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WO2001078748A2 - Methodes d'immunomodulation utilisant des carbohydrates antigeniques - Google Patents

Methodes d'immunomodulation utilisant des carbohydrates antigeniques Download PDF

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Publication number
WO2001078748A2
WO2001078748A2 PCT/US2001/012365 US0112365W WO0178748A2 WO 2001078748 A2 WO2001078748 A2 WO 2001078748A2 US 0112365 W US0112365 W US 0112365W WO 0178748 A2 WO0178748 A2 WO 0178748A2
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lnnt
cells
mice
agent
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WO2001078748A3 (fr
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Donald A. Harn
Okano Mitsuhiro
Luis Ignacio Terrazas
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Harvard University
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Harvard University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]

Definitions

  • IgE has several roles in immunity against helminth parasites. Ag-specific IgE, especially anti- adult worm Ag, is involved with the resistance to reinfection (Hagan, P. et al. (1991) Nature vol. 349 pp. 243-245; Viana, J-RC. et al. (1995) Parasite Immunol, vol. 17 pp. 297-304), and cell-mediated cytotoxicity against parasites (Capron, A. et al. (1980) Am. J. Trop. Med. Hyg. vol. 29 pp. 849-857; Gounni, AS. et al. (1994) Nature vol. 367 pp. 183-186; Cutts, L. et al.
  • Two recombinant filarial proteins are capable of inducing polyclonal IgE production in vitro, however, they also induce Ag-specific IgE (Garrud, O. et al. (1995) J. Immunol, vol. 155 pp. 131-1325).
  • Lacto- ⁇ -fucopentaose m found on adult worm and egg of S. mansoni has been found to be an antigenic determinant (KO, AL et al. (1990) Proc. Natl. Acad. Sci. USA vol. 87 pp. 4159-4163).
  • LNFIH stimulates splenic B cells from parasite-infected mice to proliferate and produce J-L-10, a cytokine that downregulates Thl immune responses (W Nelupillai, P. et al. Proc. Natl. Acad. Sci. USA vol. 91 pp.
  • the instant invention provides novel methods of regulating the immune response.
  • the invention is based, at least in part, on the discovery of the functional characteristics of L ⁇ nT, a nonfucosylated homologue of L ⁇ FHt, that is converted to L ⁇ FIH by ⁇ l-3 fucosyltransf erase in adult worms.
  • Multivalent L ⁇ nT induces polyclonal Ig ⁇ production and - 3 - HUI-038CPPC
  • cytokine production e.g., IL-10, JJ -5, IL-4, IL-13 and TGF- ⁇
  • This molecule can be used to modulate the IgE response, not only to parasite antigens, but to environmental allergens by saturating Fc ⁇ receptors on the effector cells.
  • the invention further pertains to other uses of LNnT, either in a multivalent form or as a free sugar (i.e., monovalent form), in the modulation of immune responses.
  • multivalent LNnT e.g., LNnT conjugated to dextran
  • LPS lipopolysaccharide
  • Con-A e.g., as measured by spleen cell proliferation and the production of the Thl -associated cytokines interleukin-12 (J-L-12), interleukin-18 (IL-18) and interferon-gamma (IFN- ⁇ )).
  • multivalent LNnT can be used to protect a subject from the effects of shock (e.g., toxic shock), for example as a pretreatment in subjects susceptible to shock (e.g., surgery patients at risk for shock).
  • shock e.g., toxic shock
  • the ability of multivalent LNnT to downmodulate Thl-associated cytokines allows for use of such agents in any clinical setting in which it is desireable to downmodulate type 1 immune responses, such as in autoimmune diseases (including inflammatory bowel disease, diabetes and rheumatoid arthritis).
  • multivalent LNnT has been found to induce a population of suppressor cells that are Grl+, CDl lb+.
  • These suppressor cells which also can be induced by sugars expressed on tumor cells, are able to suppress T cell proliferative responses and furthermore can make cytokines that may downregulate immune responses or that may promote angiogenesis (such as TGF ⁇ ),
  • angiogenesis such as TGF ⁇
  • the invention provides for the use of free (i.e., unconjugated, monovalent) LNnT to inhibit the generation of these Grl+, CDl lb+ suppressor cells, for example in the treatment of cancer.
  • Grl+, CDllb+ suppressor cell population in other clinical settings it may be desireable to induce this Grl+, CDllb+ suppressor cell population, for example in situations where one wants to downmodulate immune responses, such as in autoimmune diseases (e.g., inflammatory bowel disease, diabetes, rheumatoid arthritis, allergic asthma, multiple sclerosis).
  • autoimmune diseases e.g., inflammatory bowel disease, diabetes, rheumatoid arthritis, allergic asthma, multiple sclerosis.
  • other embodiments of the invention provide for use of multivalent LNnT to induce (e.g., stimulate, recruit) the generation of Grl+, CDllb+ suppressor cells, for example in the treatment of autoimmune diseases, and - 4 - HUI-038CPPC
  • suppression of the immune system is desireable (e.g., transplantation, allergy)
  • Figures la-lc Serum Ig production in BALB/C mice following JP immunization.
  • Figure 1(a) Female BALB/C mice were JP immunized with saline, HSA, Le v -HSA, LNnT- HSA or HSA- Alum. All the antigens were inoculated at the dose of 10 ⁇ g protein weight of HSA. 6 and 5 days following first (dotted bar) and second (closed bar) boosting immunization, respectively, blood samples were taken and serum total IgE was measured. Results shown are the mean + 1 SE of four individual serum.
  • Figure 1(b) Course of serum total IgE following JP immunization.
  • mice were JP immunized with saline (open square), HSA (lO ⁇ g; open triangle) or LNnT-HSA (10 ⁇ g of HSA; closed circle) at day 0. Two weeks later, the boosting immunization was followed in a same fashion. Blood was taken at day 10, 20, 27, 41, and 69, and serum total IgE was determined. Results shown are the mean + 1 SE of four individual serum.
  • Figure 1(c) Serum IgG isotypes 5 days following third JP immunization with saline (dotted bar), HSA (open bar), or LNnT-HSA (closed bar). Results shown are the mean + 1 SE of four individual serum. All the results are representative of three experiments.
  • Figures 2a-2d Antigen-specific Ab production following JP immunization with multivalent sugars.
  • Female BALB/C mice were immunized and bled as described in Fig. 1.
  • HSA-specific IgE Figure 2a
  • HSA-specific IgG Figure 2b
  • LNnT-HSA-specific IgE Figure 2c
  • LNnT-HSA-specific IgG Figure 2d
  • Specific IgE were determined as the absorbance at 450 nm of sera diluted 4 times.
  • Specific IgG were determined as the endpoint titer. Results shown are the mean + 1 SE of four individual serum. Results are representative of three experiments. - 5 - HUI-038CPPC
  • Figures 3a-3d Serum total IgE in CBA/J ( Figure 3a) and C57BL/6 ( Figure 3b) mice following JP immunization with saline, HSA or LNnT-HSA. 6 and 5 days following first (dotted bar) and second (closed bar) boosting immunization, respectively, blood samples were taken and serum total IgE were determined. Results shown are the mean + 1 SE of four individual serum.
  • Figure 3c Course of serum total IgE following SC immunization.
  • BALB/C mice were SC immunized with saline (open square), HSA (10 ⁇ g; open triangle) or LNnT-HSA (10 ⁇ g of HSA; closed circle) at day 0. Two weeks later, the boosting immunization was followed in a same fashion. Blood was taken at day 10, 20, 27, 41, and 69, and serum total IgE was determined. Results shown are the mean + 1 SE of four individual serum.
  • Figure 3D Serum total IgE in CBA/CaJ (open bar) and CBA/CaJ xid (hatched bar) following second boosting JP immunization with saline, HSA or LNnT-HSA. Results shown are the mean + 1 SE of four individual serum. Results are representative of two experiments.
