WO1998039362A1

WO1998039362A1 - Cytotoxic immunoglobulins from mammals

Info

Publication number: WO1998039362A1
Application number: PCT/EP1998/001112
Authority: WO
Inventors: Alberto Bartorelli
Original assignee: VANDER WAY Ltd
Current assignee: VANDER WAY Ltd
Priority date: 1997-03-04
Filing date: 1998-02-27
Publication date: 1998-09-11
Anticipated expiration: 1999-09-04
Also published as: AU6399998A; ITMI970474A1; IT1290018B1

Abstract

Natural Immunoglobulins M and G, extracted from mammals, particularly rabbits and pigs, proved to be cytotoxic in vitro and in vivo against tumour cells.

Description

CYTOTOXIC IMMUNOGLOBULINS FROM MAMMALS

The present invention relates to i munoglobulins of the M type (IgM) and immunoglobulins of the G type (IgG) from mammals, having cytotoxic activity against ^"human tumour cells. The invention further relates to the use of said IgM and IgG for the preparation of antitumour medicaments .

The physiological functions of immunoglobulins have been known for a long time: IgG are specifically responsible for the long-term immunological protection, whereas IgM are responsible for the early immune response. In Immunol. Lett. 30(2), 1994, 187-94, human natural auto-antibodies from foetal B lymphocytes against human tumour cells were described. IgG have been already used in the therapy or in the specific or aspecific prevention of infectious diseases, such as chickenpox, pertussis, hepatitis, tetanus, rubella, measles etc..

On the other hand, no therapeutical applications for IgM are known.

Now it has been found that Ig and IgM isolated from serum of healthy mammals have marked cytotoxic activity against human tumour cells.

The main cytotoxicity percentages are related to IgM, but IgG of the selected animals have interesting cytotoxic properties as well.

It is believed that antitumour cytotoxic IgM and IgG can be extracted from any mammals, in particular from bovineε, equines, swine, rabbits. Preferred sources are swine, in that the sera of all the tested pigs turned out to be cytotoxic, whereas in the other species a percentage of positive (cytotoxic) sera ranging from

10 to 50% was obtained. About 20% of the tested rabbits gave, for example, cytotoxic serum.

Anyway, whether a certain animal is suitable as a source of the immunoglobulins of the invention can be easily determined by those skilled in the art by means of conventional citotoxicity tests in vitro . For this purpose, cultures of JURKAT (fractions of Burkitt Lympho a), HT-29 (colon adenocarcinoma ) or MCF-7 (breast adenocarcinoma) cells can be used advantageously. Using, for example, the methods described in J. Immunol. Methods, 65, 1983, 55-63; ibidem, 119, 1989, 203-210; ibidem, 130, 1990, 149-151. 10 μl of serum of the tested animal, diluted 1:10, are distributed into wells of cell culture plates containing 40,000 JURKAT cells per well. After incubation for 30 minutes at 37"C, the percentage of survived cells are evaluated by means of known methods.

The serum of the animals which turned out to be positive can then be subjected to conventional treatments for the separation and the purification of the IgM and IgG fractions. For example, the serum, after decomplementation at 56^βC for 30 minutes, is subjected to gel filtration on a TSK SW 3000 preparative column (Toso Haas) which is eluted with 0.3 M PBS pH 7 under a pressure of 250 p.s.i. and with a flow of 2 ml/min. , to obtain a first fraction, mainly consisting of IgM, and a second fraction, mainly consisting of IgG. IgG can be further purified passing them through a G protein column (Hi

Trap ProtG) and discarding the unbound fraction.

IgM from the first fraction can be further purified as well by conventional methods, for example by affinity chromatography using anti-IGM antibodies.

The cytotoxicity of the serum is maintained in the two IgM and IgG fractions, the first having variable values higher than 60%, the second having lower values of about 30 to 40%. The cytotoxic IgM and IgG fractions, injected to nude mice or to tumor-bearing rats (HT-29), cause the tumor growth to be stopped and regressed.

For the envisaged therapeutical uses, the immunoglobulins Ig and IgM of the invention will be administered to patients suffering from tumors, in the form of pharmaceutical formulations suitable for the parenteral, preferably subcutaneous or intramuscular, administrations .

The doses depend of course on the conditions of the patient (weight, sex and age) and the severity of the pathology, but they will range from about 10 mg to about 200 mg/day. The treatment can be optionally carried out in combination with other conventional immunotherapeutic or chemotherapeutic treatments.

In order to decrease the risks of allergic reactions, the natural cytotoxic immunoglobulins obtained from serum of animals such as pigs or rabbits can be substituted with the corresponding monoclonal or humanized immunoglobulins, which can be prepared according to well-established methods. EXAMPLE

Iniectable formulation in vial

Immunoglobulins IgM from pig serum 00 mg

Sodium chloride 9 mg

Glycine 22 mg

Sodium ethylmercurithiosalicylate 0 . 1 mg

Apirogenic sterile water 1 ml .

Claims

1. Immunoglobulins M or G from mammals, having cytotoxic activity against human tumour cells.

2. Immunoglobulins according to claim 1 from pig or rabbit.

3. Immunoglobulins M according to claim 1 or 2 having an at least 60% cytotoxicity against JURKAT cells after incubation for 30 minutes at 37°C.

4. The use of the immunoglobulins of claims 1-3, for the preparation of antitu or medicaments.

5. A process for the preparation of the immunoglobulins of claims 1-3 which comprises: a) selection of sera of animals by a cytotoxicity test in vitro; b) gel-filtration of the selected sera and purification of the IgM and IgG fractions.