Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
  • C07K14/70564—Selectins, e.g. CD62
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
  • C07K14/70514—CD4
• • 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
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C07K14/70542—CD106
• • 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
  • C07K14/70546—Integrin superfamily
  • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• • 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
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • 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
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• This invention relates to methods of inhibiting cellular interactions, particularly interactions leading to inflammation or pathogenic infection.
• lymphocyte function-associated antigen 1 LFA-1
• IAM- 1 intercellular adhesion molecule 1
• Antibodies that prevent interaction of these counterreceptors can block homotypic adhesion (Mentzer, S.J. et al. J. Immunol . , 135:9-11, 1985; Rothlein, R. et al., J. Exp. Med . , 163:1132-1149, 1986) and T-lymphocyte-mediated killing (Davignon, D. et al., J . Immunol . , 127:590-595, 1981; Sanchez-Madrid, F. et al., J. Exp. Med. , 158:1785-1803, 1983) , suggesting the importance of this adhesion pathway for normal immunity.
• lymphocyte binding to endothelium is augmented by preexposure of the endothelial cells to inflammatory cytokines, and the enhanced adhesion is due, in part, to a quantitative increase in ICAM-l expression by the endothelial cells (Dustin, M.L.
• T lymphocytes activation of T lymphocytes by T-cell receptor crosslinking results in a marked but transient enhancement of lymphocyte LFA-1 avidity for ICAM-l, without increasing levels of LFA-1 expression (Dustin, M.L. et al., Nature (London) , 341:619-624, 1989).
• lymphocyte adhesion can also be viewed as involving inhibitory elements that limit inconsequential cell-cell interactions.
• the extensively sialylated (and thus highly negatively charged) lymphocyte membrane glycocalyx likely plays a role (Bell, G.I. et al. , Biophys . J., 45:1051-1064, 1984) .
• the negatively charged glycocalyx repulses cells bearing like charge and, thus, functions to limit cell-cell adhesive interactions (Brown, W.R.A. et al., Nature (London) , 289:456-460,
• CD43 sialophorin or leukosialin
• CD43 is a candidate molecule to regulate adhesion between lymphocytes because it is a major integral membrane component of the lymphocyte glycocalyx (Brown, W.R.A. et al., Nature (London) , 289:456-460, 1981; Remold-O'Donnell et al., J. Biol . Chem . , 261:7526-7530, 1986; Carlsson, S.R. et al. , J. Biol . Chem . , 261:127 '9-127'86, 1986).
• CD43 protein when presented on a cell surface, blocks interaction of that CD43-bearing cell with other cells which present either CD43 or very likely any other negatively-charged protein on their cell surfaces. Because the CD43 inhibitory effect appears to be relatively non-specific, any number of therapeutic CD43 chimeric molecules may be constructed and used to disrupt deleterious intercellular interactions.
• Such chimeras include two functional domains: (a) a cell targeting domain which directs specific binding of the chimeric molecule to a molecule (e.g., a receptor, ligand, or counterreceptor) on the surface of one or both of the cells whose interaction is to be disrupted and (b) a CD43 extracellular domain bearing a net negative charge at physiological pH which antagonizes the intercellular interaction.
• a cell targeting domain which directs specific binding of the chimeric molecule to a molecule (e.g., a receptor, ligand, or counterreceptor) on the surface of one or both of the cells whose interaction is to be disrupted
• a CD43 extracellular domain bearing a net negative charge at physiological pH which antagonizes the intercellular interaction.
• CD43 chimeric molecules can be used to disrupt the intercellular interactions leading to inflammation.
• a negatively charged CD43 extracellular domain may be covalently bonded to the leukocyte cell surface molecules lacto-N- fucopentaose III (LNF III) or sialyl Lewis x .
• P-selectin PADGEM, GMP140, CD62
• ELAM-1 proteins which are expressed on the surface of endothelial cells which are activated during an inflammatory response
• CD43:LNF III or CD43:sialyl Lewis x Upon administration of CD43:LNF III or CD43:sialyl Lewis x , these chimeric proteins bind and "coat" the endothelial cell surface, blocking interaction between the endothelium and the immune cells — both by blocking access of the immune cell ligand to the endothelial cell - A - receptor and also by non-specifically repelling the immune cell, probably as a result of CD43's high negative charge.
