was determined. Migration of natural killer cells through a Boyden chamber or similar apparatus indicates that the cells have been activated.
  • Cells were prepared from buffy coat samples from healthy blood donors (blood samples were obtained from the Finnish Red Cross Transfusion Service) .
  • Mononuclear cells were isolated by Ficoll-Isopaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradient centrif gation and subsequent filtration through nylon wool columns in RPMI 1640 Medium supplemented with 0.29 mg/ml glutamine (Gibco) , 100 IU/ml penicillin, 10 ⁇ g/ml streptomycin, and 5% heat-inactivated fetal calf serum (Gibco) .
  • Natural killer cells were further enriched by discontinuous four-step density gradient centrifugation on Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) as described in Timonen, et al . , J . Immunol . Meth . , .36 : 285-291 (I960), incorporated by reference herein, using the four uppermost gradients instead of seven.
  • the cell composition was phenotyped by flow cytometry.
  • the migration assay was conducted in a Boyden chamber as described by Axelsson, et al . , J . Immunol . Meth . , 46:251-258 (1981) .
  • Polycarbonate filters with 3 ⁇ m diameter pores (Nuclepore Corp. ,
  • Pleasonton, NY were placed between the upper and lower compartments of the Boyden chamber.
  • the lower compartment was filled with 400 ⁇ L RPMI 1640 buffer supplemented with 0.5% human AB serum and 0.5% human serum albumin (both obtained from the Finnish Red Cross Transfusion Service) .
  • Natural killer cells were added to the upper compartment of the chamber. The cell number was adjusted to 10 xlO 6 cells/ml and 200 ⁇ l of cell suspension was added to the upper compartment.
  • ICAM-2 peptides or control peptides were added in amounts varying from 0 to 100 ⁇ g/10 xlO 6 cells/ml to natural killer cell fractions 30 minutes prior to the assay and were left in the supernatants during the assay.
  • ICAM-2 As shown in Figure 20, migration of natural killer cells was dependent upon the ICAM-2 peptide concentration. ICAM-2 began to activate natural killer cells at approximately 3 hours of incubation as shown in Figure 21, wherein shaded circles represent results obtained with the control peptide, P7, and unshaded circle represent results obtained with PI. The number of natural killer cells which migrated through the filters during four hours of incubation was significantly (p ⁇ 0.001) higher than the number of control-peptide-treated natural killer cells which migrated during the same period.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:2: lie Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser Thr Ser Cys

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Abstract

L'invention concerne des peptides et des analogues de ICAM-1 et ICAM-2 efficaces dans la prévention de l'aggrégation ou de l'adhésion de leucocytes ou de lymphocytes à des cellules endothéliales. Ces peptides et analogues peuvent également s'utiliser pour amplifier l'activité de leucocytes vers des cellules cibles. La prévention de l'aggrégation ou de l'adhésion de leucocytes ou de lymphocytes contribue à la prévention de réponses immunes indésirables, telles que le rejet de transplantations.
PCT/FI1993/000480 1992-11-18 1993-11-15 Peptides de icam-2 et icam-1 chez l'homme et leurs analogues s'utilisant en therapie et diagnostic WO1994011400A1 (fr)

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US7314938B2 (en) 2003-11-05 2008-01-01 Sunesis Pharmaceuticals, Inc. Modulators of cellular adhesion
US8080562B2 (en) 2008-04-15 2011-12-20 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
US8084047B2 (en) 2005-05-17 2011-12-27 Sarcode Bioscience Inc. Compositions and methods for treatment of eye disorders
US8378105B2 (en) 2009-10-21 2013-02-19 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
US9085553B2 (en) 2012-07-25 2015-07-21 SARcode Bioscience, Inc. LFA-1 inhibitor and methods of preparation and polymorph thereof
US10960087B2 (en) 2007-10-19 2021-03-30 Novartis Ag Compositions and methods for treatment of diabetic retinopathy

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US6130202A (en) * 1990-07-20 2000-10-10 Bayer Corporation Antiviral methods
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EP0977585A4 (fr) * 1997-01-24 2001-04-11 Blood Res Center Procedes de diagnostic et de traitement de troubles lies au poids corporel chez l'animal
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US8080562B2 (en) 2008-04-15 2011-12-20 Sarcode Bioscience Inc. Crystalline pharmaceutical and methods of preparation and use thereof
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