Outline of B-hybridoma production

2.2 Outline of B-hybridoma production

Spleen cells, prepared from immunized mice or rats, are induced to fuse with murine plasmacy- toma cells using polyethylene glycol.

Many cells show cytoplasmic fusion; a lower proportion complete the nuclear fusion required to produce tetraploid hybrids (or a greater ploidy, depending upon the number of fusing cells). This procedure results in a heterogeneous mixture of fused and unfused cells, although there is a preferential association of ontogenetically similar cells: plasmacytoma cells tend to ‘rescue’ large, recently activated B lymphocytes.

After dispensing into culture wells, the cell mixture is cultured in a selective medium that positively selects for fusion hybrids. Culture supernatants are tested for antibody activity after 1–3 weeks. Positive cultures are cloned by conventional cell cloning techniques.

2.2.1 Basis of fusion and selection

To understand the choice of cells and manipulation of the system, consider the contribution of each component of the hybrid cell:

1 The plasmablast parent is terminally differentiated and dies in culture but provides the genetic information for the required antibody.

2 The plasmacytoma parent confers potential immortality on the hybrid cell, but will itself grow in culture. Once plasma cells from an appropriately immunized animal have been fused with tumour

cells in vitro it is necessary to eliminate unfused tumour cells (or tumour–tumour hybrids), then select the hybrid cells secreting antibody of the required specificity.

2.2.2 Elimination of plasmacytoma cells

This is done by the use of a plasmacytoma cell line deficient in the enzyme responsible for incorporation of hypoxanthine into DNA.

Briefly, cells can synthesize DNA in two ways, either by de novo synthesis or via the so-called ‘salvage’ pathway using exogenous or endogenous sources of preformed bases, as summarized in Fig. 2.1(a). If plasmacytoma cells are grown in the presence of a purine analogue, for example 8-azaguanine or 6-thioguanine, the hypoxanthine guanine phosphoribosyltransferase (HGPRT) enzyme catalyses the incorporation of the purine analogue into DNA where it interferes with normal protein synthesis and so the cells die. Gene coding for the HGPRT enzyme is on the X chromosome, so only a single copy per cell is expressed. Cells will arise that are deficient in the HGPRT gene and therefore do not incorporate the purine analogue, i.e. HGPRT-deficient cells

2.2OUTLINE OF B-HYBRIDOMA PRODUCTION

(a) De novo synthesis

Salvage

(b)

N pathway 5 N 10 Methylene

Block salvage tetrahydrofolate

Block de novo

Deoxyuridylate

pyrimidine

pathway of

synthesis

pyrimidines and purines by selecting

for plasmacytoma Tetrahydrofolate

Aminopterin A

blocks the enzyme

fusion partners

dihydrofolate

that lack key

reductase

enzymes HGPRT 1

and TK Dihydrofolate

Thymidylate

Thymidine

Key: 2 DNA Pyrimidine

1 Dihydrofolate synthesis reductase

Hypoxanthine

DNA synthesis Supply of key enzymes HGPRT and 2 Thymidine

3 TK from normal lymphocyte kinase

fusing with mutant plasmacytoma 3 Hypoxanthine guanine

allows synthesis of pyrimidines and

purines from hypoxanthine H phosphoribosyl transferase

Purine

and thymidine T present in HAT medium (HGPRT)

synthesis

(c)

Key: Plasmacytoma (HGPRTase deficient) but immortal Normal lymphocyte (has HGPRTase)

but has normal growth control

Fusion mixture

Polyethylene glycol

Tumour/tumour Tumour/lymphocyte lymphocyte

Unfused Lymphocyte/lymphocyte

HGPRT and immortality

Lots of HGPRTase

Message for

but no immortality

immortality but

not HGPRT

Die in HAT

Die in HAT

Tumour–lymphocyte hybrids proliferate in HAT medium. Functional selection now required to find cells producing the required antibody

42 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation 42 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

de novo synthesis (Fig. 2.1b).

A selective medium containing aminopterin (or amethopterin, methotrexate), hypoxanthine and thymidine (HAT medium) is used. Aminopterin (analogue of folic acid) binds folic acid reductase and blocks the coenzymes required for de novo synthesis of DNA. To grow in this medium a cell must make DNA via the ‘salvage’ pathway. If HGPRT-deficient plasmacytoma cells are fused with normal lymphoid cells and then placed in HAT medium, only the hybrids between plasmacytoma and normal cells will grow (plasmacytoma cell provides immortality; plasma cell provides the HGPRT enzyme aFig. 2.1c).

2.2.3 Origin of plasmacytoma lines for fusion

The majority of fusion experiments have been performed using sublines of P3/X63-Ag8 which is an 8-azaguanine-resistant subline of the plasmacytoma MOPC 21 (induced in a BALB/c mouse by the injection of mineral oil). Moreover, it tends to fuse spontaneously (with itself ) and can grow at very low cell densities, thus facilitating the recovery of fusion hybrids. It also has the disadvant-

age that it synthesizes and secretes the MOPC 21 myeloma protein (a fully sequenced IgG 1 , κ) and so hybrid cells will secrete myeloma and antibody molecules, as well as inactive hybrid molecules.

Spontaneous variants of P3/X63-Ag8 have been selected that neither synthesize nor secrete immunoglobulin molecules, but retain the ability to rescue normal antibody-producing cells. These are listed below; all are resistant to 8-azaguanine.

1 NS1-Ag4-1 synthesizes, but does not secrete, κ light chain. Hybrids can still secrete a mixed molecule of antibody heavy chains with myeloma light chains.

2 P3/X63-Ag8-6.5.3 does not synthesize or secrete immunoglobulin chains.

3 SP2/0-Ag14 is a non-synthesizing and non-secreting variant of a hybrid cell formed by the fusion of a lymphoid cell (secreting antisheep erythrocyte antibody) with P3/X63-Ag8. We would recommend that you use one of the non-synthesizing, non-secreting variants for

any fusion work; these may be acquired from commercial suppliers or a friendly research laborat- ory doing routine fusions. It should be noted that cells from commercial suppliers are generally cloned and passaged through 6-thioguanine to maintain their HAT sensitivity.

Fig. 2.1 (opposite) DNA synthesis and the selection of HAT-sensitive mutants. (a) Most cells can make DNA either by de novo synthesis or via the so-called ‘salvage’ pathway, using an endogenous or exogenous source of preformed bases. (b) In order to select cells which are solely the result of fusion with a normal antibody- forming cell with a myeloma cell, de novo DNA synthesis is partially stopped with aminopterin A. This reagent blocks the enzyme dihydrofolate reductase, while the salvage pathway is blocked by preselecting for the myeloma cells that can survive in the presence of the lethal purine analogues, 8-aza- or 6-thioguanine. These cells lack the key salvage pathway enzymes HGPRT and TK. These enzymes are then supplied by the normal antibody-forming partner cell. (c) The tumour ‘parent’ used in the hybridoma technique (× 63) is HGPRT deficient and so is unable to grow in HAT medium; HAT contains either the folic acid analogue aminopterin or the amination inhibitor, azaserine, which block de novo synthesis; as these cells cannot use hypoxanthine they die. Hybrid cells grow out from HAT medium because DNA from the normal partner provides the information to synthesize HGPRT and the tumour cell DNA provides the ‘message’ for unrestricted proliferation.

2.2OUTLINE OF B-HYBRIDOMA PRODUCTION