Human immunosuppressive protein

Details Human immunosuppressive protein Human immunosuppressive protein

C - Chemistry, Metallurgy

07

K

C07K 1/00 (2006.01) C07K 14/47 (2006.01) C07K 14/52 (2006.01) A61K 38/00 (2006.01)

Patent

CA 2492654

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-Imonocytic leukemia cells. HISP can maintain T cells in a quiescent G~0/G~1 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.

L'invention concerne un proc~d~ de purification d'une prot~ine immunosuppressive (HISP) consistant ~ obtenir un surnageant ~ partir des cellules hNT; ~ exposer le surnageant ~ une ~lectrophor­se sur gel de polyacrylamide de pr~paration afin d'obtenir 20 fragments iso~lectriques, y compris un fragment iso~lectrique actif num~ro 10; ~ placer le fragment iso~lectrique actif sur une colonne Bleu-S~pharose afin de lier l'albumine; et ~ recueillir le fragment libre contenant la HISP concentr~e isol~e. Fait ~galement l'objet de cette invention une m~thode de traitement des inflammations utilisant une quantit~ efficace d'HISP. L'HISP est anionique, pr~sente un poids mol~culaire de 40 ~ 100 kDa, un point iso~lectrique d'environ 4,8 et provient du surnageant de cellules hNT, mais non pas de cellules de carcinome embryonnaire NCCIT, de cellules de glioblastome T98G ou de cellules de leuc~mie monocytique THP-1. L'HISP peut maintenir les lymphocytes T dans un ~tat quiescent G´0?/G´1? sans abaisser leur viabilit~. L'HISP perd son activit~ lorsqu'elle est thermotrait~e, pH2, pH11, ou m~lang~e ~ de la trypsine ou ~ une carboxypeptidase, mais non pas ~ une raminidase. L'HISP peut supprimer la prolif~ration de cellules sanguines mononucl~aires p~riph~riques r~pondeuses contenues dans des cultures mixtes lymphocytaires allog~niques; l'HISP peut supprimer la prolif~ration de lymphocytes T et la production d'IL-2 en r~ponse au phorbol 12-myristate 13-ac~tate (PMA), ~ l'ionomycine et ~ la concanavaline-A. L'HISP ne se lie pas au gel h~parine-s~pharose CL-B; ou ~ la r~sine de liaison de l'albumine Bleu-S~pharose. L'HISP est concentr~e par ultrafiltration YM10. L'HISP n'agit pas ~ travers le complexe du r~cepteur CD3 des lymphocytes T ou via des cellules ~ signaux accessoires alt~r~es. L'invention concerne en outre une m~thode de traitement d'inflammations consistant ~ administrer une quantit~ efficace de cellules neuronales hNT.

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