Human immunosuppressive protein

• US 7,388,076 B2
• Filed: 07/16/2003
• Issued: 06/17/2008
• Est. Priority Date: 07/16/2002
• Status: Expired due to Fees

• Current Assignee: University of South Florida (State University System of Florida)
• Original Assignee: University of South Florida (State University System of Florida)
• Inventors: Gower, William R., Engelman, Robert W., Sanberg, Paul R.
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/621,061
• Publication Number:
  US 20040058871A1
• Time in Patent Office: 1,798 Days
• Field of Search: None
• US Class Current: 530/350
• 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 a human immunosuppressant protein (HISP) the method comprising:

a) obtaining supernatant from hNT cells;

b) exposing the supernatant to preparative polyactylamide 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 method of claim 1 wherein step a is followed by and step b is preceded by the step of i. concentrating using ultrafiltration.

3. The method of claim 1 wherein step a is followed by and step b is preceded by the step of i. fractionating the supernatant on a Sephacryl gel.

4. The method of claim 3 wherein the next step is ii. combining groups of five collected fractions and optionally determining the peak active fraction.