  • FIG. 4 Proliferative responses of splenocytes.
  • BALB/C mice were JP immunized with saline, HSA ( 10 ⁇ g) or LNnT-HSA ( 10 ⁇ g weight of HSA). 2 and 3 weeks later, boosting immunization was done in the same fashion. 5 days following the final immunization, spleens were removed. 2.5 x lO 0 ⁇ splenocytes were stimulated with ConA (2 ⁇ g/ml; dotted bar), HSA (lO ⁇ g/ml; hatched bar), LNnT-HSA ( lO ⁇ g/ml of HSA; closed bar) or no restimulation (open bar). Results shown are the mean cpm (experimental-medium control) + 1 SE of four individual mice per group. Results are representative of three experiments.
  • Figures 5a-5h B7-1 and B7-2 expression on B220+ cells.
  • Splenocytes from mice JP immunized with saline Figures 5a, 5d
  • HSA Figures 5b, 5e
  • LNnT-HSA Figures 5c, 5f
  • Cell pellets were stained with PE- conjugated mAb against either B7-1 ( Figures 5a, 5b, 5c) or B7-2 ( Figures 5d, 5e, 5f). Numbers expressed in upper right quadrant show the mean + 1SE of percentage of cells expressing both B220 and B7 molecules from six individual sample.
  • Figures 5g, 5h Effect - 6 - HUI-038CPPC
  • FIG. 6 Levels of total IgE (ng/ml) in mice treated two (open bars) or three (closed bars) times intraperitoneally with the LNnT-dextran conjugate LNnT35.
  • Figure 7 Levels of total IgE (ng/ml) in mice treated first with RMPI (saline), ovalbumin, the LNnT-dextran conjugate LNnT45, or dextran followed by booster treatment with ovalbumin.
  • Figure 8 Levels of ovalbumin-specific IgE in mice treated first with RMPI (saline), ovalbumin, the LNnT-dextran conjugate LNnT45, or dextran followed by booster treatment with ovalbumin.
  • Figure 9 Levels of total IgE (ng/ml) over time (up to 70 days post-immunization) in mice treated with either saline, HSA or LNnT-HSA.
  • Figures lOa-lOc Levels of Th2-type cytokine production by total spleen cells of mice immunized with vehicle (dextran) or LNnT-dextran conjugate.
  • Figure 10a shows JL-13 levels (pg/ml).
  • Figure 10b shows JL-4 levels (pg/ml).
  • Figure 10c shows JL-10 levels (pg/ml).
  • Figure 11 Level of production of the Thl -type cytokine interferon-gamma (pg/ml) by total spleen cells of mice immunized with vehicle (dextran) or LNnT-dextran conjugate. - 7 - HUI-038CPPC
  • Figure 12 Proliferative responses of total spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with LPS in vitro.
  • Figure 13 Level of production of the Thl -type cytokine interferon-gamma (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A in vitro.
  • Figure 14 Level of production of the Thl-type cytokine JL-12 (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with LPS or LNnT-dextran in vitro.
  • Figure 15 Level of production of the Th2-type cytokine JL-13 (pg/ml) by spleen cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A or LNnT-dextran in vitro.
  • Figure 16 Level of production of the Th2-type cytokine JL-13 (pg/ml) by CD4+ cells from mice treated with vehicle (dextran) or LNnT-dextran conjugate in vivo, followed by stimulation of harvested spleen cells with Con A in vitro.
  • FIG 17 Proliferative responses of naive spleen cells stimulated with anti-CD3 in the presence of peritoneal exudate cells (PECs) from mice treated with vehicle (Dex) or LNnT-dex conjugate (LNnT) or in the presence of PECs from LNnT-dex injected mice which had the Grl+ cells removed from the PEC population (LNnT(-)).
  • PECs peritoneal exudate cells
  • FIG. 18 Proliferative responses of CD4+ cells from Balb/c mice stimulated with anti-CD3 in the presence of peritoneal exudate cells (PECs) from mice treated with vehicle (Dex), saline (control), or LNnT-dex conjugate (LNnT) or Grl+ enriched PECs (Grl+) from LNnT-dex injected mice.
  • PECs peritoneal exudate cells
  • CD4 cells stimulated with plate-bound anti-CD3/CD28 antibodies, (a) PECs from C57BL/6
  • mice were obtained 2h or (b) from BALB/c mice 18h post-injection of LNnT-Dex or dextran, co-cultured at different ratios with 1X10 5 previously stimulated naive CD4 + cells. 72 h later
  • H-Thymidine was added to the cultures and after 18h cells were harvested and processed for
  • CD4 naive cells (a) or placed in a separate transwell plate separated by a 0.4 ⁇ m membrane
  • FIG. 23 PECs recruited by LNnT-Dex modify the cytokine profile of CD4 + cells
  • dextran were co-cultured with CD4 + naive cells, after 72h the J-FN- ⁇ (a) and IL-13 production
  • CD4 naive cells were previously stimulated with plate
  • mice and show mean + SE of triplicate cultures of 4 mice assayed individually. *p ⁇ 0.05
  • peritoneal cells Twenty hours after being injected with Dextran or LNFPJH-Dex, peritoneal cells were
  • CD4 + T cells CD4 + T cells.
  • Naive splenocytes were labeled with CFSE dye (as in Materials and Methods)
  • FIG. 28 Nitric oxide (NO) production is associated with suppression of splenocyte - 11 - HUI-038CPPC proliferation by PECs from LNFPUI-Dex injected mice.
  • mice Histograms show CFSE fluorescence intensity in T cells co-cultured with PECs from
  • mice left hand panel
  • LNFPUI-Dex injected mice center
  • LNFPJU-Dex left hand panel
  • This invention provides immunomodulatory methods in which a cell (e.g., a human immune cell) is contacted with an agent which modulates an immune response (e.g., nonspecific polyclonal IgE production, immune cell mitogenesis, or production by the cell of one or more cytokines).
  • a cell e.g., a human immune cell
  • an agent which modulates an immune response e.g., nonspecific polyclonal IgE production, immune cell mitogenesis, or production by the cell of one or more cytokines.
  • the invention is based, at least in part, on the discovery that when animals are immunized with multivalent lacto-N-neotetraose (LNnT), a carbohydrate that is putatively expressed on helminth parasite Schistosoma mansosi, both BALB/C and CB A J mice produced significantly higher amounts of total serum IgE following two intraperitoneal (JP) immunizations with multivalent LNnT conjugated to human serum albumin (LNnT- HSA) compared to groups immunized with saline or HSA alone.
  • LNnT lacto-N-neotetraose
  • HSA human serum albumin
  • LNnT conjugated to dextran also stimulates polyclonal IgE responses and that pretreatment with multivalent LNnT (e.g., LNnT conjugated to dextran), prior to immunization with an antigen, inhibits the production of antigen-specific IgE responses.
  • multivalent LNnT e.g., LNnT conjugated to dextran
  • the enhanced levels of polyclonal IgE stimulated by multivalent LNnT are persistent (e.g., sustained for at least 70 days).
  • JL-4 the - 13 - HUI-038CPPC
  • Th2-type cytokines JL-10 and IL-13 is stimulated by treatment with LNnT conjugated to dextran, whereas levels of the Thl-type cytokine interferon gamma are inhibited by treatment with LNnT conjugated to dextran.
  • the methods of the invention allow for IgE production to be modulated (e.g., stimulation of non-specific IgE and/or inhibition of antigen-specific IgE), as well as allowing for modulation of cytokine production.
  • the immunomodulatory methods of the invention allow for an immune response to be biased towards a specific cytokine secretion profile, for example, a Th2 response.