• the therapeutic proteins of the invention reduce or eliminate inflammation.
• a negatively charged CD43 extracellular domain may be fused to the receptors VCAM-1 and ICAM-l to produce chimeric proteins CD43:VCAM-1 and CD43:ICAM-l; such proteins bind and coat the surface of certain types of leukocytes, blocking interaction with their endothelial counterreceptors and therefore the intracellular interaction between the immune cells and the endothelium which leads to inflammation.
• CD43 chimeras are also useful for inhibiting interactions necessary for pathogenic infection.
• both rhinovirus and the malarial Plasmodium protozoans have been shown to bind ICAM-l and thus a CD43:ICAM-l chimeric molecule which blocks rhinovirus or Plasmodium binding may prove to be a useful therapeutic for treating or preventing the common cold or malaria.
• a CD43:CD4 chimera can be used to coat the exterior of the human immunodeficiency virus, thereby interfering with host cell infectivity.
• a similar approach using a negativiely charged CD43 extracellular domain covalently linked to a toxin molecule e.g., a Staphylococcus or E. coli toxin
• a negatively charged CD43 extracellular domain may be covalently linked to an antibody molecule which is specific for a particular cell surface molecule.
• the extracellular CD43 domain may be linked to a published anti-PADGEM antibody (see, e.g., Larsen et al., Cell 63:467, 1990) to produce a CD43 chimeric molecule which blocks binding of leukocytes to endothelium and thereby prevents inflammation (as described above) .
• the invention features a method of inhibiting an interaction between a first and a second cell in a mammal, involving providing a soluble chimeric molecule which includes (a) a cell targeting molecule which is capable of specifically recognizing and binding to a molecule on the surface of the first cell covalently bonded to (b) a CD43 extracellular domain bearing a net negative charge at physiological pH; and administering the chimeric molecule to the mammal to inhibit the intercellular interaction.
• the second cell bears a negatively-charged molecule (for example, CD43) on its cell surface; the CD43 extracellular domain is glycosylated; the CD43 extracellular domain includes the N-terminal 180 amino acids of the CD43 protein sequence; the cell targeting molecule is a P-selectin-binding portion of an LNF III receptor protein; the cell targeting molecule is an ELAM-1-binding portion of a sialyl Lewis x receptor protein; the cell targeting molecule is an LFA-1-binding portion of an ICAM-l receptor protein; the cell targeting molecule is a VLA-4- binding portion of a VCAM-1 receptor protein; the cell targeting molecule is a human immunodeficiency virus- binding portion of CD4; the cell targeting molecule is a rhinovirus-binding portion of ICAM-l; the cell targeting molecule is a Plas-modium-binding portion of ICAM-l; and the first cell is an activated endothelial cell or an infective pathogen (for example, CD43) on
• the invention features a chimeric molecule which includes (a) a cell targeting molecule which is capable of specifically recognizing and binding to a molecule on the surface of a cell joined to (b) a CD43 extracellular domain bearing a net negative charge at physiological pH.
• the CD43 extracellular domain is glycosylated; the CD43 extracellular domain includes the N-terminal 180 amino acids of the CD43 protein sequence; the cell targeting molecule is a P- selectin-binding portion of an LNF III receptor protein; the cell targeting molecule is an ELAM-1-binding portion of a sialyl Lewis x receptor protein; the cell targeting molecule is an LFA-1-binding portion of an ICAM-l receptor protein; the cell targeting molecule is a VLA-4- binding portion of a VCAM-1 receptor protein; the cell targeting molecule is a human immunodeficiency virus- binding portion of CD4; the cell targeting molecule is a rhinovirus-binding portion of ICAM-l; the cell targeting molecule is a Plasmodium-binding portion of ICAM-l; the cell targeting molecule specifically recognizes and binds to a molecule on the surface of an activated endothelial cell; and the cell targeting molecule specifically recognizes and binds to an infective pathogen.
• the invention features a method of reducing inflammation in a mammal, which involves providing a chimeric molecule which includes (a) a cell targeting molecule which is capable of specifically recognizing and binding to a molecule on the surface of an activated endothelial cell joined to (b) a CD43 extracellular domain bearing a net negative charge at physiological pH; and administering the chimeric molecule to the mammal to inhibit intercellular interaction between the activated endothelial cell and white blood cells.
