WO2006005940A2

WO2006005940A2 - Monoclonal anti-glut2 antibody

Info

Publication number: WO2006005940A2
Application number: PCT/GB2005/002727
Authority: WO
Inventors: Peter Collins; Archie Roberston
Original assignee: RIVERSIDE BIOSCIENCES Ltd
Current assignee: RIVERSIDE BIOSCIENCES Ltd
Priority date: 2004-07-13
Filing date: 2005-07-13
Publication date: 2006-01-19
Anticipated expiration: 2007-01-13
Also published as: WO2006005940A3; GB0415644D0

Abstract

A monoclonal antibody that recognises glucose transporter 2 (GLUT2) is described, as well as a hybridoma that produces the antibody. The antibody may be used to identify or isolate embryonic or fetal cells, for example in an in vitro method of prenatal diagnosis.

Description

Monoclonal Antibody

This invention relates to a monoclonal antibody (Mab 1C32H52F7) that recognises glucose transporter 2 (GLUT2), or a fragment or derivative of Mab 1C32H52F7 that retains GLUT2 binding activity. The invention also relates to a hybridoma producing the antibody, and to use of the antibody, fragment or derivative to identify or isolate embryonic or fetal cells, for example in an in vitro method of prenatal diagnosis.

Conventional prenatal testing for disorders such as Down's syndrome is carried out on fetal cells obtained by amniocentesis or chorionic villous sampling (CVS). Amniocentesis is normally performed around 16 weeks of gestation, and involves insertion of a needle into the amniotic sac of the fetus to remove amniotic fluid. CVS requires a small biopsy to be taken from the placenta of the 8-12 week old fetus. Both of these invasive procedures are associated with a risk of inducing a spontaenous abortion.

Non-invasive methods have been developed in which fetal nucleated cells are isolated from a peripheral blood sample of the mother. Such methods rely on binding of a binding moiety, such as a monoclonal antibody, to a fetal marker present on fetal cells but absent from maternal cells in the sample. One such marker is glucose transporter 2 (GLUT2). UK Patent No. 2,326,943 describes methods of testing for a variety of fetal abnormalities using the GLUT2 marker to identify or isolate fetal red blood cells from a maternal blood sample.

According to the invention there is provided a monoclonal antibody, Mab 1C32H52F7, that is specific for GLUT2.

A hybridoma that produces Mab 1C32H52F7 has been deposited under accession no. 04070801 on 8^th July 2004 at the European Collection of Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom, in accordance with the Budapest Treaty 1977. A further deposit of a hybridoma that produces Mab 1C32H52F7 (GLUT2 IC32H52F7 MUTQNE HYBRIDOMA) has been made under accession no. 04112301 on 23^rd November 2004 at the European Collection of Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom, in accordance with the Budapest Treaty 1977.

Mab 1C32H52F7 is obtainable from the deposited hybridoma having accession no. 04070801, or 04112301.

There is also provided according to the invention a fragment or derivative of Mab 1C32H52F7 that retains GLUT2 binding ability. The fragment or derivative may be any fragment or derivative that can be derived from Mab 1C32H52F7 by a person skilled in the art using conventional methods.

There is also provided according to the invention a method of obtaining Mab 1C32H52F7, which comprises isolating the antibody from supernatant of hybridoma having accession no. 04070801, or 04112301. Preferably the Mab is isolated by binding of its Fc domains to a protein G column. A method by which. Mab 1C32H52F7 may be isolated from the deposited hybridoma is described in the example below.

Mab 1C32H52F7, or fragment or derivative thereof, may be used to identify or isolate embryonic or fetal red blood cells from a sample comprising maternal blood cells and embryonic or fetal red blood cells. This may be achieved, for example, by any of the methods described in GB 2,326,943.

Typically the blood cells will be human blood cells. However, it is believed that Mab 1C32H52F7 will also recognise non human GLUT2, and so could be used to identify or isolate non human embryonic or fetal red blood cells from a sample comprising non human maternal blood cells and non human embryonic or fetal red blood cells. Identification or isolation of embryonic or fetal red blood cells using Mab 1C32H52F7, or a fragment or derivative thereof, may be used in an in vitro method of determining an abnormality of an embryo or fetus. Examples of fetal abnormalities that may be determined using a monoclonal antibody to GLUT2 are described in GB 2, 326,943. Particularly preferred examples are chromosomal abnormalities, such as Down's syndrome, trisomy 18, trisomy 13, sex chromosomal abnormalities, and diseases or disorders caused by mutations in individual genes, such as cystic fibrosis. The Mab, or fragment or derivative may alternatively be used to establish a condition, such as the sex, of the embryo or fetus.

