Human immunosuppressive protein

• US 20040058871A1
• Filed: 07/16/2003
• Published: 03/25/2004
• Est. Priority Date: 07/16/2002
• Status: Active Grant

• Current Assignee: University of South Florida (State University System of Florida)
• Original Assignee: University of South Florida A Non-Profit Institution
• Inventors: Sanberg, Paul R., Gower, William R., Engelman, Robert W.
• Granted Patent: US 7,388,076 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A method for purifying an immunosuppressant protein (HISP) has the steps of obtaining supernatant from hNT cells; exposing the supernatant to preparative polyacrylamide gel electrophoresis to produce 20 isoelectric fractions, including active isoelectric fraction #10; placing the active isoelectric fraction on a Blue Sepharose column to bind albumin; and collecting the free fraction containing the concentrated, isolated HISP. Also disclosed is a method of treating inflammation, using an effective amount of an HISP. The HISP is anionic, has a molecular weight of 40-100 kDa, an isoelectric point of about 4.8 and is obtained from the supernatant of hNT cells, but not from NCCIT embryonal carcinoma cells, T98G glioblastoma cells or THP-1 monocytic leukemia cells. HISP can maintain T cells in a quiescent G₀/G₁ state without lowering their viability. HISP loses activity when treated with heat, pH2, pH11, or mixed with trypsin or carboxypeptidase, but not with neuraminidase. HISP can suppress proliferation of responder peripheral blood mononuclear cells in allogeneic mixed lymphocyte cultures; HISP can suppress T-cell proliferation and IL-2 production in response to phorbol 12-myristate 13-acetate (PMA), ionomycin and concanavalin-A. HISP does not bind to heparin-sepharose CL-B gel; or to albumin-binding resin Blue Sepharose. HISP is concentrated with YM10 ultrafiltration. HISP does not act through the T-cell receptor-CD3 complex or via altered accessory signal cells. A method of treating inflammation comprises administering an effective amount of hNT neuronal cells.

Claims

1. A method for purifying an immunosuppressant protein (HISP) the method comprising:

a) obtaining supernatant from hNT cells;

b) exposing the supernatant to preparative polyacrylamide gel electrophoresis to produce 20 isoelectric fractions, including active isoelectric fraction #10;

c) placing the active isoelectric fraction on a Blue Sepharose column to bind albumin; and

d) collecting the free fraction containing the concentrated, isolated HISP.

2. The compound produced by claim 1.

3. A method of treating inflammation, the method comprising administering an effective amount of an immunosuppressant protein (HISP).

4. An isolated immunosuppressant protein (HISP), said protein comprising an anionic protein

having a molecular weight of 40-100 kDa;

having an isoelectric point of about 4.8;

being obtained from hNT cell supernatant;

not being obtained from NCCIT embryonal carcinoma cells, T98G blioblastoma cells or THP-1 monocytic leukemia cells;

losing activity when treated with heat, pH 2, pH 11, or mixed with trypsin or carboxypeptidase;

losing no activity when incubated with neuraminidase;

being capable of suppressing proliferation of responder peripheral blood mononuclear cells in allogeneic mixed lymphocyte cultures;

being capable of suppressing T-cell proliferation and IL-2 production in response to phorbol 12-myristate 13-acetate (PMA), ionomycin and concanavalin-A;

being capable of maintaining T cells in a quiescent G₀/G₁ state without lowering their viability;

not binding to heparin-sepharose CL-B gel;

not binding to albumin-binding resin Blue Sepharose;

concentrating with YM10 ultrafiltration; and

not acting through the T-cell receptor-CD3 complex or via altered accessory signal cells.

5. A method of treating inflammation, said method comprising administering an effective amount of hNT neuronal cells.