• the cell targeting molecule is a P-selectin-binding portion of an LNF III receptor protein; and the cell targeting molecule is an ELAM-1-binding portion of a sialyl Lewis x receptor protein.
• the invention features a method of treating pathogen infection in a mammal, which involves providing a chimeric molecule which includes (a) a cell targeting molecule which is capable of specifically recognizing and binding to a molecule on the surface of the pathogen joined to (b) a CD43 extracellular domain bearing a net negative charge at physiological pH; and administering the chimeric molecule to the mammal to inhibit intercellular interaction between the pathogen and the mammalian cell it normally infects.
• a “cell targeting molecule” is meant a molecule which binds with specific and high affinity to a binding partner present on the surface of the cell whose intercellular interaction one desires to inhibit.
• a cell targeting molecule includes, without limitation, any cell surface ligand, receptor, counterreceptor, cell adhesion molecule, enzyme, or substrate, or any antibody which is specific for a molecule on the surface of the target cell.
• the cell targeting molecule may be proteinaceous or non-proteinaceous (for example, a non- peptide cofactor or a carbohydrate recognized by a cell adhesion molecule) .
• the cell targeting molecule may bind to a proteinaceous or non-proteinaceous molecule on the target cell surface.
• negatively charged is meant that the cell surface molecule bears a negative charge at physiological pH.
• an “activated endothelial cell” is meant that the endothelial cell, in response to antigen stimulation, has initiated the cellular events (e.g., expression of cell surface proteins) characteristic of an inflammatory response.
• the CD43 chimeric molecules of the instant invention provide a number of therapeutic advantages. For example, the specificity with which the CD43 chimeric molecule interacts with its appropriate cell surface binding partner allows targeting of the therapeutic protein to the affected area (e.g., the inflamed endothelium) ; such targeted therapy allows highly effective inhibition of deleterious cell interactions with minimal side effects.
• the CD43:LNF III and CD43:sialyl Lewi ⁇ x chimeric molecules bind receptors present in significant numbers on inflamed, but not healthy, vascular endothelium.
• CD43:LNF III and CD43:sialyl Lewis x accummulate at the site of vascular damage, maximizing the potency of the inflammation treatment without affecting healthy vascular endothelium and importantly without generally immunosuppressing the patient.
• FIG. 1 is a series of graphs showing CD43 expression by HeLa cell tran ⁇ fectants and human T lymphocytes. Cells were stained either with control normal mouse sera (Left) or the anti-CD43 monoclonal antibody anti-Leu 22 (-Right) as described herein.
• the level of CD43 expression by the phytohemagglutinin (PHA)-treated T lymphocytes is representative of the level of expression in peripheral blood T cells and all T-cell lines used in the experiments described herein.
• FIG. 2 is a series of bar graphs showing the effect of CD43 on T-lymphocyte adhesion to HeLa cell transfectants.
• Carboxyfluorescein-labeled lymphocytes were allowed to adhere to HeLa cell transfectants in microtiter wells as described herein.
• Human lymphocytes tested were peripheral blood T lymphocytes (PBL-T) ; PHA- activated T cells maintained in IL-2 (PHA-T) ; thymocytes (THY) ; a T-cell lymphoma line (SupTl) ; a human T- lymphotropic virus type 1-transformed T-cell line (C91/PL) ; and an IL-2-dependent T-cell clone (JL89) .
• PBL-T peripheral blood T lymphocytes
• PHA-T PHA- activated T cells maintained in IL-2
• THY thymocytes
• SupTl T-cell lymphoma line
• C91/PL human T
• FIG. 3 is a series of bar graphs showing the effect of CD43 on LFA-1-mediated T-lymphocyte adhesion. Lymphocytes were preincubated with either normal mouse sera, or monoclonal antibody ascites to LFA-1 (TS1/22) or to CD2 (TS2/18) (all at 1:400 dilution) for 30 min on ice and then allowed to adhere to HeLa cell transfectants for 30 min at room temperature.
• TS1/22 monoclonal antibody ascites to LFA-1
• CD2 TS2/18
• FIG. 4 is a series of bar graphs showing the effect of CD43 on adhesion of PMA-treated T lymphocytes.
