WO2000025808A9 - Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonists - Google Patents
Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonistsInfo
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- WO2000025808A9 WO2000025808A9 PCT/US1999/025501 US9925501W WO0025808A9 WO 2000025808 A9 WO2000025808 A9 WO 2000025808A9 US 9925501 W US9925501 W US 9925501W WO 0025808 A9 WO0025808 A9 WO 0025808A9
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- psgl
- cells
- mice
- antagonist
- selectin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- P-selectin is a cell adhesion molecule expressed, among other places, on vascular endothelium. Interaction of P-selectin with its ligand, PSGL (also known as "PSGL-1", which is expressed, among other places, on neutrophils), causes cells circulating in the vasculature which express PSGL to attach to the endothelium, where other adhesion molecules mediate extravasation into the surrounding tissues. Thus, the P-selectin/PSGL interaction has been a well- documented step in the development of inflammatory and immune responses.
- PSGL has been cloned and well-characterized as described in International Application No. WO98/08949 (which is incorporated herein by reference). Such application discloses polynucleotides encoding various forms of PSGL, including numerous functional soluble forms of PSGL.
- PSGL is a well-characterized molecule, the soluble forms of which are particularly amenable to administration as therapeutics.
- soluble PSGL or antibodies directed to PSGL will inhibit the differentiation of activated proliferating T-cells into cytotoxic lymphocytes.
- soluble PSGL, PSGL antibodies and other PSGL antagonists will inhibit such differentiation and the attendant immune and inflammatory responses resulting therefrom.
- these antagonists can be used to treat diseases and other conditions which result from undesirable or over-aggressive immune and inflammatory responses, such as, for example, in allergic reactions and autoimmune conditions.
- the present invention provides a method of inhibiting the differentiation of an activated T-cell into a cytotoxic lymphocyte in a mammalian subject, said method comprising administering to said subject a therapeutically effective amount of a PSGL antagonist.
- inventions provide for a method of treating or ameliorating an autoimmune condition, said method comprising administering to said subject a therapeutically effective amount of a PSGL antagonist.
- Yet other embodiments provide for a method of treating or ameliorating an allergic reaction, said method comprising administering to said subject a therapeutically effective amount of a PSGL antagonist.
- PSGL antagonist is preferably selected from the group consisting of a soluble form of PSGL, an antibody directed to PSGL, an antibody directed to sLe x , an antibody directed to sulfated tyrosine, sLe x , mimetics which inhibit sLe x binding and a small molecule inhibitor of PSGL binding. Soluble forms of PSGL and antibodies directed to PSGL are most preferred.
- soluble forms of PSGL those preferred are soluble forms of PSGL comprising the first 19 amino acids of the mature amino acid sequence of PSGL, with forms comprising the first 47 amino acids of the mature amino acid sequence of PSGL being more preferred. In certain other preferred embodiments, such 47 amino acids are fused to the Ig portion of an immunoglobulin chain.
- PSGL soluble forms of PSGL, including fusion proteins comprising PSGL sequence, are disclosed in International Application No. WO98/08949. Soluble forms of PSGL can be made in accordance with the methods disclosed therein and other methods known to those skilled in the art.
- antibody includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, a CDR-grafted antibody, a humanized antibody or fragments thereof which bind to the indicated protein. Such term also includes any other species derived from an antibody or antibody sequence which is capable of binding the indicated protein.
- Antibodies to a particular protein can be produced by methods well known to those skilled in the art.
- monoclonal antibodies can be produced by generation of antibody-producing hybridomas in accordance with known methods (see for example, Goding. 1983. Monoclonal antibodies: principles and practice. Academic Press Inc., New York; Yokoyama. 1992. "Production of Monoclonal Antibodies” in Current Protocols in Immunology. Unit 2.5. Greene Publishing Assoc. and John Wiley & Sons).
- Polyclonal sera and antibodies can be produced by inoculation of a mammalian subject with the relevant protein or fragments thereof in accordance with known methods.
- Fragments of antibodies, receptors or other reactive peptides can be produced from the corresponding antibodies by cleavage of and collection of the desired fragments in accordance with known methods (see for example, Goding, supra; Andrew et al. 1992. "Fragmentation of Immunoglobulins” in Current Protocols in Immunology. Unit 2.8. Greene Publishing Assoc. and John Wiley & Sons).
- Chimeric antibodies and single chain antibodies can also be produced in accordance with known recombinant methods (see for example, 5,169,939, 5,194,594 and 5,576,184).
- Humanized antibodies can also be made from corresponding murine antibodies in accordance with well known methods (see for example, U.S. Patent Nos. 5,530,101, 5,585,089 and 5,693,762).