  • the ability to influence the production of non-specific, polyclonal IgE using the immunomodulatory methods of the invention can be used in the prevention of detrimental host reaction against parasite infection and is further applicable to the protection against environmental allergens by saturating Fc ⁇ Rs on effector cells.
  • the ability to influence the development of, for example, a Th2 response using the immunomodulatory methods of the invention is applicable to the treatment of a wide variety of disorders, including cancer, infectious diseases (e.g., HJN and tuberculosis), allergies and autoimmune diseases.
  • multivalent L ⁇ nT is the treatment or prevention of shock.
  • administration of multivalent L ⁇ nT e.g., L ⁇ nT conjugated to dextran
  • LPS type 1 cytokines
  • type 1 cytokines such as JL-12 and J-F ⁇ - ⁇ .
  • multivalent L ⁇ nT treatment can be used to inhibit the shock response in a patient, either in a patient suffering from shock or, more preferably, in a patient at risk of (susceptible to) shock, such as a surgery patient who is susceptible to shock.
  • a multivalent L ⁇ nT composition of the invention for at risk patients, these patients can be pretreated with a multivalent L ⁇ nT composition of the invention to render them less susceptible to shock.
  • Yet another aspect of the invention pertains to methods for inhibiting the induction of suppressor cells, in particular a population of Grl+, CDl lb+ suppressor cells, by blocking their induction using monovalent (free, unconjugated) L ⁇ nT.
  • administration of multivalent L ⁇ nT induces this suppressor cell population, which is capable of inhibiting T cell proliferative responses.
  • This suppressor cell population may be induced - 14 - HUI-038CPPC
  • the induction of such a suppressor population could promote tumor growth and expansion, by suppression of immune responses against the tumor and/or by production by the suppressor population of factors (such as TGF- ⁇ ) that promote tumor angiogenesis. Accordingly, inhibition of the induction of this suppressor population can be used in the treatment of cancer, by adminstration of monovalent LNnT to thereby competitively block the induction of the suppressor population.
  • factors such as TGF- ⁇
  • LNnT lacto-N-neotetraose
  • multivalent lacto-N-neotetraose (multivalent LNnT) is intended to refer to a form of LNnT comprising multiple moieties of the carbohydrate, such as a form in which multiple LNnT carbohydrates are conjugated to a carrier molecule.
  • monovalent lacto-N-neotetraose (monovalent LNnT) is intended to refer to a form of LNnT comprising a single moiety of the carbohydrate, such as a free, unconjugated form of the sugar.
  • the term "agent comprising LNnT” is intended to refer to a molecule or molecules that includes the LNnT carbohydrate moiety.
  • the agent comprises LNnT with the proviso that the agent is not an antigen from S. mansoni, or an antigen from Toxocara canis, or is not Ascaris body fluid, or is not a filarial protein capable of inducing polyclonal IgE.
  • LewisY oligosaccharide refers to a lacto type U carbohydrate comprising the structure: ⁇ Fuc( l-2)Gal( ⁇ l-4)[Fuc( ⁇ l-3)]GlcNac ⁇ .
  • human immune cell is intended to include cells of the human immune system which are capable of producing cytokines.
  • human immune cells include human T cells, human macrophages and human B cells. - 15 - HUI-038CPPC
  • T cell i.e., T lymphocyte
  • T lymphocyte is intended to include all cells within the T cell lineage, including thymocytes, immature T cells, mature T cells and the like, from a mammal (e.g., human or mouse).
  • Th2 response refers to a response by CD4+ T cells that is characterized by the production of one or more cytokines selected from IL-4, JL-5, IL-6 and JL-10, and that is associated with efficient B cell "help” provided by the Th2 cells (e.g., enhanced IgGl and/or IgE production).
  • a cytokine that regulates development of a Th2 response is intended to include cytokines that have an effect on the initiation and/or progression of a Th2 response, in particular, cytokines that promote the development of a Th2 response.
  • Preferred cytokines that are produced by the methods of the invention are JL-4, JL-5 and J-L-10.
  • the various forms of the term “modulation” are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • the term “contacting” i.e., contacting an agent with a cell
  • incubating the agent and the cell together in vitro e.g., adding the agent to cells in culture
  • administering the agent to a subject such that the agent and cells of the subject are contacted in vivo.
  • a cell e.g., a human immune cell, macrophage or T cell
  • an agent such that an immune response is modulated.
  • the agent itself comprises LNnT, as described in further detail below.
  • the agent stimulates production by the cell of at least one cytokine (e.g., a cytokine that regulates development of a Th2 response).
  • the agent stimulates production of IL-4.
  • the agent stimulates cellular proliferation (e.g., B cell proliferation).
  • the agent stimulates production of non-specific polyclonal IgE.
  • the agents of the invention stimulate cytokine production by cells, stimulate production of non-specific polyclonal IgE by cells, and/or stimulate proliferation of cells.
  • the agent is a stimulatory form of a compound comprising LNnT.
  • a "stimulatory form of a compound comprising LNnT" typically is one in which the carbohydrate structure (e.g., the LNnT) is present in a multivalent, crosslinked form.
  • the stimulatory form of a compound comprising LNnT is a conjugate of a carrier molecule and multiple carbohydrate molecules (e.g., the LNnT).
  • carbohydrate molecules can be conjugated to a protein carrier, such as a conjugate of human serum albumin (HSA).
  • HSA human serum albumin
  • the carrier protein should be selected such that an immunological reaction to the carrier protein is not stimulated in the subject (e.g., a human carrier protein should be used with a human subject, etc).
  • multivalent LNnT can be conjugated to other carrier molecules, for example carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. Other preferred carriers include polymers, such as carbohydrate or polysaccharide polymers. A preferred carbohydrate polymer is dextran. - 17 - HUI-038CPPC
  • the degree of stimulatory ability of the conjugate is influenced by the density of sugars conjugated to the carrier.
  • the sugar molecules comprise at least 10% of the conjugate by weight, more preferably at least 15% of the conjugate by weight, even more preferably at least 20% of the conjugate by weight and even more preferably at least 25% of the conjugate by weight or at least 30% of the conjugate by weight or at least 35% of the conjugate by weight or at least 40% of the conjugate by weight or at least 45% of the conjugate by weight.
  • the sugar molecules comprise about 10-25 % of the conjugate by weight, about 15-25% of the conjugate by weight or about 20-25% of the conjugate by weight or about 30-35% by weight or about 35-40% by weight or about 40-45% by weight.
  • the stimulatory form of a compound comprising LNnT is a conjugate of multiple carbohydrate molecules. More preferably, the conjugates comprise 10-11, 12-13, 14-15, 6-17, 18-19, or 20 or more sugars/conjugate.
  • Agents for use in the methods of the invention can be purchased commercially or can be purified or synthesized by standard methods. Conjugates of LNnT and a carrier protein (e.g., HSA) are available from Accurate Chemical and Scientific Corporation, Westbury, NY.
  • conjugates comprising LNnT described above another form of a stimulatory agent comprising LNnT is an isolated protein that naturally expresses LNnT in a form suitable for stimulatory activity.
  • an agent of the invention to stimulate production by immune cells of a cytokine can be evaluated using an in vitro culture system such as that described in the Examples.
  • Cells are cultured in the presence of the agent to be evaluated in a medium suitable for culture of the chosen cells. After a period of time (e.g., 24, 48, 72, or 120 hours), production of the cytokine is assessed by determining the level of the cytokine in the culture supernatant.
  • the cytokine assayed is IL-4.
  • IL-2, JL- 5, JL-10, JL-13 and/or JPN- ⁇ levels can be assessed.
  • Cytokine levels in the culture supernatant can be measured by standard methods, such as by an enzyme linked immunosorbent assay (ELISA) utilizing a monoclonal antibody that specifically binds the cytokine.