Relevant information available to the applicant on the characetristics of hybridoma having accession no. 04070801 or 04112301 is detailed below and in the following example:

Cell line deposited - GLUT2 1C32H52F7 Murine Hybridoma

Brief description - produces GLUT 2 monoclonal antibody

Identification/Name in full - GLUT2 1C32H52F7 Murine Hybridoma

Species and strain - Mouse BALBc

Organ/Tissue - B - Cell

Immunogen - GLUT2 Recombinant protein

Immunocyte donor - BALBc Mouse B cell

Immortal partner - SP2 cell

Product Specificity - GLUT2 monoclonal antibody

Ig class/subclass - IgGl

Screening Assay - ELISA

Cell products/characteristics - produces GLUT 2 monoclonal antibody

Morphology - circular

Passage No - 5

Growth as suspension/attached line - see below

Cell concentration - 5 X 10 ⁶ /ampoule

Composition of medium - 10% DMSO, 20% FCS, 70% growth medium

Growth medium - DMEM with L- Glutamine

% Serum and type - 10% Foetal Calf Serum Supplements (& cone) - 1% HT, 100U/ml penicillin, 100ug/ml Streptomycin

Temperature - 37°C

Gaseous phase - 95% humidified air, 5% CO₂

Split ratio (attached) - lit 1:8 twice weekly (40-75% confluence)

The methods by which Mab 1C32H52F7 was produced and purified are described in the following example, with reference to the accompanying drawings in which: Figure 1 shows a three dimensional representation of the structure of GLUT2; Figure 2 shows the structure of the human Glut 2 gene, and the amino acid sequence of an 89 amino acid region encoded by the gene; and

Figure 3 shows hydrophilicity plots of the full length Glut 2 protein (Figure 3A), and the 89 amino acid region (Figure 3B).

Example

Production and purification of recombinant GLUT2

Primers were designed around the 89 amino acid Glut 2 specific region shown in Figure 2. The targeted region was amplified by polymerase chain reaction (PCR) from the template DNA. The PCR was performed using a number of protocols simultaneously including Touchdown, Gradient & at optimal annealing temperatures.

The PCR products of the amplified Glut 2 regions were cloned into expression vectors and transformed into E. coli. The positive clones were screened for expression performance, and positive expressing clones were checked for integrity by DNA sequencing to ensure that the clones contain the correct Glut 2 sequence.

Growth conditions were optimised for the clones with the highest expression yields. Clones were grown in a large-scale flask to achieve optimal expression yields of GLUT2 recombinant protein. The 89 amino acid recombinant GLUT2 protein fragment was purified by ion metal affinity chromatography (IMAC) techniques.

Development of hybridoma producing monoclonal antibodies against GLUT2

5 BALB/c mice were immunised subcutaneously with the purified 89 amino acid recombinant GLUT2 protein fragment. Seven inoculations of 50μl of the antigen mixed with 50μl of adjuvant were given over a ten-week time course. Test bleeds were taken at intervals and positive immunisation was confirmed by western blot. Two days after final inoculation, the mouse spleen cells were fused with SP2 myeloma cells. HAT media was used to select for hybridoma cells. Selected hybridoma cells were maintained in DMEM supplemented with Glutamax 1, 10% Fetal Calf Serum, lOOU/ml penicillin and lOOμg/ml streptomycin.

ELISA Screening

Microtitre plates were coated with the GLUT2 protein (50ng/well) together with a control. Plates were treated with a blocking buffer to avoid false positives. All actively growing hybridoma cells supernatants were screened by ELISA for specificity to GLUT2. Those giving high readings were selected for cloning by limiting dilutions. This procedure was repeated twice to ensure that individual hybridoma cells producing GLUT2 monoclonal antibody had been isolated. An ECL of supernatant was performed as a final control of their specificity. After initial characterisation, the GLUT2 antibody selected for further study was the '1C32H52F7' monoclonal antibody.

Monoclonal antibody production and purification

Freeze Down of Hybridoma Cells:

Viable cells in the log phase of growth were frozen down at a high density.

Vials of 1C32H52F7 hybridoma cells were frozen down in 10% DMSO (Sigma, UK),

20% fetal bovine serum (Gibco, UK), and 70% maintenance medium (see below).

The vials were frozen slowly overnight at -80°C and then transferred to liquid nitrogen after 24-48 hours. Break Out Instructions:

It is preferable to break the cells out in a small volume of media (~ 3mls of media over 2 wells on a 24 well plate. Let the cells rest for 18-24 hrs and then gently remove the media. This should allow the removal of non viable cells while leaving the viable adherent cells in place. Re-feed the cells and pass as required).