- sLe x is sialyl Lewis x, a carbohydrate involved in PSGL binding (see, WO98/08949). Methods of making sLe x are known to those skilled in the art. "Mimetics which inhibit sLe x binding” include carbohydrate and peptido/carbohydrate species which bind to determinants which bind sLe x in such a manner to inhibit sLe x binding (see, for example, U.S. Patent No. 5,614,615). Other methods for making such mimetics are known in the art. The ability of such species to perform in the methods of the present invention can be determined by testing such species in the models described herein for testing of soluble PSGL and PSGL antibodies.
- Small molecules which inhibit PSGL binding can also be identified by testing of candidate materials in the models described herein. Numerous compounds are available for testing to determine which perform in accordance with the present invention.
- compositions containing a PSGL antagonist which are useful in practicing the methods of the present invention may also contain pharmaceutically acceptable carriers, diluents, fillers, salts, buffers, stabilizers and/or other materials well-known in the art.
- pharmaceutically acceptable means a material that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered. The characteristics of the carrier or other material will depend on the route of administration.
- the various pharmaceutical compositions should contain about 0.1 micrograms to about 1 milligram per milliliter of the active ingredient.
- Administration can be carried out in a variety of conventional ways. Intraperitoneal injection is the preferred method of administration. Intravenous, cutaneous or sub-cutaneous injection may also be employed.
- the PSGL antagonist will preferably be administered in the form of pyrogen-free, parenterally acceptable aqueous solutions. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
- the amount of PSGL antagonist used for treatment will depend upon the severity of the condition, the route of administration, the reactivity of the antagonist or the activity of the antagonist, and ultimately will be decided by the treatment provider.
- a therapeutically effective amount of a PSGL antagonist is administered.
- the term "therapeutically effective amount” means the total amount of each active component of the method or composition that is sufficient to show a meaningful patient benefit (e.g., curing, ameliorating, inhibiting, delaying or preventing onset of, preventing recurrence or relapse of).
- One common technique to determine a therapeutically effective amount for a given patient is to administer escalating doses periodically until a meaningful patient benefit is observed by the treatment provider.
- a therapeutically effective dose of a PSGL antagonist in this invention is contemplated to be in the range of about 0.05 mg/kg to about 25 mg/kg, preferably about 1 mg/kg to about 20 mg/kg, more preferably about 2 mg/kg to about 10 mg/kg.
- the number of administrations may vary, depending on the individual patient and the severity of the autoimmune condition.
- Example 1 ⁇ (l,3) fucosylation of carbohydrate moieties on selectin ligands is required for selectin binding and therefore, mice doubly deficient for ⁇ (l,3)-fucosyl transferase IV and VII (FT-/-) lack functional selectin ligands on endothelial cells and T cells 1 2 .
- FT-/- mice doubly deficient for ⁇ (l,3)-fucosyl transferase IV and VII
- T cells 1 2 When infected with vaccinia virus (vv), FT -II mice do not develop viral-specific cytotoxicity, although their CD8+ T cells are capable of vigorous viral-specific proliferation and interferon- ⁇ (IFN- ⁇ ) production.
- Selectins and their ligands are surface molecules reciprocally expressed on endothelial cells and leukocytes, which through their interactions initiate leukocyte rolling, the first step required for leukocyte migration through the vascular endothelium 6"8 .
- the lectin domain of selectins is recognized by sialyl Lewis x (sLex) related carbohydrates presented on cellular protein scaffolds and the oligosaccharide modifications on sLex moieties by glycosylation, sialylation, fucosylation and sulfation determine the fine specificity of the selectin-ligand interaction 9"13 .
- Vaccinia virus induces an acute infection in mice resulting in the generation of a robust T cell mediated immune response and viral-specific cytotoxicity can be demonstrated directly from freshly isolated splenocytes and PEL without restimulation in vitro 14 .
- vv infection provides a convenient acute infection model to study the generation of T cell response in vivo. We studied the T cell response of FT -/- mice using this model.
- Wild type and FT -/- mice were infected with vv via a peripheral (subcutaneously at base of the tail (sc)) or a systemic route (intraperitonially (Ip)) and viral-specific cytotoxicity was assessed using peritoneal exudate lymphocytes (PEL) and/pr splenocytes obtained on day 10 (sc route) or 7- (lp route) post infection (pf). Wild type mice showed high levels of cytotoxicity, whereas splenocytes and PEL from FT -/- mice exhibited no detectable cytotoxicity, irrespective of the route of infection (Fig. la).
- vaccinia infection elicits natural killer (NK) cell function and ⁇ interferon production by NK cells, CD4+ and CD8+ T cells as well as a strong humoral immune response 15 .
- NK natural killer
- CD8+ CTL response may be a major mediator of protection in normal animals 16 17
- mice lacking CD8+ T cells as well as mice deficient in an important component of CTL machinery, perform are able to clear vv suggesting that NK cells function, ⁇ - interferon secretion and normal antibody response can compensate for the lack of anti-viral CTL 15 ' 18"20 .