  • ELISA enzyme linked immunosorbent assay
  • the ability of the agent to stimulate cytokine production is evidenced by a higher level of cytokine (e.g., JL-4) in the supernatants of cells cultured in the presence of the agent - 18 - HUI-038CPPC
  • an agent of the invention to stimulate production of non-specific polyclonal IgE by cells can be evaluated in vitro utilizing methods such as those described in the Examples.
  • serum isolated from a subject can be analyzed by sandwich ELISA for the presence of total, as well as antigen-specific, IgE. Briefly, plates are coated with anti-IgE antibodies, washed extensively, blocked to prevent non-specific adsorption of reagents to the plate, then incubated with serum samples isolated from subjects.
  • Labeled antibody e.g., biotinylated anti-IgE antibody
  • the reactions can be subsequently developed using, for example, tetramethyl-benzidine substrate.
  • Such methods are further useful for detection of, for example, Ag-specific IgG, HSA-specific IgE, LNnT-HS A-specific IgE, as well as specific IgG subtypes, by altering the specificity of the primary antibody (e.g., that used in initial coating of the plate).
  • the ability of an agent of the invention to stimulate proliferation of cells e.g., proliferation responses
  • spleen cells can be isolated from sacrificed mice, cultured in vitro in appropriate culture medium, and labeled with ⁇ H thymidine as an indicator of DNA replication.
  • the inhibitory agents of the invention can inhibit induction of a Grl+, CDl lb+ suppressor cell population that recruited by multivalent LNnT treatment.
  • the agent is an inhibitory form of a compound comprising LNnT.
  • An "inhibitory form of a compound comprising LNnT" typically is one in which the carbohydrate structure (e.g., the LNnT) is present in a monovalent, non-crosslinked form.
  • LNnT is commercially available (e.g., as a custom order from GlycoTech, Rockville MD).
  • compositions of the agents e.g., stimulatory agents
  • the pharmaceutical compositions of the invention typically comprise an agent of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the type of carrier can be selected based upon the intended route of administration. I-n various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, transdermal or oral administration. In a preferred embodiment, the composition is formulated such that it is suitable for intraperitoneal administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • the modulators can be administered in a time release formulation, for example in a - 20 - HUI-038CPPC
  • composition which includes a slow release polymer.
  • the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the agent may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent.
  • the agent can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan, et al., (1984) J.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the active agent in the composition i.e., a stimulatory or inhibitory agent of the invention
  • a stimulatory or inhibitory agent of the invention preferably is formulated in the composition in a therapeutically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as the production of sufficient levels of non-specific polyclonal IgE to thereby influence the therapeutic course of a particular disease state.
  • a therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • the active agent is formulated in the composition in a prophylactically effective amount.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, for example, influencing the production of sufficient levels of non-specific polyclonal IgE for prophylactic purposes. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • a non-limiting range for a therapeutically or prophylactically effective amounts of a stimulatory or inhibitory agent of the invention is 0.01 nM-20 mM.
  • a stimulatory or inhibitory agent can be used in an amount between 500 ⁇ g to 100 mgs.
  • dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • the amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. - 22 - HUI-038CPPC
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • An agent of the invention can be formulated into a pharmaceutical composition wherein the agent is the only active compound therein.
  • the pharmaceutical composition can contain additional active compounds.
  • two or more agents may be used in combination.
  • an agent of the invention can be combined with one or more other agents that have immunomodulatory properties.
  • a stimulatory agent may be combined with one or more cytokines or adjuvants.
  • a pharmaceutical composition of the invention comprising a stimulatory or inhibitory agent of the invention, can be administered to a subject to modulate immune responses (e.g., production of non-specific polyclonal IgE) in the subject.
  • immune responses e.g., production of non-specific polyclonal IgE
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • a pharmaceutical composition of the invention can be formulated to be suitable for a particular route of administration.
  • a pharmaceutical composition of the invention can be suitable for injection, inhalation or insufflation (either through the mouth or the nose), or for intranasal, mucosal, oral, buccal, parenteral, rectal, intramuscular, intravenous, intraperitoneal, and subcutaneous delivery.
  • a pharmaceutical composition of the invention can be packaged with instructions for using the pharmaceutical composition for a particular purpose, - 23 - HUI-038CPPC
  • the invention provides immunomodulatory methods that can be used modulate various immune responses.
  • a cell is contacted with an agent (e.g., an agent comprising LNnT) with the cell such that the immune response is modulated (e.g.., stimulated).
  • an agent e.g., an agent comprising LNnT
  • the methods of the invention can be practiced either in vitro or in vivo.
  • cells can be obtained from a subject by standard methods and incubated (i.e., cultured) in vitro with an agent of the invention to modulate, for example, the production of a cytokine, the production of non-specific, polyclonal IgE, proliferation of an immune cell (e.g., a splenocyte), or the development of a Th2 response.
  • an agent of the invention to modulate, for example, the production of a cytokine, the production of non-specific, polyclonal IgE, proliferation of an immune cell (e.g., a splenocyte), or the development of a Th2 response.
  • PBMCs peripheral blood mononuclear cells
  • Specific cell populations can be depleted or enriched using standard methods.
  • monocytes/macrophages can be isolated by adherence on plastic.
  • T cells or B cells can be enriched or depleted, for example, by positive and/or negative selection using antibodies to T cell or B cell surface markers, for example by incubating cells with a specific mouse monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using anti- mouse-Ig coated magnetic beads.
  • mAb mouse monoclonal antibody
  • Monoclonal antibodies to cell surface markers are commercially available.
  • an agent is administered to a subject in a pharmacologically acceptable carrier (as described in the previous section) in amounts sufficient to achieve the desired effect, such as to modulate, for example, the production of a cytokine, the production of non-specific, polyclonal IgE, proliferation of an immune cell, or the development of a Th2 response in the subject or to prevent a detrimental host reaction against parasite infection or to protect against environmental allergens by saturating Fc ⁇ Rs on effector cells in the subject or to inhibit a disease or disorder (e.g., an allergy or an autoimmune disease) in the subject.
  • a pharmacologically acceptable carrier as described in the previous section
  • One preferred route of administration for the agent is intraperitoneal. Another preferred route of administration is orally. Yet another preferred route of administration is intravenous.
  • Application of the methods of the invention to the treatment of disease conditions may result in cure of the condition, a decrease in the type or number of symptoms associated with the condition, either in the long term or short term (Le., amelioration of the condition) or simply a transient beneficial effect to the subject.
  • the methods of the invention can be used to stimulate production cytokines (such as JL-4) in vitro for commercial production of these cytokines (e.g., cells can be cultured with a stimulatory agent in vitro to stimulate IL-4 production and the IL-4 can be recovered from the culture supernatant, further purified if necessary, and packaged for commercial use).
  • cytokines such as JL-4
  • cells can be cultured with a stimulatory agent in vitro to stimulate IL-4 production and the IL-4 can be recovered from the culture supernatant, further purified if necessary, and packaged for commercial use.
  • Another aspect of the invention pertains to use of a stimulatory agent of the invention in the treatment or prevention of shock in a subject.
  • administration of multivalent LNnT e.g., LNnT conjugated to dextran
  • LNnT e.g., LNnT conjugated to dextran
  • type 1 cytokines such as JL-12 and J-FN- ⁇ .
  • multivalent LNnT treatment can be used to inhibit the shock response in a patient.
  • these patients can be pretreated with a multivalent LNnT composition of the invention to render them less susceptible to shock.
  • the invention provides a method for inhibiting or preventing shock in a subject comprising administering an agent comprising multivalent lacto-N-neotetraose (LNnT), such that shock is inhibited or prevented in the subject.
  • the subject may be a patient already - 25 - HUI-038CPPC
  • the multivalent LNnT agent can be administered to the patient at least 1 hour prior to a time when shock may develop in the patient or at least 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours or more prior to a time when shock may develop in the patient.
