Recombinant Dna Technology Monoclonal Antibodies

FREDERICK J.ROWELL and JAMES R.FURR

CONTENTS 12.1 RECOMBINANT DNA TECHNOLOGY

12.1.1 Introduction

12.1.2 Principles of recombinant DNA technology

12.1.2.1 Identification and isolation of the required gene

12.1.2.2 Modification of the isolated gene prior to insertion

12.1.2.3 Insertion into the cloning vector

12.1.3 Production of polypeptides using recombinant DNA technology

12.1.3.1 Somatostatin

12.1.3.2 Insulin

12.1.3.3 Human growth hormone (HGH)

12.1.3.4 Lymphokines and monokines

12.1.3.5 Purity of genetically engineered proteins 499

12.1.3.6 Gene therapy 12.2 MONOCLONAL ANTIBODIES

12.2.1 Introduction

12.2.1.1 The immune system

12.2.1.2 Antigens

12.2.1.3 Antibody structure and classes

12.2.2 Monoclonal antibodies

12.2.3 Application of monoclonal antibodies

12.2.3.1 Analytical

12.2.3.2 Diagnostic

12.2.3.3 Therapeutic

500 500

501 501 501 506 506 506 506

12.2.3.4 Humanised antibodies and alternative production methods 507

FURTHER READING

12.1 RECOMBINANT DNA TECHNOLOGY 12.1.1 Introduction

For hundreds of years mankind has utilised micro-organisms to produce a whole range of natural products which we can use (e.g. ethanol, organic acids, dextrans, antibiotics). Micro-organisms are extremely easy to cultivate and large scale culture results in high yields of the product required which can then be purified and utilized. Natural products can also be extracted from plant tissue. Biologically active compounds from animals can be isolated from the appropriate organ or tissue but as these are extremely potent compounds, they are normally only present in small quantities and large amounts of the appropriate tissue are required to obtain useful quantities of the product. This is a particular problem with compounds of human origin due to lack of cadavers and to the possibility of contamination of the resulting product with human viruses such as hepatitis and the AIDS virus.

For proteins extracted from animals and used in humans such as insulin derived from pigs, since the protein is not chemically identical to the equivalent human protein, its use may evoke an immune response leading to sensitisation of the patient. Somatostatin, a hormone that inhibits the secretion of pituitary growth hormone, required half a million sheep brains to be processed to yield about 5 mg of the product. Today, using recombinant DNA technology, the same amount of hormone corresponding to the human protein can be harvested from 9 litres of a culture medium in which has been grown a micro-organism possessing the inserted human somatostatin gene. It is therefore now possible to produce, in large quantities, a whole range of biologically active polypeptides of identical composition to those found naturally in humans or any other living organism using recombinant DNA technology (often termed genetic engineering).

12.1.2 Principles of recombinant DNA technology

The insertion of a human gene into, say, a bacterial cell can only be achieved through techniques that enable the gene to replicate within the cell so that all the progeny derived from the original cell possess the inserted gene and that the product defined by the genetic code from the inserted gene is produced via the normal processes of transcription and translation within the cells of the recipient or host cells. The technique is to insert the gene into extranuclear DNA molecules such as plasmids (extrachromosomal loops of DNA) and bacteriophages (viruses that utilise bacteria as their hosts). They are easily isolated from cells and opened up so that the new gene can be covalently attached to the open strand of DNA, the loop reformed, and the plasmid or bacteriophage containing the new gene re-inserted into the bacterium or other host cell. The agents which open and reform the DNA loops are specific enzymes termed endonucleases and ligases respectively.

The six main steps in the genetic engineering process (see Figure 12.1) are therefore;

(1) Isolating the gene for the protein to be synthesised.

(2) Opening the cloning vector (the plasmid or bacteriophage).

(3) Covalently attaching the DNA corresponding to the new gene into the open loop of the cloning vector.

(4) Reforming the bonds within the enlarged DNA to regenerate the loop.

(5) Re-inserting the enlarged vector into the host.

(6) Culturing the mutant or chimaeric host cell to enable it to replicate and produce the required protein which is isolated from the culture medium.

