GLYCOSYLATION AND CARBOHYDRATE THERAPEUTICS

Synthetic Core 2-like Branched Structures Containing GalNAc-lewis.sup.x and Neu5Ac.alpha.2-2Gal.beta.1- 3GalNAc Sequences as Novel Ligands for Selectins

U.S. Patent Number: 5,972,907
Issue Date: October 26, 1999

Summary: Compounds which bind to selectin receptors and thus may modulate the course of inflammation, cancer and related processes by intervening with cell-cell adhesion events. Further, such compounds can be used for identification and analysis of such receptors. In this regard the invention is directed to compounds of formula (I). ##STR1## wherein R.sup.1 is independently H, alkyl, aryl, an aryl alkyl, alkenyl or one or more additional saccharide residues; R.sup.2 =H or OH provided that when R.sup.2 is H, R.sup.3 is OH; R.sup.3 =H or OH provided that when R.sup.3 is H, R.sup.2 is OH; X=H, SO.sub.3.sup.- or PO.sub.4.sup.- ; Y is independently H, OH, OR.sup.4 or NHCOR.sup.4, wherein R.sup.4 is alkyl, and Z is an organic acid residue. .alpha.-L-Fucose residue can be modified or replaced with suitable bioisosters or a different saccharide residue such as D-mannose. Modification of L-fucose may include replacement of each or all of the hydroxyl groups with H or OR' wherein R' can be methyl, ethyl or allyl groups.

Detail: The invention relates to compounds useful in the treatment of inflammation, allergic reactions, autoimmune diseases, cancer, and similar other conditions that are cell adhesiondependent. More specifically, the invention concerns compounds, containing GalNAc lewis.sup.x as a mucin Core 2 branched structure, which have the ability to bind selectin receptors. Such structures have not been reported to be part of any O-linked glycoproteins. The invention is also concerned with pharmaceutical compositions containing such compounds. It is also directed to methods useful for the synthesis of such compounds and analogs derived therefrom.

Fluorinated Glucosamine Analogs Useful for Modulating Post-Translational Glycosylation of Cells

U.S. Patent Number: 7,098,195
Issue Date: August 29, 2006

Summary: The invention provides compositions and methods for inhibiting cell migration, e.g., lymphocytes and inflammation. The invention also provides an improved process for preparing fluorinated N-acetylglucosamines.

Detail: The invention features methods of inhibiting cell migration, cell proliferation or cell differentiation by contacting a cell with a fluorinated N-acetylglucosamine (F-GlcNAc), e.g., 2-acetamido-2-deoxy-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose or 2-acetamido- 2-deoxy-1,4,6-tri-O-acetyl-3-deoxy-3-fluoro-D-glucopyranos- e in an amount sufficient to inhibit cell migration, proliferation or differentiation.

Also provide by the invention is a method of decreasing an amount of HECA-452 epitope on a glycoprotein, e.g., PSGL-1 or CD44 on a cell, by contacting the cell with a fluorinated N-acetylglucosamine. The amount of the glycoprotein on the cell in the presence of the fluorinated N-acetylglucosamine as compared to in the absence of the fluorinated Nacetylglucosamine differs by less than 10%, 5% or 1%.

In another aspect the invention features a method of inhibiting inflammation in a tissue, e.g., dermal tissue of a subject by administering to the subject a fluorinated N-acetylglucosamine. The inflammation is for example, chronic inflammation, e.g., DTH, acute inflammation, cutaneous inflammation, psoriasis, inflammatory bowel disease, colitis or Crohn's disease. The fluorinated N-acetylglucosamine is administered prior to an inflammatory event. Alternatively, the fluorinated N-acetylglucosamine is administered after an inflammatory event. Administration is, intraperitoneal, subcutaneous, nasal, intravenous, oral, topical and transdermal delivery.

The cell is a leukocyte such as a lymphoid cell, e.g., T-cell or a hematopoietic cell. Alternatively, the cell is a cancerous cell such a leukemic cell or a lymphoma, e.g., cutaneneous lymphoma. The cell is further contacted with a chemotherapeutic agent such as daunorubicin (DNR), cytarabine (ara-C), idarubicin, thioguanine, etoposide, and mitoxantrone or an anti-inflammatory agent such as aspirin, ibuprofen, naproxen sodium, celecoxib, prednisone, prednisolone, and dexamethasone.

