Human monoclonal antibodies

2.8 Human monoclonal antibodies

Monoclonal antibody production from human B lymphocytes is more difficult than from rodent cells. Antigens which may ethically be used to immunize people are largely limited to standard vaccines. In many cases cells spontaneously produce interesting immunoglobulins, such as auto- antibodies which are likely to be of a low frequency within the B lymphocyte pool. It is not gener- ally possible to remove the spleen to obtain a large number of lymphocytes! Therefore peripheral blood must be used instead, but this is not a very good source of antibody-secreting cells.

To increase the number of B lymphocytes for immobilization some researchers transform the cells with Epstein–Barr virus (EBV), but this can lead to selection of a subset of B lymphocytes. B

56 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation 56 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

e.g. X63-Ag8.653, SP2/0-Ag14, or a pregenerated mouse/human hybridoma partner such as K6H6/B5 or SPAZ4 cells. The procedures for fusion, selection and cloning are then identical to the protocol for mouse–mouse hybridomas.

2.8.1 Generation of Epstein–Barr virus-immortalized human B-cell lines

MATERIALS AND EQUIPMENT RPMI 1640 Fetal calf serum (FCS) Cyclosporin A Absolute ethanol (Analar) B95-8 cells (Marmoset cell line) Hank’s balanced salt solution Phosphate-buffered saline (PBS)

30 ml heparinized blood Ficoll–Hypaque 75-cm 2 tissue culture flasks for 25 ml cultures

Preparation of the culture medium

METHOD

1 Prepare a culture solution of RPMI 1640 medium containing 20% v/v FCS.

2 In a separate flask, prepare a stock solution of cyclosporin A in ethanol to concentration of

1 mg/ml.

3 Supplement the culture medium with the solution prepared in step 2 to make a final concentration of 1 µg/ml of cyclosporin A, i.e. 1 : 1000 dilution.

4 Inoculate the prepared culture medium with 1 × 10 6 B95-8 cells/ml and incubate for 3 days

at 37°C in a humidified 5% CO 2 incubator.

Preparation of human lymphocytes

METHOD

1 Make a 1 : 2 dilution of the heparinized blood with PBS.

2 Underlay 15 ml of the diluted blood with 15 ml of Ficoll–Hypaque and centrifuge at room temperature for 10 min at 1500 g.

3 Transfer the buffy coat (at the interface) into another tube, add 30 ml of PBS and centrifuge at room temperature for 15 min at 300 g.

4 Discard the supernatant and resuspend the pellet in 10 ml of Hank’s balanced salt solution.

5 Centrifuge at room temperature for 10 min at 300 g.

6 Repeat steps 8 and 9.

7 Following the two washes in Hank’s balanced salt solution, resuspend the pelleted cells in the 3 ml of modified RPMI 1640 culture medium (prepared in steps 1–3).

2.8HUMAN MONOCLONAL ANTIBODIES

Note: Cyclosporin A is added to inactivate the cytotoxic lymphocytes in the preparation isolated from heparinized blood which would otherwise kill the EBV-infected B lymphocytes.

Infection and culture of the B lymphocytes

METHOD

1 Count the mononuclear cells.

2 Place 1 × 10 7 cells in a tissue culture flask and add 3 volumes of modified RPMI 1640 culture medium containing the B95-8 cells following the 3 days of culture (steps 1–4).

3 Incubate at 37°C in a humidified 5% CO 2 for 3 weeks.

4 Change the culture medium every 3 days by making a 1 : 3 dilution of the cells in supplemented RPMI 1640 culture medium.

Note: B95-8 cells are a biohazard (category II) as they are a source of live EBV, and should only be handled by personnel known to be EBV-immune.

2.8.2 Preparation of human B-cell hybridoma

This technique may be used on B lymphocytes isolated from spleen, peripheral blood or EBV-transformed lines (see above). It is a modification of the method described by Lu et al. (1993).

MATERIALS AND EQUIPMENT EBV-transformed cells from the section above, or non-transformed B cells

RPMI 1640 containing 20% fetal calf serum (FCS) Heterohybridoma cell line, e.g. K6H6/B5 Dulbecco’s modified Eagle’s medium Polyethylene glycol 1500 (PEG-1500) Dimethyl sulphoxide (DMSO) Growth medium: RPMI 1640 containing 10% FCS, 1% non-essential amino acids, 1 m M sodium

pyruvate, 10 m M HEPES buffer, 2 m ML -glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin

Growth medium containing 16 µ M thymidine, 100 µ M hypoxanthine, 1 µg/ml azaserine Culture plate, 96 microwells

METHOD

1 Prepare a culture of K6H6/B5 cells to around 2 × 10 6 cell/ml in an equal volume of modified RPMI 1640 culture medium and Dulbecco’s modified Eagle’s medium.

2 Mix 5 × 10 6 EBV-transformed cells with 2.5 × 10 6 K6H6/B5 cells in a conical flask, and centrifuge at 250 g and room temperature for 5 min.

3 Discard the supernatant and resuspend the cell pellet in 1 ml of RPMI 1640 containing 35% w/v PEG and 7.5% DMSO.

4 Slowly add an additional 3 ml of RPMI 1640, then make up to a volume of 120 ml with growth medium containing 16 µ M thymidine, 100 µ M hypoxanthine, 1 µg/ml azaserine.

5 Add 1 × 10 4 cells per well to 96-microwell culture plate.

Place all the plates in a humidified 37°C incubator with 5% CO 2 .

Continued

58 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

6 Feed the cells once a week with fresh medium (growth medium containing 16 µ M thymidine, 100 µ M hypoxanthine, 1 µg/ml azaserine).

7 At 2 weeks, microscopically examine the wells for the presence of hybrid colonies.

8 Test the tissue culture supernatant for the presence of antibodies of interest.

9 Clone positive wells by the same method as the mouse hybridomas. TECHNICAL NOTES

• It is possible to expand the B-cell clones by growing in SCID mice. However, this technique

has the same reservations as applied to growth of mouse hybridomas in whole animals and wherever possible in vitro cell culture should be used.

• To expand hybridomas it may be expedient to invest in a hollow fibre system that allows bulk production of monoclonal antibodies.