Methods of making monoclonal antibodies

General

While the generation of a mAb is a fairly simple process, the generation of a useful one is often more complex. The fusion process itself takes only a few minutes and involves the incubation of the two types of cell together with a fusogen which is generally polyethylene glycol (PEG) (Figure 1). Hybrids which secrete antibodies of the desired specificity are detected by screening assays that are as close as possible to the final anticipated application of the antibody, although if this is to be a complex application such as in vivo use fully relevant screening is not possible.

Cells to be fused

The main requirements for the generation of a good monoclonal antibody are 1) a source of large numbers of suitably activated B lymphocytes from a hyperimmune subject and 2) a fusion partner from (ideally) the same species which is immortal and which has plasmacytoma/myeloma characteristics, i.e. a well-developed endoplasmic reticulum and the capacity to make large amounts of the required antibody after fusion, but which lacks the capacity to make antibody coded by its own genes. This last point is important since otherwise the fused cells make not only antibody from both parents, but also hybrid antibodies involving the combination of the different chains of the two and the capacity of the fused cells to make the required monoclonal in large amounts is corrupted.

The fusion partner must also be made sensitive to a drug such as aminopterin or azaserine so that those cells which do not acquire the drug resistance genes by fusion can be eliminated; this is a comparatively simple process.

In rodent systems the above conditions are fairly readily achieved. The spleen from a hyperimmune animal is probably the best source of B lymphocytes and nonsecreting, drug-sensitive myeloma lines of high fusion efficiency from both the mouse and rat are readily available. In the BALB/c mouse, the most commonly used cell lines are the P3-X63-Ag8 6.5.3 and Sp2/0 cell lines and in the rat the Lou Y3 Ag 1.2.3 line is the most successful (although this retains the capacity to make a kappa light chain). Lympho cytes from the Armenian or Syrian hamster can also be fused with mouse myeloma lines to yield stable hybrids.

A small number of groups have been working on generating monoclonal antibodies from bovine, ovine or rabbit sources in more recent years but the rodent systems remain dominant.

Selection and cloning

After fusion, the cells are grown in selective medium. The usual medium for rodent fusions is hypoxan-thine, aminopterin and thymidine (HAT). Only cells which have the enzyme hypoxanthine phos-phoribosyl transferase (HPRT) and thymidine kinase (TK) can grow in this medium, and the myeloma cells, which have been selected to lack one of these two enzyme activities, therefore die if they have not acquired them by fusion. Spleen cells generally only survive for 10-14 days in culture and thus only the hybrid fused cells survive.

Small clones of hybridomas emerge from the debris of the dead spleen and myeloma cells 5-20 days after the fusion and are assayed to detect those which make antibody of the required specificity. These are then cloned on feeder cells at a density of one hybridoma cell per well in 96-well tissue culture plates so that the final antibody is indeed monoclonal and that the cell secreting it is stable. The antibody-producing cells can then be grown in bulk in the ascitic fluid of rodents or in large culture vessels depending on the quality and quantity required. Antibody purification from the other components of the culture medium is accomplished by standard biochemical or immunochemical purification systems.

Methods for generating human monoclonal antibodies

Human monoclonal antibodies are required for most therapeutic uses as rodent ones are immunogenic and thus ultimately ineffective in humans. They have, however, proved difficult to generate, mainly because it is usually not possible for ethical reasons to hyper-immunize human subjects and frequently the only available source of B lymphocytes is peripheral blood in which the state of activation of the cells is less suitable for fusion.

There are three main ways in which this problem may be overcome (Figure 2):

(a) A conventional mouse hybridoma is made from an ordinary hyperimmunized BALB/c mouse, and the antibody-coding genes are then manipulated so that the constant regions are of human rather than murine origin. A further sophistication of this technique is to also 'humanize' the

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