WO1986000928A1

WO1986000928A1 - Cell treatment

Info

Publication number: WO1986000928A1
Application number: PCT/GB1985/000341
Authority: WO
Inventors: Selby John Starkie; Gwynfor Rees Williams
Original assignee: Axon Healthcare Ltd.
Priority date: 1984-07-31
Filing date: 1985-07-30
Publication date: 1986-02-13
Also published as: EP0190244A1; GB8419459D0; JPS61502797A

Abstract

A process for producing antibodies in vitro, which comprises sensitising cells by exposure to an antigen, and culturing the sensitised cells so that they proliferate and synthesise antibody which can bind with epitopes on the antigen.

Description

CELL TREATMENT FIELD OF THE INVENTION

This invention relates to cell treatment, with the intention that treatment should stimulate the growth of cells making a particular antibody or induce cells to make a particular antibody. BACKGROUND OF THE INVENTION

B lymphocytes proliferate in vivo, on stimulation caused by the presence of an antigen. The proliferating cells in a subject exposed to the antigen yield both plasma cells which actively synthesise and secrete antibody against the specific antigen and also memory cells which are effectively inert until stimulated by the presence of the antigen. Memory cells thus confer on a subject protection against reinfection by the specific antigen. OBJECT OF THE INVENTION

It is an object of the invention to produce antibodies ^n vitro. STATEMENT OF THE INVENTION

According to the present invention, B lymphocyte cells are sensitised by exposure to an antigen jLn vitro, under conditions such that the B lymphocytes are induced to make antibody specific for the antigen. DETAILED DESCRIPTION OF THE INVENTION

The cells may be virgin B lymphocytes, or the sensitisation may be a secondary effect, or stimulation, with respect to already-sensitised B lymphocytes. Thus, in the invention, B lymphocytes (which may or may not be making immunoglobulin) and antigen are contacted (in circumstances under which the B lymphocytes are able to grow) in such a way that the B lymphocytes are induced to make antibody specific for the sensitising antigen, or B lymphocytes which are sensitised to the antigen (B memory cells) are induced to proliferate. If possible, isolated B memory cells may be used for specific purposes; such cells have the advantage that proliferation and antibody production are immediately consequent on exposure to the antigen. The sensitised cells are treated so that they proliferate in culture and synthesise antibody which binds with epitopes on the antigen, e.g. as described in the International Patent Application entitled "Antibody Production" filed in the names of Axon Healthcare Ltd., Starkie and Williams on 30th July 1985.

The conditions suitable for conducting the method of the invention comprise any suitable B lymphocyte culture medium. This may be supplemented, as necessary or desired, with one or more agents which induce proliferation, e.g. feeder cells such as mouse macrophages.

The following Examples illustrate the invention. ("Sepharose" is a registered Trade Mark) . Example 1 30 ml whole blood were taken from a healthy adult man, and defibrinated. 10 ml aliquots of the defibrinated blood were layered over 15 ml aliquots of Hypaque and centrifuged at 400 g for 20 minutes. Serum was removed from the upper layer, and stored at 4 C. White blood cells were removed from the interface of serum and Hypaque. They were washed three times in RPMI 1640 growth medium (to which 10% foetal calf serum had been added) and resuspended in 30 ml growth medium containing 24 ml RPMI 1640, 2.4 ml foetal calf serum, 0.48 ml L-glutamine, 0.48 ml of penicillin and streptomycin, 0.24 ml sodium pyruvate and 2.4 ml of the serum from the healthy adult man. One ml of cell suspension was placed in each of 3 rows of 10 culture wells. Mouse macrophages were obtained by injecting 10 ml growth medium (at 37 C) into the peritoneal cavities of three freshly killed mice, and then withdrawing the suspensions of mouse macrophages in growth medium. One ml of the suspension obtained was added to each of the 3 rows of 10 culture wells described above.

Tumour cells from the oligodendroglioma of a human subject were diced and macerated with scalpel blades. The tissue debris was homogenised in a Potter-Elvehjelm homogeniser. The antigenic material was centrifuged at 600 x g for 20 minutes, and then resuspended in sterile saline (0.85 g/1) to 2 g/1.

Doubling dilutions were prepared of the tumour antigen resuspension, and 0.1 ml of each dilution was added to one well of each of the 3 rows of culture wells containing mouse macrophages; in each row, therefore, each well received twice as much antigen as the well immediately to the right, and so that the 3 rows were 3 replicates of 10 culture wells. The cultures were incubated at 37 C, 100% rh in 5% carbon dioxide for 9 days. At intervals of 48 hours, 0.1 ml of growth medium (containing 40 μl foetal calf serum, 40 μl of serum from the healthy adult man, 8 μl of penicillin and streptomycin, 8 μl of L-glutamine and 4 μl of sodium pyruvate) was added to each culture well.

The culture obtained after 9 days may be treated by transformation with Epstein-Barr virus as described in the International Patent Application to which reference has already been made. Example 2

The procedure of Example 1 was repeated, except that tissue was obtained from ovarian carcinoma tissue from a human subject. Example 3

The antigen Pseudomonas aeruginosa (serotype 11) was cultured by a standard bacteriological method. The organisms were harvested, and suspended in sterile saline. They were washed three times, by centrifugation and sterile aspiration, in sterile saline, and

7 resuspended in sterile saline to a concentration of 10 organisms per millilitre. The organisms were killed by repeatedly freezing to the temperature of liquid nitrogen and warming to 37 C, until a test sample of the suspension contained no live organisms. Six cycles of freezing and thawing were usually enough.

Dilutions were prepared of the killed organism, in culture medium, and 0.1 ml of each dilution was added to one well of each of three rows of culture wells containing mouse macrophages, prepared as in Example 1.

The further procedure of Example 1 was then followed.

Example 4

The procedure of Example 3 was repeated, but using Branhamella catarrhalis as the antigen.

Example 5

The procedure of Example 3 was repeated, but using

Shigella sonnii as the antigen.

Example 6 Mouse IgG was obtained by affinity chromatography of mouse serum using a goat antibody against mouse gamma chain attached to Sepharose CL6B.

The mouse IgG was used as the antigen in the procedure of Example 1.

Claims

-5-CLAIMS

1. A process for producing antibodies in vitro, which comprises exposing B lymphocyte cells to an antigen, and culturing the cells under conditions such that they proliferate and synthesise antibody which can bind with epitopes on the antigen.

2. A process according to claim 1, in which the cells are human virgin B lymphocytes.

3. A process according to claim 1, in which the cells are human B memory cells which have previously been exposed to the antigen (or are derived from human B lymphocytes which have previously been exposed to the antigen) .