• the JL89 T lymphocytes were preincubated with either normal mouse sera or TS1/22 monoclonal antibody as described above for FIG. 3 and then allowed to adhere to the HeLa cell transfectants for 1 hr at 37°C.
• PMA was used at 50 ng/ml where indicated.
• FIG. 5 is a series of bar graphs showing the effect of neuraminidase on CD43 interference with T- lymphocyte adhesion.
• JL89 T lymphocytes were preincubated with normal mouse sera or TS1/22 monoclonal antibody as described above for FIG. 3 and then allowed to adhere to untreated or neuraminidase-treated HeLa cell transfectants. Vibrio cholera neuraminidase was used at 0.02 units per ml.
• HeLa cells were chosen to generate stable, CD43- expressing lines because they naturally express ICAM-l and LFA-3, the counterreceptors for the leukocyte adhesion molecules LFA-1 and CD2, respectively.
• the PEER-3 cDNA clone encoding CD43 (Pallant, A.S. et al., Proc . Natl . Acad . Sci . USA, 86:1328-1332, 1989) was first subcloned into the CDM8 expression vector (Seed, B. , Nature (London) 329:840-842, 1987) as described in Ardman, B.A. et al. (J. Exp. Med . , 172:1151-1158, 1990).
• HeLa cells (4 X 10 6 ) were then cotransfected by electroporation with 2 ⁇ g of the pSV2neo plasmid (encoding neomycin resistance; Southern et al., J . Mol . Appl. Genet . 1:327, 1982) and 20 ⁇ g of the CDM8-CD43 plasmid, or transfected alone with pSV2neo.
• cells were treated with G418 sulfate at 400 ⁇ g/ml (Geneticin; GIBCO, Grand Island, NY) to select for cells stably transfected by pSV2neo.
• the cotransfected cells were screened by immunofluorescence for CD43 expression, using an anti-CD43 monoclonal antibody, termed anti-Leu 22 (Becton Dickinson, Lincoln Park, NJ) . All CD43-positive HeLa cell clones selected by this method consistently expressed CD43 for the duration of the experiments (>6 mo) .
• the degree of CD43 expression by the positive clones was assessed by flow cytometry and found equivalent to CD43 expression by human T lymphocytes (Fig. 1) .
• the CD43-positive and CD43-negative phenotypes of the transfectants were confirmed by anti-Leu 22 immunoprecipitation of solubilized membranes from radiolabeled cells. Specifically, HeLa cell transfectants were surface labeled with Na 125 I (Amersham, Arlington Heights, IL) by using bovine milk lactoperoxidase (Calbiochem-Behring, San Diego, CA) . Solubilized cell lysates were then immunoprecipitated as described in Ardman, B.A. et al. (J. Exp . Med.
• the immunoprecipitates were resolved by 10% SDS/PAGE under reducing conditions, and the dried gel was autoradiographed at -70°C using an intensifying screen.
• CD43 protein was observed in the CD43-positive, but not the CD43-negative, HeLa cells.
• neuraminidase was treated with neuraminidase before SDS/PAGE analysis. Follwing neuraminidase treatment, the CD43 band shifted to a higher M r , a characteristic feature of CD43.
• CD43-positive HeLa cells were immunophenotyped. Transfectants were stained with either monoclonal antibodies to LFA-1 ⁇ subunit (TSI/22), ⁇ 2 integrin subunit (TSI/18) , ICAM-l (RR1/1) , or LFA-3(TS2/9) (Rothlein, R. et al. , J. Immunol . ,
• Coulter 541 flow cytometer (Coulter) .
• the CD43-positive and CD43-negative HeLa cells expressed virtually identical levels of ICAM-l (Table 1) .
• LFA-3 expression by the HeLa cell transfectants was substantially less than ICAM-l expression, and between the transfectants, the CD43-positive cells expressed 25% less LFA-3 than did the CD43-negative cells (Table 1) .
• No LFA-1 or 3 2 -integrin chain expression was detected in either HeLa cell transfectant. Because ICAM-l expression by the different HeLa cell transfectants was similar, the effect of CD43 on LFA-1-mediated, T- lymphocyte binding to the HeLa cells could be tested.
• Lymphoid cells (5 x 10 6 ) were washed twice with RPMI 1640 medium, labeled with 2' ,7'-bis(2-carboxyethyl)-5,6- carboxyfluorescein, acetoxymethyl ester (BCECF; Molecular Probes) in 1 ml of RPMI 1640 medium for 20 min at room temperature, washed, and then resuspended to a concentration of 5-6 x 10 5 cells per ml in RPMI medium.