- These parameters are not defective in FT -/- mice (not shown).
- FT -/- mice could clear vv similar to wild type mice (not shown). These results indicate that ⁇ (l,3)-fucosyl transferase deficiency selectively affects the generation of effector CTL.
- FT -/- mice are severely compromised for lymphocyte homing to peripheral lymph nodes 1,21 , suggesting the possibility that failure to find anti-viral CTL in the FT -/- mice may be due to defective T cell priming in the peripheral of visceral lymph nodes, leading in turn to diminished levels of vv-specific CTL in the spleen.
- non-specific T cells may be activated and traffic to the site of invention 2223 .
- recent data using TCR transgenic mice and MHC- peptide tetramers indicate that most activated cells are indeed antigen-specific 24"26 .
- cells from infected mice were immunomagnetically depleted of CD4+ T cells and NK cells, and tested for vv-specific proliferation. Both wild type and FT -/- CD8+ T cells proliferated comparably and specifically in response to vv stimulation (Fig. 2c).
- mice triply deficient for L-, P- and E-selectins for their ability to generate antiviral CTL after vv infection.
- Triple selectin deficient mice like wild type mice and unlike FT-/- mice, exhibited a robust CTL activity (Fig. 3).
- the defect in effector CTL generation in the FT -/- mice is unrelated to a defective selectin function but is a consequence of a selectin ligand function.
- Activated Tal cells but not resting T cells or activated Th-2 cells, bind P-selectin, although PSGL-1 is expressed in equivalent amounts of all of these cell types 29 .
- Carbohydrate modifications which confer binding ability to HECA 452, an antibody directed against the cutaneous lymphocyte antigen (CLA), modulate PSGL-1 binding to E-selectin 30 .
- CLA cutaneous lymphocyte antigen
- FT -/- mice are severely compromised in generating viral-specific effector CTL, but have virtually normal LAK and SEA induced CTL activity, la.
- Splenocytes from wild type or FT -/- mice infected with vv sc, and Splenocytes & PEL from mice infected ip were tested for cytolysis of vv infected MC57G (H2b) targets by 4 h Cr release assay, lb.
- Splenocytes from ip infected mice were restimulated in vitro by incubation with vv infected autologous splenocytes for 5 days and tested for antiviral cytoxicity.
- FT -/- mice generate activated CD8+ T cells which proliferate and produce cytokines in a viral-specific manner.
- Splenocytes and PEL from vv infected mice stained with FTTC-conjugated and mouse Thy 1.2, CD4 or CD8 monoclonal antibodies (2a) or doubly stained with CD8 FTTC or PE and CD62-L FTTC, CD1 l ⁇ FTTC or CD44 PE (2b) were analyzed by flow cytometry.
- splenocytes from vv infected mice were immunomagnetically depleted of CD4+ T cells and NK cells and stimulated with vv as described in Fig. lc.
- FT -/- but not selectin -/- mice fail to generate viral-specific effector CTL.
- Wile type mice and mice deficient for L-, P-, and -E selectins were infected with vv and their splenocytes were tested for antiviral cytotoxicity on day 7 pi as described in Fig. 1.
- Soluble PSGL-1 and anti-murine PSGL-1 antibody inhibits development of effector CTL by primed wild type CD8+ T cells in vitro.
- Splenocytes harvested from wild type mice on day 7 post vaccinia infection were stimulated with vv in the absence or presence (20 ⁇ g/ml) of soluble recombinant PSGL-1 or non fucosylated PSGL-1 (dead PSGL-1) (4a) or of anti-murine PSGL-1 antibody, 2 PH-1, or anti -human PSGL-1 antibody, PL-1 (4b). Viral-specific cytotoxicity was measured 5 days later. 4c.
- FT -/- APC abrogates and wild type APC restores effector CTL generation.
- Wild type and FT -I- mice were infected with vv and on day 7 pi, CD8+ T cells (responders) were positively selected and stimulated with vv infected and ⁇ -irradiated wild type or FT -/- APC (T cells depleted splenocytes). Viral-specific cytotoxicity was assayed 5 days later.
- Vaccinia viral infection FT IV and VII -/-, L-, P- and E-selectin -/- mice and their wild type counterparts were maintained under SPF facility at the Center for Blood Research. Mice 6 - 8 weeks of age and matched for sex were used for the studies. Mice were infected with WR strain of vv (ATCC) either sc at the base of the tail or lp (10 5 pfu/mice in 0.2 ml PBS).