  • Yet another aspect of the invention pertains to a method of inhibiting induction of Grl+, CDl lb+ suppressor cells in a subject.
  • This method can be used to inhibit induction of the suppressor cells in a clinical setting where such inhibition is desirable, such as in the treatment of cancer.
  • the metho comprises: administering to the subject an agent comprising monovalent lacto-N-neotetraose (LNnT), such that induction of Grl+, CDllb+ suppressor cells in the subject is inhibited.
  • LNnT monovalent lacto-N-neotetraose
  • administration of multivalent LNnT induces this suppressor cell population, which is capable of inhibiting T cell proliferative responses.
  • This suppressor cell population may be induced in a clinical setting in a patient by tumor cells that express multivalent LNnT, and the induction of such a suppressor population could promote tumor growth and expansion, by suppression of immune responses against the tumor and/or by production by the suppressor population of factors (such as TGF- ⁇ ) that promote tumor angiogenesis. Accordingly, inhibition of the induction of this suppressor population can be used in the treatment of cancer, by adminstration of an agent comprising monovalent LNnT to thereby competitively block the induction of the suppressor population.
  • the agent is administered intraperitoneally. I-n another embodiment, the agent is administered intravenously.
  • the method for inhibiting induction of the suppressor population is carried out in a subject suffering from cancer.
  • the stimulatory methods of the invention i.e., methods using the stimulatory agents of the invention
  • autoimmune diseases that are associated with Thl -type dysfunction
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and that promote the production of cytokines and autoantibodies involved - 26 - HUI-038CPPC in the pathology of the diseases.
  • modulation of T helper-type responses can either have a beneficial or detrimental effect on an autoimmune disease.
  • EAE experimental allergic encephalomyelitis
  • stimulation of a Th2-type response by administration of IL-4 at the time of the induction of the disease diminishes the intensity of the autoimmune disease (Paul, W.E., et al. (1994) Cell 76:241-251).
  • Th2-specific cytokines Koury, S. J., et al. (1992) J. Exp. Med. 176:1355-1364.
  • T cells that can suppress EAE secrete Th2-specific cytokines (Chen, C, et al. (1994) Immunity 1: 147-154). Since stimulation of a Th2-type response in EAE has a protective effect against the disease, stimulation of a Th2 response (and/or downmodulation of a Thl response) in subjects with multiple sclerosis (for which EAE is a model) may be beneficial therapeutically.
  • RA rheumatoid arthritis
  • a stimulatory agent of the invention e.g., an agent comprising multivalent LNnT
  • a stimulatory agent of the invention can be administered to the subject, for a variety of therapeutically beneficial purposes, including downmodulating the production of the Thl- associated cytokines IL-12 and J-FN- ⁇ , and induction of Grl+, CDl lb+ suppressor cells.
  • the stimulatory agent can be used alone, or in combination with one or more additional agents that promote Th2 responses (e.g., Th2-promoting cytokines, such as JL-4 or JL-10), and/or downmodulate Thl responses (e.g., antibodies to Thl-promoting cytokines such as anti-IL-2, anti-IL-12, anti-JEN- ⁇ ).
  • the stimulatory agent may be administered - 27 - HUI-038CPPC either systemically or locally.
  • the agent may be administered directly into the joints.
  • the stimulatory agent preferably is administered intravenously.
  • autoimmune diseases may be treated by an ex vivo approach.
  • cells e.g., T cells, macrophages, B cells, peritoneal exudate cells
  • a stimulatory agent of the invention for example, to stimulate generation of the Grl+, CDl lb+ suppressor cell population and/or to inhibit production of Thl -associated cytokines (e.g., JL-12, IFN- ⁇ ) and/or to stimulate production of Th2-associated cytokines (e.g., IL-13), followed by readministration of the cells to the subject.
  • Thl -associated cytokines e.g., JL-12, IFN- ⁇
  • Th2-associated cytokines e.g., IL-13
  • Non-limiting examples of autoimmune diseases and disorders having an autoimmune component that may be treated according to the invention include diabetes mellitus, inflammatory bowel disease, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sj ⁇ gren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vag
  • the invention also provides pharmaceutical compositions for carrying out the methods of the invention.
  • the invention provides a pharmaceutical composition comprising an agent comprising multivalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition as a modulator of IgE responses in a subject.
  • LNnT multivalent lacto-N-neotetraose
  • a pharmaceutical carrier packaged with instructions for use of the pharmaceutical composition as a modulator of IgE responses in a subject.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent comprising multivalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment or prevention of shock in a subject.
  • the agent can comprise LNnT conjugated to a protein carrier, such as human serum albumin, or LNnT conjugated to a carbohydrate polymer, such as dextran.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent comprising monovalent lacto-N-neotetraose (LNnT) and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition for the treatment of cancer in a subject.
  • LNnT monovalent lacto-N-neotetraose
  • mice Young adult (7-9 weeks old) CBA/J, BALB/C, and C57BL/6 strain female mice were purchased from Harlan (Indianapolis, IN). Female CBA/CaJ xid and age-matched control female CBA CaJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). JL-4 deficient BALB/C mice were generated as described (). This deficient mice were bred and maintained at Harvard School of Public Health according to the guidelines set forth by the Harvard Medical Area Research Committee. Antigens and inoculations
  • HSA Human serum albumin
  • Multivalent LNnT or Lewis ⁇ were conjugated with HSA (LNnT-HSA and LeY-HS A) by Accurate Chemical and Scientific Corporation (NY). In both neoglycoproteins, 13 molecules - 29 - HUI-038CPPC
  • HSA Sigma Chemical Co., MO
  • HSA adsorbed to alum Intergen Company, NY; HSA-alum
  • Dulbecco's PBS Gibco BRL, NY
  • Groups of four to six mice were immunized intraperitoneally or subcutaneously with Ags (lO ⁇ g of HSA) or saline.
  • First and second boosting immunization were performed in the same fashion 2 and 3 weeks later, respectively.
  • Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, JL).
  • Immunized mice were bled from the tail 10, 6, and 5 days following primary, first, and second boosting immunization, respectively.
  • Total and HSA-specific IgE were determined by sandwich ELISA.
  • ELISA plates (Corning Inc., NY) were coated overnight at 4°C with 100 ⁇ l of 5 ⁇ g/ml rat anti -mouse IgE mAb (Biosource, CA) in carbonate-bicarbonate buffer, pH 9.6. After washing four times with PBS containing 0.05% Tween20 (PBS-T), plates were blocked with 200 ⁇ l PBS containing 10% FCS and 0.3% Tween20 for 2 hrs at 37°C.
  • PBS-T PBS containing 0.05% Tween20
  • Ag-specific IgG ELISA were determined. Briefly, ELISA plates were coated with lOO ⁇ l Ag (2 ⁇ g/ml) overnight at 4°C in carbonate-bicarbonate buffer, and blocked as described above. Then plates were incubated with samples from individual serum in two-fold serial dilution from 100 times for 2 hrs at 37°C, followed by goat anti-mouse IgG mAb- peroxidase conjugate (Boehringer-Mannheim, J- ) for 1 hr at 37°C. Thereafter, plates were developed and terminated as described above. Finally the absorbance at 450 nm was measured using a UNMax automatic microplate reader.
  • Results were expressed as endpoint titers where the endpoint was determined as the final serum dilution which yields a higher absorbance than twice of the background absorbance.
  • Optimum dilutions of anti-mouse IgG mAb-horseradish peroxidase-labeled conjugates were determined to be 1/1000.
  • Plasma IgE specific for L ⁇ nT-HS A was also tested for in the same fashion as this method using L ⁇ nT- HSA (2 ⁇ g/ml) as a coating Ag and biotinylated anti-mouse IgEmAb (1 ⁇ g/ml, Pharmingen) followed by avidin-peroxidase conjugate (Sigma) as a detection Ab.