• BCECF Molecular Probes
• Lymphoid cells (100 ⁇ l) were allowed to adhere to the HeLa cells for 30 min at room temperature, and then the wells were washed four times by alternating four-corner vacuum aspiration (25-gauge needle) with gentle addition of 200 ⁇ l of phosphate-buffered saline per well using a multichannel pipettor. The cells were then fixed in 0.37% formalin, and the plates were read on an automated microfluorimeter (Pandex) . All assays were done in quadruplicate and repeated three times. Student's t test for unpaired samples was used for statistical calculations.
• Cells were obtained from the following sources.
• Peripheral blood from normal donors was used to isolate mononuclear cells by Ficoll/Hypaque centrifugation.
• Peripheral blood mononuclear cells were enriched for T lymphocytes by plastic adherence to remove monocytes and nylon fiber filtration to remove B lymphocytes.
• T-cell blasts were generated by growing peripheral blood mononuclear cells in phytohemagglutinin at 5 ⁇ g/ml (Sigma) for 3 days and 100 units of recombinant interleukin 2 (IL-2) (Cetus, Norwalk, CT) for 10-17 days.
• IL-2 interleukin 2
• Normal human thymocytes were obtained from infants undergoing corrective cardiac surgery at the New England Medical Center.
• Human lymphocyte cell lines used were SUPT1 (T cell lymphoma) , JL89 (IL-2-dependent T-cell clone) , and C91/PL (human T-lymphotropic virus type 1- transformed T-cell line) .
• the HeLa cells and the lymphocyte cell lines were grown in RPMI 1640 medium/10% fetal bovine serum (HyClone)/2 mM L-glutamine/penicillin and streptomycin at 100 ⁇ g/ml.
• Lymphocytes were preincubated for 30 minutes on ice with normal mouse serum or with anti-LFA-l antibody (TS1/22) to block LFA-l-mediated adhesion (Marlin et al., Cell 51:813-819, 1987) or anti- CD2 (TS2/18) to block CD2-mediated adhesion at a final dilution of 1:200 in RPMI 1640 medium.
• the lymphocytes then were allowed to adhere to the CD43-positive or CD43- negative HeLa cells.
• the effect of CD43 on LFA-1- mediated lymphocyte adhesion was determined by comparing the percentage of lymphocytes that were blocked from binding to the different HeLa cell transfectants.
• lymphocytes were allowed to adhere to HeLa cells in the presence of PMA (Sigma, St. Louis, MO) at 50 ng/ml (final concentration) for 60 min at 37°C in a humidified incubator with 5% C0 2 . Binding of the JL89 T cell clone to the CD43-negative and CD43-positive HeLa cells was then compared before and after T-lymphocyte activation by PMA. To prevent cell clumping during the coincubation period, 50% of the usual number of T cells (i.e., 25 x 10 3 cells) were added to each well.
• PMA Sigma, St. Louis, MO
• CD43 A notable feature of CD43 is its extensive substitution with negatively charged, sialic acid residues (Remold et al., J . Biol . Chem . 261:7526, 1986; Carlsson et al., J . Biol . Chem . 261:12779, 1986). It has been proposed that these sialic acid residues confer an anti-adhesion function to CD43 by providing a net negative charge to cell surfaces, resulting in repulsion between CD43-positive cells (Brown et al., Nature (London) 289:456-460, 1981) .
• the HeLa cell transfectants were treated with neuraminidase before testing for T- lymphocyte adhesion.
• HeLa cells were incubated with Vijbrio cholerae neuraminidase (0.02 unit per ml;
• Neuraminidase treatment of the HeLa cells increased T-cell adhesion to both the CD43-negative and CD43-positive transfectants (Fig. 5) .
• HeLa cells was significantly greater than the increase in lymphocyte binding to the CD43-negative cells (18.5% vs. 8.8%, respectively; see Fig. 5).
• Preincubation of the neuraminidase-treated lymphocytes with the anti-LFA-1 antibody TS1/22 resulted in 80% inhibition of T-cell binding to both sets of HeLa cell transfectants.