- Cytotoxicity assays To test viral-specific cytotoxicity, on day 7 pi, peritoneal exudate cells were harvested by flushing with 3 mis of PBS and/or spleens were collected. Splenocytes and PEL were depleted of RBC by lysis in 0.17 M ammonium chloride and the cells were tested for killing of 51 Cr labeled, MC57G targets uninfected or infected with vv as described earlier 31 .
- splenocytes from normal mice were cultured in the presence of 200 IU/ml recombinant IL2, and 3 days later, cells were tested for killing of 51 Cr labeled Yac- 1 targets.
- SEA induced cytotoxicity assay For SEA induced cytotoxicity assay, splenocytes were cultured in the presence of lO ⁇ g/ml SEA (Sigma) and CD8 T cells were selected (see later) and tested for lysis of Raji cells coated with SEA (100 ng/ml for 30 min before the assay). Cytotoxicity was defined as (test release-spontaneous release)/(maximum release-spontaneous release) X 100. Percent killing of uninfected targets (vv cytotoxicity) or uncoated target (SEA induced cytoxicity) was subtracted from that of infected/coated targets to calculate viral-specific cytotoxicity.
- splenocytes and PEL were stained singly with FTTC-conjugated anti-mouse CD3, CD4 or CD8 monoclonal antibodies (Pharmingen).
- Activated CD8+ T cells defined as L-selectin low, LFA-1 high and CD44 high, were assayed by dual staining with PE CD8 X FTTC Mel- 14, FTTC CD1 l ⁇ or FTTC CD8 X PE CD44 (Pharmingen).
- CD4+ T cells and NK cells For depletion of CD4+ T cells and NK cells, cells were stained with purified rat anti -mouse CD4 and NK 1.1 antibodies, washed and incubated with goat anti-rat Ig G coated magnetic beads (Dunai, 10 beads/cell). The depleted population contained ⁇ 3% CD4 or NK cells as determined by flow cytometry.
- splenocytes harvested 6-7 days post vaccinia were depleted of T cells using anti-CD3 coated Dynal beads and infected with vv (10 pfu/cell, 2 h at 37°C), irradiated (400 rads) and UV-treated as described in 32 .
- 5 X 10 6 infected cells were cultured with 5 X 10 6 autologous uninfected splenocytes in 24 well culture plates for 4-5 days before testing for CTL activity.
- CD4+ T cells and NK cells were depleted as described above.
- CD8+ cells were positively selected using CD8+ milteny beads according to manufacturer's instructions.
- PSGL-1 Ig chimera in vitro stimulation, soluble recombinant PSGL-1 Ig chimera, its non-fucosylated variant (dead PSGL-1) (gifts of Genetics Institute, Cambridge, MA), anti-murine PSGL-1 antibody, 2 PH-1, anti-human PSGL-1 antibody, PL-1 (gift of%), and anti-murine L-selectin antibody, Mel-14 (gift of .7) were added at a final concentration of 20 ⁇ g/ml.
- Lymphocyte proliferation and IFN- ⁇ assay 2 X 10 5 splenocytes, depleted of CD4+ T cells and NK cells as described above, were cultured with equal numbers of ⁇ -irradiated splenocytes that were uninfected or infected with vv in triplicate wells of 96 well trays. Three days after stimulation, 50 ⁇ g supematants were harvested for IFN- ⁇ assay and the cultures were pulsed with 3 H thymidine (0.5 ⁇ Cl/well) for 6-8 h, harvested and counted for 3H incorporation as described in 27 . Supematants were assayed for IFN- ⁇ using IFN- ⁇ mini assay kit (Antigen, MA, USA) calibrated with and IFN- ⁇ standard according to manufacturers protocol.
- IFN- ⁇ mini assay kit Antigen, MA, USA
- CD8+ T cells correlating cytotoxicity in vitro are more efficient in anti-vacccinia protection than CD4+ dependent interleukins. J. Immunol. 146, 4301-4307 (1991).
- Counting antigen-specific CD8 T cells A ree valuation of bystander activation during viral infection. Immunity 8, 177-187 (1998).
- P-selectin ligand glycoprotein ligand- 1 is broadly expressed in cells by myeloid, lymphoid and dentritic lineage and in some nonhematopoietic cells. Blood 88, 3010-21 (1996).
- mice that are doubly deficient in the ⁇ (l,3)-fucosyltransferases, FT-TV and FT-V ⁇ (FT-/- mice), lack functional selectin ligands on leukocytes and endothelial cells.
- FT-/- mice developed markedly fewer cytotoxic T cells as compared to wild-type mice, although comparable numbers of CD8+ T cells accumulated at the site of infection in both strains and were capable of vigorous viral- specific proliferation.
- Cytotoxic T lymphocytes are critical mediators of antigen-specific host defense against viral infections.
- naive CD8 + T cells must first encounter viral antigen on professional antigen- presenting cells (APCs) in secondary lymphoid organs.
