Reproduced by kind permission from Old and Primrose 1985 p

cleavage site is at one end of the gene and a Bam HI cleavage site at the other, thereby providing cohesive termini to facilitate its insertion at these sites in the plasmid vector.

The plasmid used is the artifically created plasmid pBR 322 which has been completely sequenced. About 20 endonucleases cleave this plasmid at known sites. Two antibiotic resistance markers are associated with this plasmid: tetracycline resistance (Tc r) and ampicillin resistance (Apr). Small peptides like somatostatin are rapidly degraded by E. coli and it is necessary to fuse the somatostatin gene to the beta-galactosidase gene for protection. This is achieved by inserting the beta-galactosidase gene together with the lac control region adjacent to the somatostatin gene. The lac control region comprises a promoter site, an operator site which "switches on" the adjacent structural genes, and the ribosomal binding site. Thus we have all the elements necessary for successful transcription and subsequent translation. The initial and final plasmid is illustrated in Figure 12.4. Note that the Bam HI site is in the Tcr marker and cutting and gene insertion results in the loss of tetracycline resistance.

The hybrid protein produced at the ribosome consisting of the beta-galactosidase protein fused to the somatostatin is treated with cyanogen bromide (CNBr). This cleaves at the methionine residue, which lies between the two molecules, yielding somatostatin plus beta-galactosidase fragments. The use of CNBr cleavage is limited to those peptides not possessing methionine as part of their sequence. The somatostatin is detected by immunoassay. Similar techniques have been used for the synthesis of other smaller peptides such as endorphins and enkephalins, which are considered to be opioid peptides.

12.1.3.2 Insulin

Insulin is an excellent example of how the problems of post-translational modification have been overcome. The protein secreted contains a signal sequence of amino acids at the N-terminus. During passage through the membrane these are cleaved off so that the pro-insulin formed consists of three chains (A, B, and C). Insulin is formed by the removal of the C chain by proteases. This leaves the A and B chains of insulin in a stable tertiary structure held together by the two disulphide bonds formed when the molecule originally folded as pro-insulin. Bacteria cannot bring about these processes. Thus the approach used is to construct separately the genes for the A and B chain, insert them separately into pBR plasmids and then add the elements of the lac control and beta-galactosidase gene, followed by cloning into the two separate bacterial strains. Thus one culture is producing a hydrid A chain and another a hybrid B chain. Separate cleavage with CNBr frees the two polypeptide chains which, after purification, can be joined by disulphide bonds. Genetically engineered human insulin has now replaced porcine insulin in use.

Emfll

Figure 12.4 Diagrammatic representation of the steps involed in generating a recombinant plasmid for the bacterial synthesis of somatostatin (Reproduced by kind permission from Emery (1984), p. 163).

12.1.3.3 Human growth hormone (HGH)

Initially, the only source of HGH was human pituitary tissue which was removed at autopsy. HGH from this source was insufficient for clinical treatment. The peptide contains 191 amino acids and although chemical synthesis of the gene is feasible, it is not an easy process. It was therefore necessary to prepare cDNA (copy DNA) by extracting mRNA from pituitary tissue. It was found that there was an Hae III cleavage site in the sequence coding for amino acids 23 and 24. Treatment with Hae III yielded the larger fragment (amino acids 24-191) which could then be combined with the smaller chemically synthesised fragment (amino acids 1-23) which was preceded by the initiating amino acid methionine. This was now inserted into the plasmid next to an appropriate promoter, etc., that is required for successful transcription and translation. The product, however, is not completely identical to the natural hormone as it contains an extra NH2-terminal, methionine, which could induce an immunological reaction. An alternative approach is to insert the entire cDNA

sequence into a SV (Simian virus) 40, using monkey kidney cell tissue culture as the host. The HGH excreted by this method is identical to that found in the pituitary gland.

12.1.3.4 Lymphokines and monokines

These are families of proteins that have been shown to exhibit antiviral effects, together with the enhancement of elements of the immune system with resultant anticancer effects. These proteins regulate the cellular parts of the immune system. Macrophages produce monokines and the T cells and B cells produce lymphokines. For example alpha, beta and gamma interferons are produced by the leukocytes, fibroblasts and activated T cells, respectively. Natural yields of lymphokines and monokines are low but gentically engineered human versions are now available.

12.1.3.5 Puriy of genetically engineered proteins

A variety of analytical methods is used to ensure that products resulting from genetic engineering are fit for human use. These include tests for:

(1) Identity: polyacrylamide gel electrophoresis, isoelectric focusing, chromatography (particularly reverse phase HPLC), peptide mapping (in which the protein is digested under controlled conditions by protease enzymes and the HPLC profile of the resulting polypeptide fragments serves as a finger print for the parent protein), amino acid analysis, and spectroscopy.

(2) Purity: chromatography, spectroscopy, assays for host DNA, assays for pyrogens and other residual cellular proteins derived from the outer membranes of the host organism, particularly for products to be administered chronically or in high doses.

(3) Potency of the product: bio-assay against a national or international reference preparation. This ensures that the product has the required biological activity.

12.1.3.6 Gene therapy

The above applications aim to counteract the deficiency of a natural protein through its substitution by injection of the protein synthesized outside the body through genetic engineering. Some diseases are due to defects in the patient's genes, and examples of such diseases are listed in Table 12.2, together with the deficient gene. This deficiency may be manifested in the lack of production of a hormone or factor, synthesis of an inactive enzyme, or synthesis of a malfunctioning receptor. It is now possible in some cases to synthesize the normal gene and to insert it into a vector using the processes described above, to produce vectors which express the functioning human gene.