• CD43 leukosialin or sialophorin
• results described above demonstrate that CD43 (leukosialin or sialophorin) , when expressed by opposing cells, functions as an anti-adhesion molecule.
• CD43 expression by HeLa cells interfered significantly with adhesion of T lymphocytes, cells that naturally express CD43. The interference occurred at physiologic levels of CD43 expression by the HeLa cell transfectants and was observed for T lymphocytes, irrespective of their source or derivation.
• the results also show that CD43 expression by opposing cells interferes with LFA-l- mediated T-cell adhesion. Under the assay conditions used, most T-cell adhesion was LFA-l-mediated (see Fig. 3) .
• LFA-1 is one of several activation-dependent intercellular adhesion molecules and PMA treatment of LFA-1-positive cells substantially enhances their binding avidity to ICAM-l (Dustin et al., Nature (London)
• CD43 Two distinctive features of CD43 are the extensive O-glycosylation and sialylation of its extracellular domain (Remod-O'Donnell et al., J. Biol . Chem . 261:7526- 7530, 1986; Carlsson et al., J. Biol . Chem . 261:12779- 12786, 1986; Pallant et al., Proc . Natl . Acad . Sci . USA 86:1328-1332, 1989; Shelly et al., Proc . Natl . Acad . Sci . USA 86:2819-2823, 1989).
• the sialic acid residues impart a negative charge to CD43 and could limit the association of CD43-positive cells with each other by repulsion of like charge.
• the results of the neuraminidase-treatment experiments in the present study suggest that sialic acid residues of CD43 contribute to, but are not completely responsible for, its anti-adhesion effect (see Fig. 5) . Because the efficacy of neuraminidase treatment was gauged by the ability to eliminate a CD43 sialic acid- dependent epitope (anti-Leu 22 reactive) from the CD43- positive HeLa cells, it is possible that other sialic acid residues on CD43 remained intact. Under these circumstances, the sialic acid contribution to the CD43 anti-adhesion effect may have been underestimated.
• CD43 structural features consistent with its ability to interfere with LFA-l-mediated T-cell binding, an interaction predicted to occur within an intermembrane distance of 25-30 nm (Springer, Nature (London) 346:425- 434, 1990).
• Other receptor-counterreceptor pairs e.g., CD2/LFA-3 are predicted to require intermembrane distances even smaller than that for LFA-l/ICAM-1, suggesting that their ability to interact would be diminished substantially by CD43 expression.
• CD43 has pro-adhesive features (Park et al., Nature (London) 350:706-709, 1991) and can bind directly to ICAM-l (Rosenstein et al.,
• the threshold for intercellular adhesion might depend, to a large degree, upon whether CD43 is expressed by one or both interacting cells.
• the binding threshold between CD43-positive lymphocytes might be expected to be greater than that between lymphocytes and CD43-negative cells (e.g., high endothelial venules) .
• CD43-negative cells e.g., high endothelial venules
• the in vivo effect(s) of CD43 depend upon the extent of its sialylation in addition to its tissue distribution. Incompletely sialylated CD43, a form naturally expressed by thymocytes (Ardman et al., J. Exp. Med . 172:1151-1158, 1990; Remold-O'Donnell et al.
• a CD43 chimeric molecule which includes (a) a cell targeting molecule which directs specific binding of the chimeric molecule to a molecule (e.g., a receptor, ligand, or counterreceptor) on the surface of one or both of the cells whose interaction is to be disrupted and (b) a CD43 extracellular domain bearing a net negative charge at physiological pH which antagonizes the intercellular interaction.
• a chimeric protein should be soluble to facilitate administration.
• LNF III and CD43:sialyl Lewis x are particularly preferred chimeric proteins.
• LNF III and sialyl Lewis x both molecules on the surface of leukocyte cells
• P-selectin and ELAM-1 interact specifically with P-selectin and ELAM-1 (respectively) , receptors present on the surface of endothelial cells activated during inflammation; such immune cell-endothelial cell interactions are required for progression of the inflammatory response.
• therapeutic CD43:LNF III or CD43:sialyl Lewis x chimeric molecules can be administered in order to coat the endothelial cell surface with CD43 protein (specifically blocking interaction with immune cells) and thereby treat or prevent inflammation.