- APCs professional antigen-presenting cells
- Antigen-activated T cells proliferate for several days and eventually migrate to the site of viral infection. Finally, they acquire effector functions, namely the ability to kill other cells that express cognate antigen on MHC class I and to produce effector cytokines, particularly interferon (IFN)- ⁇ .
- IFN interferon
- Antigen-laden APC must initially migrate from the site of infection to organized lymphoid tissues. Here, they stimulate naive T cells, which home to these organs from the blood. Subsequently, activated T cells must find their way back into the blood stream and from there into infected peripheral tissues.
- Leukocyte migration to many lymphoid and non-lymphoid organs requires the concerted action of one or more of the three selectins (L-, E- and P-selectin, CD62) and their ligands, which are reciprocally expressed on endothelial cells and leukocytes (1-3).
- Selectins mediate leukocyte rolling in microvessels by binding to sialyl-Lewis x (sLeX) and related carbohydrates that are frequently presented on sialomucin scaffolds such as PSGL-1 (4,5).
- a critical aspect of selectin-binding carbohydrates is ⁇ (l,3)-fucosylation of one or more N-acetyl-glucosamine residues in sialylated core-2 glycans.
- FTs ⁇ (l,3)- fucosyltransferases
- vaccinia virus intraperitoneally (i.p.) into FT -/- mice and animals that were triply deficient in L-, E- and P-selectin (selectin -/-) (10).
- Vaccinia virus has been shown to induce an acute infection in wild-type mice resulting in the generation of a robust T cell-mediated immune response and viral-specific cytotoxicity can be demonstrated directly from freshly isolated splenocytes and peritoneal exudate lymphocytes (PEL) without restimulation in vitro (11).
- vaccinia infection elicits natural killer (NK) cell function and IFN- ⁇ production by NK cells, CD4 + and CD8 + T cells as well as a strong humoral immune response (11-17).
- NK natural killer
- CD8 + CTL are the principal mediators of protection in normal animals (13), mice lacking CD8 + T cells as well as mice deficient in perforin, an important component of the CTL machinery, can clear vaccinia infections (12,15,17). Therefore, normal viral clearance in mice that are deficient in FTs or selectins does not exclude that these molecules have a role in the generation, migration or function of anti-viral CTL.
- Table 1 shows that equivalent fractions of T cells in the blood, spleen as well as in PEL expressed activation markers (L-selectin 1 ⁇ and CD44 Hl ) suggesting that antigen-specific priming of T cells can occur normally in the absence of selectins or their ligands.
- activation markers L-selectin 1 ⁇ and CD44 Hl .
- TCR transgenic mice and MHC-peptide tetramers indicate that most activated cells are indeed antigen-specific (21-23).
- mice that were deficient in FT-IV or FT-VII alone were deficient in FT-IV or FT-VII alone. Both strains had significantly reduced CTL activity compared to wild-type mice, but the reduction was more notable in the FT-VII -/- than in the FT-IV -/- mice (not shown). The most striking reduction of CTL activity was seen in the FT-IV / FT-VII doubly deficient mice suggesting that both enzymes may be necessary to elicit optimal CTL activity.
- FT -/- T cells might be incapable of detecting or responding to vaccinia antigen.
- antigen-specific FT -/- T cells might exist and get activated, but they may not be able to kill target cells.
- splenocytes were immunomagnetically depleted of CD4 + T cells and NK cells, and tested for vaccinia virus-specific proliferation.
- CD8 + T cells from primed mice proliferated rapidly and specifically upon antigen challenge (Fig. 5B).
- FT-/- mice There was no difference between CD8 + T cells from FT-/- mice compared to cells from selectin -/- or WT animals. Thus, FTs are not required for the proliferative T cell response to antigen, but may be necessary later when activated CD8 + T cells give rise to effector CTL.
- FT -/- CD8 + cells lacked at least two distinct qualities of effector cells; CTL activity and IFN- ⁇ production.
- Cytolytic activity was reproducibly induced in both wild-type and FT -/- T cells that encountered vaccinia antigen presented by wild-type APC, whereas FT-/- APC were incapable of eliciting CTL activity on CD8 + cells from either wild-type or FT -/- mice (Fig. 6).
- PSGL-1 This sialomucin is expressed on the surface of myeloid and lymphoid cells and can be modified by FTs on many leukocytes including dendritic cells (reviewed in 5). PSGL-1 protein is expressed at normal levels on FT -/- leukocytes, but it is functionally deficient because it lacks the fucosylation needed to serve as a selectin ligand (8 and data not shown) .
- Recombinant PSGL-1/Ig was either generated in cells that had been cotransfected with core-2 enzyme and FT- VII (to generate PSGL-1/Ig decorated with sLeX-like carbohydrates or from cells that expressed only core-2 enzyme, but not FT-VII (mimicking non-fucosylated PSGL-1 in FT -/- mice).