  • Serum total IgG isotypes were also determined by ELISA. Plates were coated with
  • JL-5 (0 to 5,000 pg/ml), JL-10 (0 to 25,000 pg/ml), JPN- ⁇ (0 to 20,000 pg/ml) (Pharmingen, CA) and EL- 4 (0 to 1,500 pg/ml) (Endogen, MA) were used in duplicate. Following overnight incubation at 4°C, the wells were washed and appropriate 50 ⁇ l/well biotinylated detection Ab at l ⁇ g/ml (rat anti-mouse IL-5, EL- 10 or JPN- ⁇ from Pharmingin and rat anti-mouse JL-4 from Endogen) were added.
  • splenocytes were resuspended and incubated on ice for 15 min with mAbs as follows: anti-CD45R/B220 (RA3-6B2), anti-B7-l (16-lOA 1), and anti-B7-2 (GL-1) or isotype-matched controls. All mAbs were either FJTC- or PE-conjugated (Pharmingen). After washing with Hanks' balanced salt solution (Gibco) containing 0.05% sodium azide, flow cytometry analysis was performed by using a FACSCalibur flow cytometer and Cell Quest software (Becton Dickinson, CA). Dead cells were excluded from analysis on the basis of propidium iodide (Molecular Probes, OR) staining. Lymphocytes were gated according to the physical characteristics of forward and side scatter and at least 10,000 events were acquired. Statistics
  • mice Following first boosting JP immunization with LNnT-HSA, BALB/C mice produced significantly higher amounts of serum total IgE than those JP immunized with saline, HSA, LeY-HSA, and HSA- Alum (Fig. la). Serum IgE was significantly elevated following second JP immunization. This elevation was not seen following primary inoculation, however, high elevation of serum IgE lasted at least 8 weeks following second JP immunization with LNnT (Fig. lb). Serum IgGl was also significantly increased in mice JP immunized with multivalent LNnT compared with those immunized with saline or HSA alone, whereas the amount of other isotypes were not significantly different (Fig. Ic).
  • HSA-specific IgE and IgG were determined in sera in mice immunized JP with HS A- Alum. On the contrary, those signals in mice immunized IP with LNnT-HSA were not detected and were statistically no different from the control groups immunized with HSA - 33 - HUI-038CPPC
  • Splenocytes of BALB/C mice were prepared 5 days following second boosting JP immunization with saline, HSA or LNnT-HSA.
  • Mice JP immunized with HSA or saline showed moderate but significant in vitro proliferative responses to LNnT-HSA compared to HSA or unrestimulation, suggesting that LNnT induces the proliferative responses in naive splenocytes.
  • Mice immunized JP with LNnT-HSA showed the significant responses even without restimulation compared to the control mice (Fig.4).
  • mice immunized JP with LNnT-HSA produced significantly more U- , JL-5, and JL-10, but not IFN- ⁇ compared to those JP immunized with saline or HSA, suggesting that LNnT skewed splenocytes into polarized Th2 responses in response to ConA stimulation.
  • B7-2 (CD86) expression on B220+ cells in LNnT-inoculated mice
  • B7-1 and B7-2 on B220 positive splenocytes were also investigated.
  • B7-1 expression was not altered in cultured splenocytes (Fig. 5a,c,e). However, following 24 hrs culture incubation without restimulation in vitro, B220 positive cells expressing B7-2 molecule were increased in mice JP immunized with LNnT-HSA compared to the control mice immunized with saline or HSA (Fig. 5b.d.f). This increase was found following 6 hr - 36 - HUI-038CPPC
  • IL-4 is required for the induction of polyclonal IgE production by multivalent LNnT Because Th-2 cytokines, especially JL-4, are involved with IgE production, the role of
  • IL-4 for the induction of polyclonal IgE production by multivalent LNnT in vivo was investigated using JL-4 gene deficient mice. JL-4 deficient mice did not induce polyclonal IgE production following repeated JP immunization with LNnT-HSA. J-n this experiment, total serum IgE. J-L-4 deficient mice or wild type BALB/.C mice were immunized with saline, HSA or LNnT-HSA. Following second boosting immunization, sera were sampled and serum total IgE was measured.
  • splenocytes from wild type or J-L-4 deficient mice were cultured without additional stimulants and the production of IL-4, IL-5, IL-10, and JEN- ⁇ were measured at 24, 72, and 120 hrs postincubation.
  • splenocytes from JL-4 deficient mice with the immunization of LNnT-HSA produced JL-5, JL-6, and JPN- ⁇ without restimulation.
  • the amounts of IL-5 and IL-6 were significantly lower than wild type BALB/C mice (Fig. 6b,c,d,e).
  • mice were injected intraperitoneally either two or three times with either dextran alone, RPMI media or LNnT conjugated to dextran in saline
  • LNnT-dextran The sugar conjugate was referred to as LNnT35, wherein the 35 refers to the degree of LNnT substitution on each dextran molecule.
  • the total amount of IgE elicited in the mice was determined. The results are shown in Figure 6, wherein the numbers after LNnT (i.e., 200, 100 or 50) refer to the amount injected (dextran weight). The results - 37 - HUI-038CPPC
  • IL-4 induces B cells to develop polyclonal IgE producing cells in mice in vivo and in vitro (Coffman, RL. et al. (1986) J. Immunol, vol. 136 pp. 949-954; Finkelman, FD et al. (1990) Annu. Rev. Immunol, vol. 8 pp. 303-333; Tepper, RI. et al. (1990) Cell vol. 62 pp. 457; Snapper, CM. et al. (1991) J.Immunol, vol. 147 pp. 1163-1170; Nakanishi, K. et al. (1995) Int. Immunol, vol. 7 pp. 259-268).
  • IL-4 derived from IL-4 producing cells such as T cells (including NK1+ CD4+ T cells), eosinophils, and cells of the mast cell/basophil lineage may be required (Coffman, RL. et al. (1997) J. Exp. Med. vol. 185 pp. 373-375; Sabin, EA. et al. (1996) J. Exp. Med. vol. 184 pp. 1871-1878).
  • at least two factors in parasite antigens may be required to induce polyclonal IgE production: B cell mitogenic activity and induction of IL-4 production.
  • nematode products contain a B-cell mitogen that polyclonally activates B cells, which is converted into a polyclonal IgE response when these stimulated B cells come under the influence of IL-4 or an IL-4 like molecule activated by other factors.
  • mice JP immunized with LNnT produced IL-4, JL-5, and JL- 10 without restimulation in vitro, although they also produced detectable amount of IL-2 and JPN- ⁇ .
  • JL-4 deficient mice did not induce polyclonal IgE production following JP immunization with this carbohydrate although they produced significant amount of JL-5, JL- 10, and JFN- ⁇ .
  • Development of IL-5 and JL-10 production following S. mansoni infection was also seen in JL-4 deficient mice (Pearce, EJ. et al. (1996) Int. Immunol, vol. 8 pp. 435- 444; King., CL. et al. (1996) Exp. Parasitol.
  • multivalent LNnT may possess at least two functions, B cell mitogenic activity and induction of JL-4 production, to induce polyclonal IgE production.
  • B cell mitogenic activity and induction of JL-4 production, to induce polyclonal IgE production.
  • JL-4 production to induce polyclonal IgE production.
  • splenocyte from control mice IP immunized with saline also showed the significant proliferative responses against LNnT, suggesting that this carbohydrate possesses the mitogenic activity.
  • CBA J and BALB/C mice produce the polyclonal IgE following second immunization
  • C57BL/6 mice produce it following third immunization.
  • host response against S. mansoni infection is strain dependent.