The invention provides an improved method for preparing fluorinated N-acetylglucosamine which comprises the intermediate step of preparing benzyl 2-acetamido-2- deoxy-3,6-di-O-benzyl-D-glucopyranoside from benzyl 2-acetamido-3-O-benzyl-4,6-benzylidene- 2-deoxy-D-glucopyranoside, the improvement comprising (i) hydrolyzing benzyl 2-acetamido-3-O-benzyl-4,6-benzylidene-2-deoxy-D-glucopyranoside under appropriate conditions to form benzyl 2-acetamido-3-O-benzyl-2-deoxy-D-glucopyranoside; (ii) reacting benzyl 2-acetamido-3-O-benzyl-2-deoxy-D-glucopyranoside with a tin compound to form a tin complex comprising benzyl 2-acetamido-3-O-benzyl-2-deoxy-D-glucopyranoside; and (iii) reacting the tin complex with a benzylating agent under appropriate conditions to form benzyl 2-acetamido-3,6-benzyl-2-deoxy-D-glucopyranoside.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Stress Protein Compositions and Methods for Prevention and Treatment of Cancer and Infectious Disease

U.S. Patent Number: 6,984,384; 7,378,096 (CIP)
Issue Date: January 10, 2006; May 27,2008 (CIP)

Summary: Pharmaceutical compositions comprising a stress protein complex and related molecules encoding or cells presenting such a complex are provided. The stress protein complex comprises an hsp110 or grp170 polypeptide complexed with an immunogenic polypeptide. The immunogenic polypeptide of the stress protein complex can be associated with a cancer or an infectious disease. The pharmaceutical compositions of the invention can be administered to a subject, thereby providing methods for inhibiting M. tuberculosis-infection, for inhibiting tumor growth, for inhibiting the development of a cancer, and for the treatment or prevention of infectious disease. The invention further provides a method for producing T cells directed against a tumor cell or a M. tuberculosis-infected cell, wherein a T cell is contacted with an APC that is modified to present an hsp110 or grp170 polypeptide and an immunogenic polypeptide associated with a tumor or with the M. tuberculosis-infected cell. Included in the invention are T cells produced by this method and a pharmaceutical composition comprising such T cells. The T cells can be contacted with a M. tuberculosis-infected cell in a method for killing a M. tuberculosis-infected cell, or with a tumor cell in a method for killing a tumor cell.

Detail: The invention provides a pharmaceutical composition comprising a stress protein complex. The stress protein complex comprises an hsp110 or grp170 polypeptide and an immunogenic polypeptide. In some embodiments, the hsp110 or grp170 polypeptide is complexed wit h the immunogenic polypeptide, for example, by non-covalent interaction or by covalent interaction, including a fusion protein. In some embodiments, the complex is derived from a tumor. In other embodiments, the complex is derived from cells infected with an infectious agent. The immunogenic polypeptide of the stress protein complex can be associated with a cancer or an infectious disease. The stress protein complex of the invention can further include additional stress polypeptides, including members of the hsp70, hsp90, grp78 and grp94 stress protein families. In one embodiment, the stress protein complex comprises hsp110 complexed with hsp70 and/or hsp25.

The invention additionally provides a pharmaceutical composition comprising a first polynucleotide encoding an hsp 110 or a grp170 polypeptide and a second polynucleotide encoding an immunogenic polypeptide. In some embodiments involving first and second polynucleotides, the first polynucleotide is linked to the second polynucleotide. The pharmaceutical compositions of the invention can further comprise a physiologically acceptable carrier and/or an adjuvant. The efficacy of a pharmaceutical composition can further comprise GM-CSF-secreting cells. Alternatively, GM-CSF-secreting cells can be co-administered with a pharmaceutical composition of the invention, by administration before, during or after administration of the pharmaceutical composition. The use of GM-CSFsecreting cells enhances the efficacy of the pharmaceutical composition.

In some embodiments, the complex is purified from a tumor or from cells infected with an infectious agent. In such embodiments, the stress polypeptide, as purified, is complexed with one or more immunogenic polypeptides. The binding of the stress polypeptide to the immunogenic polypeptide can be altered and/or enhanced by stress, such as by exposure to heat, anoxic and/or ischemic conditions, or proteotoxic stress. In particular, a stress protein complex of the invention can comprise a stress polypeptide complexed with an immunogenic polypeptide, wherein the complex has been heated. Such heating, particularly wherein the stress polypeptide comprises a heat-inducible stress protein, can increase the efficacy of the stress protein complex as a vaccine. Examples of heatinducible stress proteins include, but are not limited to, hsp70 and hsp 110.