- FT- VII to generate PSGL-1/Ig decorated with sLeX-like carbohydrates or from cells that expressed only core-2 enzyme, but not FT-VII (mimicking non-fucosylated PSGL-1 in FT -/- mice).
- Coincubation with the fucosylated PSGL-1/Ig partially blocked the generation of viral-specific CTL, whereas non-fucosylated PSGL-1/Ig had no effect (Fig. 7B).
- inhibitors of PSGL-1 were only effective when they were present during T cell stimulation by APC.
- Neither anti-PSGL-1 nor fucosylated PSGL-1/Ig inhibited target cell
- Wild-type, FT -/-, and selectin -/- mice (6-8 weeks of age and matched for sex) were infected with the WR strain of vv (ATCC) either sc at the base of the tail or ip (10 5 pfu/mice in 0.2 ml PBS).
- vv WR strain of vv
- PEL were harvested by flushing with 3 mis of PBS and /or spleens were collected.
- Splenocytes and PEL were depleted of RBC by lysis in 0.17 M ammonium chloride and tested for killing of 51 Cr labeled, MC57G targets, uninfected or infected with vv in a standard chromium release assay. Cytotoxicity was defined as (test release- spontaneous release)/ (maximum release-spontaneous release) X 100%. Percent killing of uninfected targets was subtracted from that of infected targets to calculate viral-specific cytotoxicity.
- CD8 + T cells were positively selected using anti-CD8 antibody-coated Miltenyi beads according to manufacturer's instructions.
- APC splenocytes were depleted of T cells using anti-CD3 coated Miltenyi beads, infected with vv (10 pfu/cell, 2 h at 37°C), irradiated (400 rads) and UV-treated as described earlier (34).
- 2X10 6 CD8 + T cells obtained from wild-type or FT-/- mice were cultured with 5X10 s wild-type and FT-/- APC in 24-well culture plates for 4-5 days before testing for CTL activity.
- mice Total leukocyte counts, T cell subset frequency and activation status of CD8 + T cells in PEL, spleen and peripheral blood of wild-type, selectin -/- and FT -/- mice.
- Mice were infected by i.p. injection of vaccinia virus (10 5 pfu/mouse) and at day 7 p.i., peripheral blood was obtained by tail bleeding and PEL and spleen were harvested. After lysing of RBC, leukocyte counts were performed on all samples using a hemocytometer.
- T cell subset proportions aliquots of cells were labeled with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 and analyzed on a flow cytometer (FACScan, Becton Dickinson) following standard procedures.
- FACScan Fluorescence-Activated Cell Sorting
- anti-CD8 FTTC and anti-L-selectin PE or anti-CD8 FTTC and anti-CD44 PE Shown are % CD8 + T cells that were L-selectin low or CD44 high. L- selectin levels are not shown for selectin-/- mice because all cells were negative for L- selectin. Mean+/- SD from 6 mice in each group are shown.
- Anti-viral CTL activity and IFN- ⁇ production but not virus-specific proliferation is markedly reduced in FT-/- mice.
- 5A CTL activity is reduced in FT-/- but not in selectin-/- mice. Wild-type, triple selectin-/- and FT-/- mice were infected with vv ip and 7 days later, their PEL were tested for lysis of vv infected 5i Cr labeled MC57G target cells (25). Scattergrams for 16 wild-type, 16 FT-/- and 10 selectin-/- mice at 4 different effector: target (E:T) ratios are shown. Each symbol represents the mean percent specific cytotoxicity (from triplicate measurements) of cells from a single animal. 5B.
- Viral-specific proliferation is comparable in selectin-/- and Fr-/- mice.
- Mice were infected with vv and 7 days later, their splenocytes were immunomagnetically depleted of CD4 + T cells and NK cells.
- 2xl0 5 depleted splenocytes were cultured with equal numbers of T cell-depleted and ⁇ -irradiated splenocytes that were uninfected or infected with vv in triplicate wells of 96-well plates. Two days after stimulation, the cultures were pulsed with 3 H thymidine (0.5 ⁇ Ci/well) for 6-8 h, harvested and counted for 3 H incorporation. Shown is the mean cpm +/- S.D.
- IFN- ⁇ _production is reduced in FT-/- mice but not in selectin-/- mice.
- PEL obtained on d7 pi were stimulated with 1 ⁇ g/ml ⁇ CD3 in the presence of Brefeldin A for 6 h, stained with anti-CD8 Cychrome, fixed, permeabilized and then stained with anti-IFN- ⁇ _PE using intracellular staining kit (Pharmingen) before analyzing in a flow cytometer. Representative results from 1 mouse for each strain (out of 3 animals tested with similar results) are shown.