  • CBA/J, C3H/HeJ, and BALB/C mice developed bigger liver granulomas and higher portal hypertension whereas C57BL/6 mice developed relatively smaller granulomas and lower portal hypertension (Fanning, MM. et al. (1981) J. Inf. Dis. vol. 144 148-153; Hernandez, NJ. et al. (1997) Eur. J. Immunol, vol. 27 pp. 666-670).
  • Immunization with LNnT seems to have a same characteristics as S.
  • peritoneal B-l cell outgrowth due to S. mansoni infection was strain dependent, occurring in CBA/J, C3H HeJ, and BALB/C mice but not in C57BL/6 mice (Palanivel, V. et al. (1996) Exp. Parasitol. vol. 84 pp. 168-177).
  • B-l cell subset is a major source of B cell JL-10 that downregulate Thl responses (Amiri, P. et al. (1992) Nature vol. 356 pp. 604).
  • the present result that peritoneal B-l cells seem to be involve in part in the induction of polyclonal IgE by multivalent LNnT (Fig.2d) is consist with the report.
  • B7-1 and B7-2 costimulatory molecules are ligands for CD28/CTLA-4 and involved in T cell activation, cytokine production, and regulation of tolerance (McKnight, AJ. et al. (1994) J. Immunol, vol. 152 pp. 5220-5225; Perez, VL. et al. (1997) Immunity vol. 6 pp. 411-417). Costimulation by B7-1 and B7-2 can differentially regulate Thl cell differentiation, although the effect of these molecules are dependent on the status of immune reaction, doses and routes of antigen inoculation, types of APC, and the experimental model of diseases (Thompson, CB. (1995). Cell vol. 81 pp. 979- 982).
  • mice were injected intraperitoneally with either media alone (RPMI), vehicle alone (dextran), 50 ⁇ g of LNnT-dextran conjugate (LNnT 50) or 100 ⁇ g of LNnT- dextran conjugate weekly for three weeks.
  • the spleen cells were then harvested from the mice and either total spleen cells or CD4+ cells were stimulated in vitro with ConA or LPS. Following in vitro stimulation, the proliferative and cytokine responses of the cells were measured. The results are illustrated in Figures 12-16.
  • Figure 12 demonstrates that the proliferative response of total spleen cells from the
  • LNnT-dextran treated mice following in vitro LPS stimulation was significantly decreased as compared to mice treated only with vehicle (dextran).
  • Figure 13 demonstrates that interferon- gamma production (after 48 or 72 hours) by total spleen cells from the LNnT-dextran treated mice (treated in vivo with either 200, 100 or 50 ⁇ g of conjugate) following in vitro ConA stimulation was significantly decreased as compared to mice treated only with vehicle (dextran).
  • mice were treated with media alone (RPMI), vehicle (dextran) or LNnT-Dex at either 100 ⁇ g or 50 ⁇ g doses.
  • the spleen cells were taken from these groups of mice and then stimulated in vitro - 43 - HUI-038CPPC
  • Figure 15 demonstrates that JL-13 production by total spleen cells from the LNnT-dextran treated mice following in vitro stimulation with either LNnT-dex or ConA was significantly greater than JL-13 production by mice treated with vehicle (dextran) alone and then stimulated in vitro with LNnT-dex or ConA.
  • Figure 16 demonstrates that IL-13 production by CD4+ cells from the LNnT-dextran treated mice following in vitro stimulation with ConA was significantly greater than JL-13 production by mice treated with vehicle (dextran) alone and then stimulated in vitro with ConA.
  • mice were treated with LNnT-dextran conjugate as described in Example 3 and peritoneal exudate cells (PECs) were recovered and analyzed by FACS analysis to characterize the surface markers expressed on this population of cells.
  • PECs peritoneal exudate cells
  • mice were injected i.p. with either LNnT-dex, dextran alone or saline and two hours later PECs were harvested.
  • Total na ⁇ ve spleen cells were planted on wells coated with anti-CD3 antibodies and then the LNnT-activated PECs were added to the culture.
  • the proliferative response of the na ⁇ ve spleen cells was determined as a measure of their activation by anti-CD3. The results are illustrated in Figures 17 and 18.
  • Figure 17 demonstrates that PECs obtained 2 hours post-injection of LNnT-dex are able to inhibit the proliferative responses of naive spleen cells stimulated with anti-CD3.
  • the data labeled CD3 + PEC LNnT(-) represents PECs from LNnT-dex injected mice which have had the Grl+ cells removed; i.e., this data represents a Grl- population of PECs).
  • Figure 18 demonstrates that anti-CD3 proliferative responses by CD4+ cells from Balb/c mice are inhibited by PECs from LNnT-dex treated mice.
  • the data labeled CD4+ aCD3 + Grl+ represents PECs from LNnT-dex injected mice which have had enriched for Grl+ cells).
  • multivalent LNnT recmits a population of cells that are Grl+, CDl lb+, but that do not require JL-4 or JL-13 for their induction and that are F4/80-, wherein this population of cells has suppressor activity as evidenced by their ability to inhibit anti-CD3 proliferative responses.
  • dextran backbone was supplied by Neose Technologies I-nc (Horsham, PA). The level of
  • LNnT-Dex substitution varied from 21 to 45 LNnT-Dex residues per molecule of dextran.
  • PECs Peritoneal exudate cells
  • peritoneal lavage with 5 ml of ice-cold Hank's balanced salt solution (HBSS, Gibco). PECs were washed 2 times and red blood cells were lysed by hypotonic shock with amonium
  • Viable cells were counted and adjusted to 5xl0 5 cells/ml. Viability measured by
  • PECs were analyzed for surface markers, cytokine production and for suppressor activity in co-cultures with naive CD4 cells. Flow Cytometric Analysis.
  • Live cells were electronically gated using forward and side scatter parameters.
  • PECs were adjusted to a concentration of 5x10 /ml, plated in 24 well plates (Costar) and
  • IL-12 JL-10 (antibodies and cytokines were obtained from Pharmingen), JL-l ⁇ , JL-18, IL-13,
  • TGF- ⁇ obtained from R&D.
  • Splenocytes were prepared from naive cells
  • CD4 + cells were plated in 96 well flat bottom
  • Thymidine (185 GBb/mmol activity, Amersham, England) l ⁇ Ci/well was added and
  • paramagnetic beads MiniMAcs, Miltenyi Biotec
  • goat anti-rat IgG isotype of the anti-Gr-1 antibody
  • PECs were plated on the superior chamber at a
  • naive CD4 + cells were added. Cultures were maintained for 72h, then l ⁇ Ci/well 3 H-
  • CD4 + cells were transferred to a 96 well plate for harvesting as described.
  • LNnT-Dex injection expands a greater number of PECs than injection with control dextran.
  • mice The peritoneal cell response in mice to the polyvalent sugar LNnT-Dex was examined
  • Gr-l+/F4/80+/ CDl lb+ is the predominant cell type expanded by LNnT-Dex.
  • PECs expanded by LNnT-Dex exhibit "Natural Suppressor Cell” activity.
  • Naive CD4 + cells were stimulated by plate-bound anti-CD3 and anti-CD28 antibodies,
  • CD4 + T cells in the absence of PECs, or in the presence of control PECs (saline or dextran)
  • mice tested (BALB/c and C57BL/6) and was characteristic of both early (2h) and
  • Gr-1 4" cells mediate suppression induced by PECs elicited by LNnT-Dex.
  • polyvalent LNnT-Dex recruits PECs that specifically inhibits
  • naive CD4 + cells proliferative response to anti-CD3/CD28 stimulation.
  • Suppression is mediated by both cell to cell contact and by soluble factors.
  • CD3/CD28 antibodies stimulated CD4 + cells (approximately 50% inhibition, Fig 4b, p ⁇ 0.05),
  • PECs expanded by LNnT-Dex secrete a different profile of cytokines than control PECs.