Maintenance of 1C32H52F7 Hvbridoma Cell line:

The 1C32H52F7 hybridoma cell line is maintained at a confluence of 30-70% and propagated in DMEM medium supplemented with Glutamax 1 (Gibco, Invitrogen Ltd. Paisley, UK), 10% fetal bovine serum (v/v) (Gibco, UK), 1% HT supplement (Gibco, UK), penicillin (Gibco, UK, 100 U per ml), streptomycin (Gibco, UK, 100 μg per ml), in an atmosphere of 95% humidified air and 5% CO₂ at 37°C. Cells are passaged every two — three days.

Alternatively, the 1C3 2H5 2F7 hybridoma cell line is maintained at a confluence of 30-70% and propagated in DMEM medium supplemented with Glutamax 1, 4500mg/L D-Glucose (Gibco, Invitrogen Ltd. Paisley, UK Cat. 61965-026), 2mM Sodium Pyruvate, 10% foetal bovine serum (v/v) (Gibco, UK), 1% HT supplement (Gibco, UK), penicillin (Gibco, UK, 100 U per ml), streptomycin (Gibco, UK, 100 mg per ml), in an atmosphere of 95% humidified air and 5% CO₂ at 37⁰C. Cells are passaged every two - three days.

Slow growing cells or recovery of cells after cryopreservation - cells can be rescued in media further supplemented with 10% Doma-Drive (Growth enhancing supplement, Immune Systems Ltd, UK Cat. T31-1003).

Production of 1C32H52F7 Monoclonal Antibody:

The 1C32H52F7 monoclonal antibody producing hybridoma cells are grown to 70% confluence as described above. Cells are then grown in DMEM medium supplemented with Glutamax II (Gibco, Invitrogen Ltd. Paisley, UK), 10 % fetal bovine serum (v/v) (Gibco, UK), 1% HT supplement (Gibco, UK), penicillin (Gibco, UK, 100 U per ml), streptomycin (Gibco, UK, 100 μg per ml), in an atmosphere of 95 % humidified air and 5 % CO₂ at 37°C. Supernatant containing the monoclonal antibody is removed after 5-7 days for antibody purification.

Monoclonal Antibody purification using a Protein G column:

The 1C32H52F7 monoclonal antibody (Isotype IgG) is purified by virtue of its Fc domains using a protein G column. The antibody is purified on an AKTA prime purifier (Amersham, UK)

Application:

1. Pipette lOOμl of Tris pH 7.5 into each of the collection tubes

2. Wash a HiTrap Protein G column with 2ml water to remove ethanol

3. Attach column to AKTA Prime

4. Set up buffer lines as follows: ^■ A1- P.B.S

^■ A8- P.B.S ^■ B- Glycine/HCl pH 3.0

5. Select programme on 3 on AKTA 1 or programme 13 on AKTA 2

6. Start Run

7. Transfer line A8 into supernatant when programme reaches 40ml.

8. At 45ml set AKTA Prime to hold until 15ml of supernatant remains.

9. Release hold

BREAK FLOW INJECTION

POINT (ml) % B (ml/mitt) FRACTIONBUFFERVALVE V.

0 0 40 0 1 Waste

35 0 1 0 1 Load

45 1 0 1

45 0 1 0 1 Inject

60 0 1 0 1 Load

65 0 1 0 1 Load

70.1 100 40 0 1 Waste

105 100 1 1 1 Load

115 100 1 0 1 Load

115.1 0 40 0 1 Waste

130 0 1 0 1 Load

135 0 1 0 1

10. Collect fractions for BCA Analysis

11. Dialyse overnight in PBS (100ml PBS for every 1ml of purified supernatant).

Storage of Purified Antibody

Purified antibody is stored in a PBS (Sigma, UK) buffer containing 0.1% Azide

(Sigma, UK). Antibody is stored at -8O⁰C.

Claims

1. Monoclonal antibody 1C32H52F7, or a fragment or derivative thereof that retains GLUT2 binding ability.

2. A hybridoma that produces an antibody according to claim 1.

3. A hybridoma having ECACC accession no. 04070801 , or 04112301.

4. A method of obtaining monoclonal antibody 1C32H52F7 which comprises isolating the antibody from supernatant of hybridoma having accession no. 04070801, or 04112301.

5. Use of an antibody, fragment, or derivative according to claim 1 to identify embryonic or fetal red blood cells in a sample comprising maternal blood cells and embryonic or fetal red blood cells.

6. Use of an antibody, fragment, or derivative according to claim 1 to isolate embryonic or fetal red blood cells from a sample comprising maternal blood cells and embryonic or fetal red blood cells.

7. Use of an antibody, fragment, or derivative according to claim 1 in an in vitro method for diagnosis of an abnormality or condition of an embryo or fetus.

8. Use according to claim 7 for diagnosis of Down's syndrome.