- Fig. 6 ⁇ (l,3)-fucosylated PSGL-1 is required on APC for the induction of CTL activity in activated CD8 + cells.
- Wild-type and FT-/- mice were infected with vv and 7 days later, their splenic CD8 + T cells were immunomagnetically selected and stimulated with vv-infected wild-type or FT-
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
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JP2000579247A JP2002528513A (en) | 1998-10-30 | 1999-10-29 | Inhibition of cytotoxic T-cell differentiation by P-selectin ligand (PSGL) antagonists |
CA002347119A CA2347119A1 (en) | 1998-10-30 | 1999-10-29 | Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonists |
MXPA01004310A MXPA01004310A (en) | 1998-10-30 | 1999-10-29 | Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonists. |
BR9914957-5A BR9914957A (en) | 1998-10-30 | 1999-10-29 | Inhibition of cytotoxic t cells by selectin-p ligand antagonists (psgl) |
IL14271799A IL142717A0 (en) | 1998-10-30 | 1999-10-29 | Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonists |
EP99964950A EP1131084A4 (en) | 1998-10-30 | 1999-10-29 | Inhibition of differentiation of cytotoxic t-cells by p-selectin ligand (psgl) antagonists |
AU30971/00A AU774419C (en) | 1998-10-30 | 1999-10-29 | Inhibition of differentiation of cytotoxic T-cells by P-selectin ligand (PSGL) antagonists |
AU2004214516A AU2004214516A1 (en) | 1998-10-30 | 2004-09-22 | Inhibition of differentiation of cytotoxic T-cells by P-selectin ligand (PSGL) antagonists |
Applications Claiming Priority (2)
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US10631598P | 1998-10-30 | 1998-10-30 | |
US60/106,315 | 1998-10-30 |
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WO2000025808A1 WO2000025808A1 (en) | 2000-05-11 |
WO2000025808A8 WO2000025808A8 (en) | 2000-10-26 |
WO2000025808A9 true WO2000025808A9 (en) | 2001-07-19 |
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US (1) | US20020058034A1 (en) |
EP (1) | EP1131084A4 (en) |
JP (1) | JP2002528513A (en) |
CN (1) | CN1342085A (en) |
AU (2) | AU774419C (en) |
BR (1) | BR9914957A (en) |
CA (1) | CA2347119A1 (en) |
IL (1) | IL142717A0 (en) |
MX (1) | MXPA01004310A (en) |
NZ (1) | NZ528610A (en) |
WO (1) | WO2000025808A1 (en) |
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US20040001839A1 (en) * | 2000-12-29 | 2004-01-01 | Avigdor Levanon | Multimers - isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof |
US20040002450A1 (en) * | 2000-12-29 | 2004-01-01 | Janette Lazarovits | Y17 - isolated molecules comprising epitopes containing sulfated moieties, antibodies to such epitopes, and uses thereof |
US7132510B2 (en) * | 2000-12-29 | 2006-11-07 | Bio-Technology General (Israel) Ltd. | Specific human antibodies for selective cancer therapy |
DE60231016D1 (en) | 2001-06-05 | 2009-03-12 | Genetics Inst Llc | Process for the purification of strongly anionic proteins |
US20040116333A1 (en) | 2001-08-03 | 2004-06-17 | Rong-Hwa Lin | Modulators of P-selectin glycoprotein ligand 1 |
US7744888B2 (en) | 2001-08-03 | 2010-06-29 | Abgenomics Cooperatief U.A. | Methods of modulating T cell or natural killer cell activity with anti-P-selectin glycoprotein ligand 1 antibodies |
EP1517923A2 (en) * | 2002-04-22 | 2005-03-30 | Recopharma AB | Compositions and methods for inhibiting microbial adhesion |
KR20050059001A (en) * | 2002-07-01 | 2005-06-17 | 사비언트 파마슈티컬즈 인코퍼레이티드 | Compositions and methods for therapeutic treatment |
US20040208877A1 (en) * | 2002-07-01 | 2004-10-21 | Avigdor Levanon | Antibodies and uses thereof |
BR0312483A (en) * | 2002-07-01 | 2005-08-09 | Savient Pharmaceuticals Inc | Antibodies and their uses |
US20040202665A1 (en) * | 2002-07-01 | 2004-10-14 | Janette Lazarovits | Compositions and methods for therapeutic treatment |
US20050152906A1 (en) * | 2003-06-30 | 2005-07-14 | Avigdor Levanon | Specific human antibodies |
US20050069955A1 (en) * | 2003-06-30 | 2005-03-31 | Daniel Plaksin | Antibodies and uses thereof |