In some embodiments, the immunogenic polypeptide is known. The immunogenic polypeptide is a known molecule, the immunogenic polypeptide can be provided in admixture with the stress polypeptide, or as a complex with the stress polypeptide. The hsp 110 or grp170 polypeptide can be complexed with the immunogenic polypeptide by non-covalent binding. Alternatively, the complex can comprise a fusion protein, wherein the stress polypeptide is linked to the immunogenic polypeptide. Examples of immunogenic polypeptides include, but are not limited to, antigens associated with cancer or infectious disease, such as the breast cancer antigen her2/neu or the Mycobacterium tuberculosis antigens Mtb8.4 and Mtb39. Where the immunogenic polypeptide is unknown, it can be obtained incidentally to the purification of the stress polypeptide from tissue of a subject having cancer or an infectious disease.

Also provided is a pharmaceutical composition comprising an antigen-presenting cell (APC) modified to present an hsp 110 or grp170 polypeptide and an immunogenic polypeptide. Alternatively, the APC can be modified to present an immunogenic polypeptide obtained by purification of hsp 110 or grp170 from disease cells, including cancer cells and cells infected with an infectious agent. Preferably, the APC is a dendritic cell or a macrophage. The APC can be modified by various means including, but not limited to, peptide loading and transfection with a polynucleotide encoding an immunogenic polypeptide.

The pharmaceutical compositions of the invention can be administered to a subject, thereby providing methods for inhibiting M. tuberculosis-infection, for inhibiting tumor growth, for inhibiting the development of a cancer, and for the treatment or prevention of cancer or infectious disease.

The invention further provides a method for producing T cells directed against a tumor cell. The method comprises contacting a T cell with an antigen presenting cell (APC), wherein the APC is modified to present an hsp 110 or grp170 polypeptide and an immunogenic polypeptide associated with the tumor cell. Such T cells can be used in a method for killing a tumor cell, wherein the tumor cell is contacted with the T cell. Likewise, the invention provides a method for producing T cells directed against a M. tuberculosis-infected cell, wherein a T cell is contacted with an APC that is modified to present an hsp110 or grp170 polypeptide and an immunogenic polypeptide associated with the M. tuberculosis-infected cell. Included in the invention are T cells produced by this method and a pharmaceutical composition comprising such T cells. The T cells can be contacted with a M. tuberculosis-infected cell in a method for killing a M. tuberculosis- infected cell The T cells can be CD4+ or CD8+.

The invention also provides a method for removing tumor cells from a biological sample. The method comprises contacting a biological sample with a T cell of the invention. In a preferred embodiment, the biological sample is blood or a fraction thereof. Also provided is a method for inhibiting tumor growth in a subject. The method comprises incubating CD4+ and/or CD8+ T cells isolated from the subject with an antigen presenting cell (APC), wherein the APC is modified to present an hsp 110 or grp 170 polypeptide and an immunogenic polypeptide associated with the tumor cell such that T cells proliferate. The method further comprises administering to the subject an effective amount of the proliferated T cells, and thereby inhibiting tumor growth in the subject. In an alternative embodiment, the method for inhibiting tumor growth in a subject comprises incubating CD4+ and/or CD8+ T cells isolated from the subject with an antigen presenting cell (APC), wherein the APC is modified to present an hsp110 or grp170 polypeptide and an immunogenic polypeptide associated with the tumor cell such that T cells proliferate, cloning at least one proliferated cell, and administering to the patient an effective amount of the cloned T cells, thereby inhibiting tumor growth in the subject.

Use of Recombinant Heat Shock Protein Complexed to Kidney Cancer Antigen

U.S. Patent: Pending
Application Number: 11/894,970

Summary: A heat shock protein in combination with carbonic anhydrase IX and a method for improving immune response to carbonic anhydrase IX in a mammal by complexing it with a heat shock protein prior to administration to the mammal.

Detail: Carbonic anhydrase IX (CA9) has been identified recently as a potential target for immunotherapy. CA9 is present in 95-100% of clear renal carcinoma cells (RCC) and it is not present in normal, nonmucosal tissue. We have now found that HSP proteins, normally found inside a cell, are powerful immune activators when found outside a cell that are capable of stimulating an immune response against proteins complexed to the HSP.