• a negatively charged CD43 extracellular domain is fused to the receptors VCAM-1 and ICAM-l to produce chimeric proteins CD43:VCAM-1 and CD43:ICAM-l; such proteins coat the surface of certain types of leukocytes, and again block the intracellular interaction between the immune cells and the endothelium which leads to inflammation.
• CD43:LNF III, CD43:sialyl Lewis x , CD43:ICAM-l, CD43:VCAM-1, or any other CD43 chimera an extracellular (soluble) portion of the CD43 protein bearing a net negative charge at physiological pH is covalently bonded to the cell targeting molecule of choice.
• the CD43 domain is not only negatively charged but also of sufficient size to physically interfere with the intercellular interaction.
• the CD43 domain includes the N-terminal 180 amino acids and, more preferably, includes the N-terminal 220-233 amino acids of the CD43 sequence (as described, e.g., in Shelley et al., Proc . Natl . Acad . Sci . USA 86:2819, 1989; Pallant et al., Proc. Natl . Acad . Sci . USA 86:1328, 1989).
• the cell targeting molecule can be chosen from ligands, receptors, counterreceptors, antibodies, carbohydrates, or any molecule which is capable of specific interaction with a surface molecule on the targeted cell.
• the cell targeting domain must also be soluble and, in addition, must be of sufficient size and composition to direct specific interaction with its binding partner on the target cell surface.
• the cell targeting domain preferably includes between the N-terminal 430 and N-terminal 452 amino acids of the ICAM-l sequence described by Staunton et al. (Cell 52:925, 1988);
• VCAM-1 the cell targeting domain preferably includes between the N-terminal 580 and the N- terminal 606 amino acids of the VCAM-1 sequence described by Osborn et al.
• the cell targeting domain preferably includes between the N- terminal 350 and the N-terminal 371 amino acids of the CD4 sequence described by Maddon et al. (Cell 42:93, 1986) and Hussey et al. (Nature 331:78, 1988).
• the relative positions of the CD43 and the cell targeting domains within the chimeric protein are not critical to the chimeric molecule's function, although the orientation, N-terminus—Cell Targeting Molecule— CD43—C-terminus is preferred.
• a proline-rich hinge region of variable length may be inserted between the two domains.
• the chimeric molecules of the invention are preferably chimeric proteins expressed from chimeric fusion genes designed and constructed using standard techniques of molecular biology (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual , 2d ed. , Cold Spring Harbor Press, Cold Spring Harbor, NY; or Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, 1989) .
• Chimeric molecules may also be produced, however, by chemical coupling of the proteins, protein fragments, or molecules of the chimera. The chemical coupling approach is the method of choice for CD43:carbohydrate chimeras such as CD43:LNF III and CD43:sialyl Lewis x . Therapy
• the CD43 chimeric molecules of the invention are generally useful for blocking deleterious intercellular interactions, and, in particular, are useful for antagonizing the endothelial cell-immune cell interactions leading to inflammation and host cell- pathogen cell interactions leading to pathogen infectivity.
• therapeutic CD43 chimeric molecules are formulated in an appropriate pharmaceutically-acceptable buffer such as physiological saline.
• the therapeutic preparation is administered in accordance with the condition to be treated. Ordinarily, it will be administered intravenously, at a dosage that provides suitable competition for the deleterious intercellular interaction; such a dosage will normally be in the range of 0.01 to 100 mg/kg/day, preferably 0.1 to 5 mg/kg/day.
• the therapeutic preparation may be injected directly into the inflamed joint.
• CD43:LNF III, CD43:sialyl Lewis x , CD43:ICAM-1, and CD43:VCAM-1 therapeutic proteins are particularly useful for blocking interactions between white blood cells and activated endothelium and are therefore useful for treating or preventing inflammatory disorders, for example, the vasculitis disorders, lupus and rheumatoid arthritis, as well as for suppressing downstream immune responses leading, for example, to organ rejection.
• CD43:ICAM-l may also be used to disrupt cell-mediated, organ-specific inflammation secondary to virus infection thus could be useful for treating disorders such as viral myocarditis.
• the methods and therapeutics described herein may be used to treat disorders in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated, the CD43 or cell targeting proteins employed in construction of the therapeutic CD43 chimeric protein are preferably specific for that species.

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  • 1993-05-13 EP EP93913851A patent/EP0650367A4/fr not_active Withdrawn
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