US20050266009A1 (en) * | 2003-06-30 | 2005-12-01 | Savient Pharmaceuticals, Inc. | Antibodies and uses thereof |
WO2005047320A2 (en) * | 2003-11-12 | 2005-05-26 | Wisconsin Alumni Research Foundation | Equine p-selection glycoprotein ligand-1 and uses thereof |
MY148646A (en) | 2004-05-10 | 2013-05-15 | Abgenomics Cooperatief Ua | Anti-psgl-1 antibodies |
NZ590478A (en) | 2004-05-11 | 2012-07-27 | Abgenomics Cooperatief Ua | Peptide motif Pro-Met-(Glu or Ser)-Ile used to identify a ligand which on binding an activated T-cell receptor induces apoptosis or cell death |
CA2609915A1 (en) * | 2005-05-31 | 2006-12-07 | President And Fellows Of Harvard College | Modulation of t cell recruitment |
US20070298034A9 (en) * | 2005-12-09 | 2007-12-27 | Angela Widom | Sulfotyrosine specific antibodies and uses therefor |
US8058229B2 (en) | 2008-08-14 | 2011-11-15 | Acceleron Pharma Inc. | Method of increasing red blood cell levels or treating anemia in a patient |
CA2838952C (en) | 2011-06-13 | 2020-11-24 | Stefan Bassarab | Anti-psgl-1 antibodies and uses thereof |
PT3166636T (en) | 2014-07-08 | 2021-06-29 | Sanford Burnham Med Res Inst | Psgl-1 modulators and uses thereof |
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IL102639A (en) * | 1991-07-25 | 1997-03-18 | Idec Pharma Corp | Compositions for inducing cytotoxic t lymphocyte responses |
US5747036A (en) * | 1991-08-28 | 1998-05-05 | Brigham & Women's Hospital | Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease |
US6277975B1 (en) * | 1992-10-23 | 2001-08-21 | Genetics Institute, Inc. | Fusions of P-selectin ligand protein and polynucleotides encoding same |
PT666914E (en) * | 1992-10-23 | 2004-04-30 | Inst Genetics Llc | NEW PROTEIN LIGAND OF SELECTIN P |
US5843707A (en) * | 1992-10-23 | 1998-12-01 | Genetics Institute, Inc. | Nucleic acid encoding a novel P-selectin ligand protein |
AU681369B2 (en) * | 1992-11-16 | 1997-08-28 | Board Of Regents Of The University Of Oklahoma, The | Glycoprotein ligand for P-selectin and methods of use thereof |
US5614615A (en) * | 1995-03-21 | 1997-03-25 | The Scripps Research Institute | Sialyl Lewis X mimetics incorporating fucopeptides |
US5659018A (en) * | 1995-08-01 | 1997-08-19 | Genetics Institute, Inc. | Mocarhagin, a cobra venom protease, and therapeutic uses thereof |
DE69630307T2 (en) * | 1995-08-03 | 2004-07-15 | The Board Of Regents Of The University Of Oklahoma, Norman | O-GLYCANINHIBITORS FROM SELECTIN-MEDIATED INFLAMMATIONS |
US5962318A (en) * | 1996-11-15 | 1999-10-05 | St. Jude Children's Research Hospital | Cytotoxic T lymphocyte-mediated immunotherapy |
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1999
- 1999-10-29 JP JP2000579247A patent/JP2002528513A/en not_active Withdrawn
- 1999-10-29 MX MXPA01004310A patent/MXPA01004310A/en unknown
- 1999-10-29 AU AU30971/00A patent/AU774419C/en not_active Ceased
- 1999-10-29 CA CA002347119A patent/CA2347119A1/en not_active Abandoned
- 1999-10-29 BR BR9914957-5A patent/BR9914957A/en not_active IP Right Cessation
- 1999-10-29 IL IL14271799A patent/IL142717A0/en unknown
- 1999-10-29 EP EP99964950A patent/EP1131084A4/en not_active Withdrawn
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- 1999-10-29 WO PCT/US1999/025501 patent/WO2000025808A1/en not_active Application Discontinuation
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Publication number | Publication date |
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AU774419C (en) | 2005-03-03 |
WO2000025808A8 (en) | 2000-10-26 |
CN1342085A (en) | 2002-03-27 |
AU3097100A (en) | 2000-05-22 |
US20020058034A1 (en) | 2002-05-16 |
WO2000025808A1 (en) | 2000-05-11 |
AU2004214516A1 (en) | 2004-10-14 |
NZ528610A (en) | 2005-03-24 |
CA2347119A1 (en) | 2000-05-11 |
JP2002528513A (en) | 2002-09-03 |
EP1131084A4 (en) | 2001-12-19 |
BR9914957A (en) | 2001-07-24 |
MXPA01004310A (en) | 2003-06-06 |
IL142717A0 (en) | 2002-03-10 |
AU774419B2 (en) | 2004-06-24 |
EP1131084A1 (en) | 2001-09-12 |
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