Cell Fusion

7.2. Cell Fusion

Fig. 2.12. vividly illustrates the underlying principle of generating monoclonal antibodies, which are duly accomplished by the fusion of an antibody forming cell (invariably a spleen cell) with a myeloma* cell particularly in the presence of one of a variety of fusing agents e.g., polyethylene glycol (PEG).

* Tumour of B-lymphocyte (or cell).

GENETIC RECOMBINATION

Immune spleen cells and HGPRT myeloma cells are fused with polyethylene glycol resulting in hybrid cells.

Immune

HGPRT

spleen cells

myeloma cells

Cells are distributed in microwell plates and grown in HAT medium. Unfused myeloma cells die because

Fuse with

they have no HGPRT to use

PEG

the salvage pathway. Unfused spleen cells are unable to

HAT grow in vitro ; only fused cells selection

grow[ ].

Fused cells (hybridomas) Antibody

grown in microwells and screening

supernate of each well tested for specific antibody production. Wells making antibody of interest are subcloned and grown in

larger volumes. Subcloning

Fig. 2.12. Production of Monoclonal Antibodies (MABs).

[Cells from an immunized mouse are fused with myeloma cells and undergo HAT* medium selection. The fused cells, called hybridomas, are then screened for their ability to react to the antigen of interest. The clones of interest are then subcloned and expanded.]

This ultimately results into the formation of ‘hybridoma’. Separation of fused hybridoma

cells from normal spleen cell population :

The separation of fused hybridoma cells from the normal spleen cell population is achieved most conveniently by virtue of the glaring fact that the spleen cells normal die off in culture after a short period of time. Interestingly, the unfused myeloma cells and the hybridoma cells are absolutely ‘im- mortal’ ; and, therefore, the dire need of an efficacious and specific method is of prime necessity to get rid of the unfused myeloma cells. It is achieved by employing myeloma cells which are killed in the presence of the drug aminopterin.

8-Azaguanine (8 AzG)

PHARMACEUTICAL BIOTECHNOLOGY

Just like a plethora of cells, myeloma cells, predominantly make use of two altogether distinct pathways for the nucleotide synthesis, namely : (a) synthetic pathway (major one) ; and (b) salvage pathway (minor one) — as depicted in Fig. 2.13.

Salient Features : The various salient features of Fig. 2.13. are as stated below : (1) Normal cells invariably synthesize nucleotides by employing both pathways. (2) The very incorporation of the drug 8-azaguanine to the normal cells, enables it to penetrate

right into the DNA through a reaction catalyzed by a specific enzyme HGPRT* via the salvage pathway. Such cells eventually prove to be fatal as they simply cannot function with an altered base.

(3) Evidently, a variant cell that cannot get along with the salvage pathway by virtue of the fact that it specifically lacks HGPRT should be 8 AzG resistant ; and, therefore, would not be killed by the said drug substance (i.e., 8 AzG).

(4) Therefore, such HGPRT – mutants may be selected particularly with this drug ; and these are the actual myeloma cells generally employed for the desired fusion.**

MAJOR SYNTHETIC PATHWAY [DE NOVO BIO SYNTHESIS]

PURINE

PYRIMIDINE

PURINE RIBONUCLEOTIDES

d UMP

SALVAGE AMINOPTERIN PATHWAY

Fig. 2.13. Aminopterin Works by Blocking the Reduction of Dihydrofolate (FH 2 ) to

Tetrahydrofolate (FH 4 ).

Explanation : In pyrimidine biosynthesis CoFH 4 is oxidized to FH 2 , thereby using up FH 4 . In purine biosynthesis CoFH 4 is converted to FH 4 non enzymatically so that FH 4 can be reconstituted to CoFH 4 . Aminopterin blocks the conversion of FH 2 to FH 4 so that no more FH 4 can be generated. As soon as it has depleted the existing FH 4 , the cell can no longer

* Hypoxanthine guanine phosphoribosyl transferase (HGPRT). ** The best fusion partners are HGPRT – cells that have also lost the ability to produce either the H or the L

chain of the immunoglobulin (Ig). These are known as non secretors.

GENETIC RECOMBINATION

function. As a consequence of the pyrimidine pathway now using up virtually all of the FH 4 , the purine pathway also stops ; however, the cell still carries out DNA synthesis via the

salvage pathway.

(5) Aminopterin acts on the major synthetic pathway (or De Novo Biosynthesis) by interfering directly with the conversion of dihydrofolate (FH 2 ) to tetrahydrofolate (FH 4 ) and also pre- venting a series of one-carbon transfers particularly. Hence, in the very presence of aminopterin the cell cannot synthesize nucleotides via the main synthetic patyway ; and,

therefore, should take the salvage pathway instead. (6) A normal cell can still grow in the presence of aminopterin, whereas a HGPRT – cell cannot,

perhaps due to the fact that the prevailing HGPRT – mutants cannot carry out the salvage pathway ; and the net effect would be the fatal fate of the ensuing HGPRT – cells in the

presence of aminopterin. (7) In a situation, when HGPRT – myeloma cells get fused with the normal B lymphocytes (or B

cells), it has been duly observed that the resulting hybridomas are capable of growing in the presence of aminopterin because the normal cell profusely contributes functional HGPRT.

(8) In another situation, when two chemical entities e.g., hypoxanthine and thymidine, which are recognized as the precursor molecules employed by the enzyme HGPRT in the salvage pathway, are duly incorporated into the medium, evidently the ensuing hybridoma is capa- ble of using the alternate pathway in the synthesis of DNA.

(9) Importantly, the unused normal spleen cells usually die as they are not capable of growing for longer span in the tissue culture, and the unfused myeloma cells get killed by the aminopterin.

(10) As a result the only fused hybridomas are able to grow adequately. This particular pheno- mena is known as the HAT selection (i.e., hypoxanthine, aminopterin, thymidine selection).

Four Important Principles of HAT-Selection :

The four most important principles of HAT selection are as enumerated under : (a) When the major synthetic pathways are blocked by the folic acid analogue aminopterin,

the cell should employ the salvage pathway. This pathway essentially contains the enzyme HGPRT.

(b) HGPRT – myeloma cells may be selected particularly because they can grow in the presence of 8-azaguanine (i.e., 8-AzG). HGPRT + cells incorporate 8-AzG into DNA. HGPRT – cells

do not incorporate the toxic molecule. Hence, HGPRT – cells can grow in its presence. (c) HGPRT – cells die in the presence of HAT because both the major synthetic pathway and

the salvage pathway are blocked virtually. (d) Fusion of the HGPRT – myeloma cells with the HGPRT + spleen cells allows growth in HAT by incorporating the missing enzyme for the salvage pathway.