12.1.2.1 Identification and isolation of the required gene

If the amino acid sequence of the protein to be synthesized by recombinant DNA technology is known then its complementary sequence of codons (triplet sequences of nucleotides, the sequence of each codon corresponding to the amino acid sequence in the protein) can be synthesized. If the protein consists of many amino acids, as is the usual case, then this approach is impractical and the approach is limited to synthesis of the sequences of codons unique to the gene for the required protein. In addition the synthesis incorporates a radioactive tag, usually in the form of 32P into the sugar-phosphate backbone of the synthetic DNA strips. These radioactive fragments will now bind to the complementary codons on the DNA corresponding to unique codon sequences of the required gene. This provides us with the means of identifying the location of the required gene within a multitude of DNA molecules obtained from synthesis of mRNA mixtures using the enzyme reverse transcriptase and nucleotides.

It is assumed that cells or tissues which are producing the required protein will also have a high concentration of the messenger RNA (mRNA) coding for the protein since this contains the transferred genetic message which is read at the ribosome during the synthesis of the protein. mRNA from these target cells or tissues is extracted and the minute amounts of mRNA thus isolated can be transformed into the genetic DNA code for the protein and finally larger quantities of this key intermediate product can be synthesised using a second enzyme called DNA polymerase.

In practice the process described is more complicated since firstly, single stranded mRNA must be used and is formed from the double stranded naturally occurring form and secondly, the mRNA coding for the required gene will be embedded within a much larger segment of mRNA coding for a variety of other genes. Hence this large mRNA fragment has to be cut into smaller pieces using specific enzymes and this process may cut the required gene into smaller segments in the process whereas only the complete intact sequence corresponding to the code for the protein is required.

12.1.2.2 Modifications of the isolated gene prior to insertion

Having isolated the DNA sequence coding for the required gene it is necessary to ensure that it is in a form which can be successfully incorporated into the DNA of the cloning vector. This requires four key preliminary modifications of the gene.

Firstly, sequences of DNA known as introns which are interruptions of the code found in mammalian genes must be removed enzymatically since their presence leads to incorrect translation of the spliced gene by the bacterial or viral cloning vector during protein synthesis.

Secondly a signal has to be incorporated at the beginning of the DNA code for protein to signal to the enzyme RNA polymerase to initiate the transformation of mRNA from the synthetic DNA code for the protein. Likewise the correct signals must be attached to the gene's DNA code to instruct the vector's ribosome to start and stop the gene's synthesis during the translation process. A diagrammatic representation of the resulting vector plasmid is illustrated in Figure 12.2.

Thirdly an ancillary DNA code for a marker gene (e.g. the gene for resistance to tetracycline) is spliced adjacent to the gene coding for the required protein. This serves as a means of detecting whether the total sequence has been successfully incorporated in to the host's DNA.

Finally the synthesized double stranded DNA carrying all the modifications listed must be treated with enzymes to expose protrusions of bases at the ends of the DNA duplex that can form bonds with complementary protrusions on the ends of the opened circle of the bacterial or viral DNA to which the foreign DNA is to be attached.

12.1.2.3 Insertion into the cloning vector

The extrachromosomal circular DNA molecules found in bacteria can be separated from the rest of the chromosomal material in the cell by agarose gel electrophoresis. They can be opened up by breaking bonds between specific pairings of nucleotides in the DNA molecule using enzymes termed restriction enzymes (or endonucleases). Different restriction enzymes break bonds selectively between different pairs of nucleotides as shown in Table 12.1. In order that the correct orientation of the bases on the end of the opened plasmid and the complementary bases on the ends of the gene to be inserted occurs, it is necessary to use the appropriate endonuclease. Production of the mutually complementary sequences at the exposed ends of the strands of vector and foreign DNA produces cohesive or "sticky ended" strands since they will tend to aggregate together due to formation of complementary hydrogen bonds between them. The nicks in the sugar phosphate backbone are now sealed using a DNA ligase so the foreign gene becomes an integral part of the plasmid's genetic material (Figure 12.1).

The next process is the insertion of the chimaeric plasmid into the host bacteria. Bacteria can take up free extracellular DNA by a process termed transformation. The rate of uptake is slow but this can be enhanced by allowing the process to take place at low temperatures (0-5°C) in the presence of calcium ions. It is necessary to identify those cells which have taken up the enlarged plasmid. This is achieved by use of the marker gene such as that for antibiotic resistance. Successful incorporation of this gene together with the gene for the protein should have produced a bacterium which exhibits resistance to the antibiotic tetracycline. Hence bacteria carrying this resistance gene will survive in a culture medium containing quantities of antibiotic which is fatal to non-resistant bacteria.

Table 12.1 Recognition sites and end products of endonuclease activity.

Enzyme Recognition site

Cleavage product

EcoR I

Hae III

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