  • the peritoneal cells were havrvested 2h or 18h after
  • PECs was different. Notably PECs expanded by LNnT-Dex produced significantly lower
  • pro-inflammatory cytokines such as IL-l ⁇ , JL-12, JL-18 and JPN- ⁇ than PECs
  • PECs:CD4 + cells were co-cultured as previously described,
  • CD4 + cells were then plated and incubated in RPMI for another 72 h (rested). CD4+ cells
  • LNnT-Dex-PECs are Th2 committed, because the greatest amounts of IL-13 are secreted
  • TGF- ⁇ TGF- ⁇
  • IL-10 was a major regulatory factor involved in the suppressive activity of PECs
  • IL-10 is a potent suppressor of cell mediated immune responses ( involved in
  • IL-10 IL-10 and TGF- ⁇ , which are capable of counterbalancing the pro-
  • CA classically activated
  • IFN- ⁇ dependent Grl + suppressors are found in the bone marrow and peripheral
  • mice Female BALB/c and SCID:SCID mice between 6 and 8 weeks of age were used in
  • LNFPJH-Dex and Dextran were obtained from Neose Technologies Inc., Horsham,
  • the glyco-conjugate consisted of 12 LNFPJU molecules conjugated to a 10 kDa
  • RPMI 1640 medium was supplemented with 10% fetal bovine serum
  • Rat anti-mouse F4/80-Cy5 mAbs was purchased from PharMingen (San Diego, CA). Rat anti-mouse F4/80-Cy5 mAbs was
  • mice were injected intraperitoneally with 50 ⁇ g of LNFPUI-Dex or Dextran in Hanks'
  • HBSS Balanced Salt Solution
  • mice were euthanized by CO 2 inhalation and peritoneal cells (PECs) were
  • Spleen cell preparations were prepared from na ⁇ ve mice. Following lysis of red blood
  • Splenocytes were cultured for three hours on antibody coated
  • L-nMMA, MnTBAP, or anti-IFN- ⁇ mAbs were analyzed by flow cytometry. In some experiments, L-nMMA, MnTBAP, or anti-IFN- ⁇ mAbs
  • C ⁇ -cultured cells or freshly isolated PECs were stained with various combinations of mAbs for 30 min on ice in the dark and washed twice in FACs - 58 - HUI-038CPPC buffer. Acquisition of cells was preformed using a FACScalibur flow cytometer (Becton
  • GrlYF4/80 + PECs in LNFPUI-Dex injected mice is shown in Figure 26B. Analysis of double- positive cells from sugar-injected mice showed two subpopulations characterized as Grl 4"
  • Grl + subpopulations express CDl lb and F4/80
  • succinimidyl ester (CFSE) before stimulation with anti-CD3 and anti-CD28 antibodies
  • CD4 + T cells were gated and analyzed their proliferation.
  • Figure 27 shows data
  • Activated macrophages often suppress T cell activity via nitric oxide (NO)
  • iNOS inducible NO synthase
  • MnTBAP did not have SOD or superoxide dismutase (SOD) mimetic. MnTBAP did not have SOD or superoxide dismutase (SOD) mimetic. MnTBAP did not have SOD or superoxide dismutase (SOD) mimetic. MnTBAP did not have SOD or superoxide dismutase (SOD) mimetic.
  • L-nMMA decreased NO in cell cultures to background level.
  • JFN- ⁇ is a critical factor for development
  • Grl hlgh positive cells are responsible for the suppression of anti-CD3/CD28-induced T cell
  • Grl + cells which also express CDl lb and/or F4/80 (Kusmartev, supra; Cauley,
  • the Grl 4" subpopulation contained 99.7% positive cells. The ability of
  • F4/80 4" cells were isolated using
  • Grl 4" cells were isolated from this same population of F4/80 4" cells by
  • F4/80 + /Grl + double-positive macrophages are responsible for the
  • resident PECs include macrophages, neutrophils and NK cells, as
  • LNFPUI-Dex or dextran were injected into SCUD mice to determine if
  • Table VI compares the percentage Grl + cells of the
  • SCIDs compared to BALB/c may be due to immuno-compensatory mechanisms in SCUD
  • LNFPUI-Dex has not previously been observed. Twenty hours after injection of LNFPUI-Dex
  • EXAMPLE 7 NOD-Model of Insulin-Dependent Diabetes Mellitus
  • mice develop pancreatitis starting around 4 weeks of age.
  • JJDDM J-nsulin-dependent diabetes mellitus
  • mice (100 ug). Eight weeks later, mice were observed for symptoms and blood glucose levels were
  • mice injected with dextran were completely wet, and the bedding soaked,
  • mice are shown in Table NU.
  • symptoms occur from 8-12 weeks post transfer.
  • mice Dex or Dextran, followed by weekly injections. Experiments were terminated when mice
  • LNnT or Dextran in BALB/c or C57BL/6 mice- PECs were obtained at 2h or 18h after
  • Nitric oxide (NO) production ( ⁇ M) by co-cultures of CD3/CD28 stimulated naive splenocytes and PECs
  • Na ⁇ ve splenocytes were preincubated for 3 hours with anti-CD3/CD28 mAbs. then PECs from control (uninjected or Dex injected) and LNFPIII-Dex injected mice were added at the above ratios. After 72 hours of co-culture- supernatants were harvested and NO products assayed.

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Abstract

L'invention concerne des méthodes de modulation des réponses immunitaires, notamment la réponse des IgE et les réponses autoimmunes. Lesdites méthodes consistent à mettre une cellule en contact avec un agent comprenant du lacto-N-néotétraose (LNnT) multivalent, lequel module la réponse immunitaire. Lesdites méthodes permettent d'accroître la production d'IgE polyclonales non spécifiques, d'inhiber la production d'une réponse par les IgE spécifiques de l'antigène, d'induire la production de cytokines et de stimuler la prolifération des splénocytes. Selon un mode de réalisation préféré, l'invention concerne des méthodes de modulation de la réponse immunitaire à un antigène (allergan) in vivo. L'invention concerne également des compositions pharmaceutiques servant à moduler les réponses immunitaires, lesquelles compositions renferment les agents selon l'invention. L'invention concerne enfin le traitement ou la prévention du choc chez un sujet à l'aide de LNnT multivalent, des méthodes de traitement d'une maladie autoimmune chez un sujet faisant intervenir le LNnT multivalent, et le traitement du cancer chez un sujet à l'aide de LNnT monovalent.
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US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
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EP1332759A1 (fr) * 2002-02-04 2003-08-06 Kyowa Hakko Kogyo Co., Ltd. Les compositions pharmaceutiques et alimentaires contenant des di- ou oligosaccharides qui élèvent la sécrétion de l'insuline
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
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US11654156B2 (en) 2010-12-31 2023-05-23 Abbott Laboratories Nutritional formulations including human milk oligosaccharides and antioxidants and uses thereof
US12280069B2 (en) 2010-12-31 2025-04-22 Abbott Laboratories Methods of using human milk oligosaccharides for improving airway respiratory health
US11207335B2 (en) 2010-12-31 2021-12-28 Abbott Laboratories Methods of using human milk oligosaccharides for improving airway respiratory health
US11975014B2 (en) 2010-12-31 2024-05-07 Abbott Laboratories Nutritional formulations including human milk oligosaccharides and antioxidants and uses thereof
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US11701376B2 (en) 2010-12-31 2023-07-18 Abbott Laboratories Nutritional compositions comprising human milk oligosaccharides and nucleotides and uses thereof for treating and/or preventing enteric viral infection
US11633412B2 (en) 2010-12-31 2023-04-25 Abbott Laboratories Nutritional formulations including human milk oligosaccharides and antioxidants and uses thereof
EP2758082A4 (fr) * 2011-09-23 2014-12-31 Harvard College Méthodes de traitement de stéatose hépatique par des composés contenant du glycane issu d'helminthe
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