Table 12.2 Some diseases possibly amenable to treatment by gene therapy.

_Disease state__Defective gene_

Cancer-melanoma HLA-B7

Cystic fibrosis cystic fibrosis transmembrane regulator Growth hormone deficiency growth hormone Haemophilia factor VIII and factor XI Hypercholesteremia low density lipoprotein receptor Phenylketonuria_phenylalanine hydroxylase_

Transplantation of this vector into the patient so that the vector produces the required gene product in the patient has been attempted with some success for cystic fibrosis. Alternatively the DNA for the normal gene could be introduced into the patient's own cells via genetic engineering.

12.2 MONOCLONAL ANTIBODIES 12.2.1 Introduction

Antibodies are proteins that are designed to bind specifically to foreign or antigenic molecules or microorganisms which invade higher living organisms. This specific binding initiates a range of in vivo processes designed to neutralise the adverse biological activity of the invading molecules or micro-organisms and expedite their elimination from the body. It is their specific binding with relatively high affinity to antigens which has found exploitation in many areas of biological sciences. It has been the ease of production of antibodies to a wide variety of antigenic species coupled with the ability to produce a single type of antibody of constant specificity and composition through monoclonal antibody technology, that has led to the use of antibodies as potent biological targeting agents for in vivo use and as diagnostic agents.

12.2.1.1 The immune system

Higher animals possess a highly sophisticated immune system. Substances that activate the immune system are known as antigens. Two kinds of effector mechanisms mediate the immune response to antigens. One response is mediated by a population of lymphocytes known as T lymphocytes (T cells). These T cells act in conjunction with a second set of lymphocytes termed B lymphocytes (B cells) to ensure that antibodies are only produced to foreign molecules and micro-organisms. It is the function of activated B cells to produce antibodies and of the T cells to police the antibody production process so that antibodies are only produced to invading foreign molecules and micro-organisms.

Each B cell has on its surface a unique receptor. A small fraction of the B cells which normally circulate in the blood and lymph will fortuitously bind to patches on the surface of the foreign molecule or micro-organism (called epitopes). The T cell screens the resulting B cell-molecular complex and if the foreign molecule does not possess the marker flags on its surface which identify it as "self" then the T cells signal to the complexed B cells to activate division of these B cells through release of activator molecules (particularly interleukins). The activated B cells now rapidly increase in number and secrete antibodies which possess antibody binding sites, the structure of which is identical to the receptors on the pre-activated cells which themselves bound to the foreign molecules or micro-organisms. Hence these antibodies will bind to the same foreign molecules or epitopes on the surface of larger invading micro-organisms. This is the basis of the humoral immune response.

It should be noted that each activated B cell will produce a series of identical daughter cells or clones each of which secretes the same unique antibody. In practice a number of B cells are activated for each antigen and consequently a range of different cloned B cells is generated and hence a variety of antibodies each recognizing different epitopes will be present in the antiserum of the animal exposed to the foreign antigen. The resulting antiserum is termed a polyclonal antiserum.

12.2.1.2 Antigens

An antigen is any substance which can elicit an immune response in an animal which possesses a functional immune response. Proteins which are foreign to the animal are generally highly immunogenic and will stimulate the production of a range of different antibodies, each being specific for a particular determinant (or epitope) associated with the surface of the protein. These antibodies will bind to that particular portion of the protein only. Polysaccharides and nucleic acids are less immunogenic than proteins, even though they may have a high molecular weight. Low molecular weight molecules (Mr<about 2000) do not elicit the formation of antibodies but when they are covalently coupled to a foreign protein molecule which acts as a carrier for these smaller molecules, a range of antibodies may be produced when the carrier-complex is used as an immunogen, some of which recognise the smaller molecules on the surface of the carrier complex. These antibodies can now also bind to the uncomplexed small molecules in solution. In this way antibodies to a range of smaller molecules including drugs, steroids, vitamins and pesticides, can be raised.

12.2.1.3 Antibody structure and classes

All antibodies have the same general structure and are symmetrical molecules made up of four polypeptide chains. Two chains contain identical sequences of 400-500 amino acid residues and are termed heavy (H) chains. The two other chains are termed light (L) chains and these contain over 200 amino acid residues. The H chains are joined together by disulphide bonds and each L chain is joined to a H chain by a disulphide bond. The sequence of amino acids in the amino terminal half of both H and L chains differs substantially between antibodies stimulated by different antigens. This region of the chain is termed the variable region (VH and VL) and within each of these variable regions lie three hypervariable segments. The VH and LH are folded in such a way as to bring together the hypervariable regions together to form a groove or cavity into which the epitope fits. The antigen-antibody binding is highly specific due to the stereochemical complementarity which is required coupled to complementary hydrophobic and/or ionic interactions between the amino acids in the binding site and the contact groups on the surface of the epitope.

The carboxyl terminal shows far less sequence variation between antibodies and is termed the constant region (C) of the antibody molecule. Each species of animal will produce identical constant regions for each subclass of antibody. Cleavage of the antibody molecule can be achieved with proteolytic enzymes such as pepsin and trypsin into fragments that retain antigen binding properties (termed Fab fragments) and fragments that do not (termed Fc fragments). The major humoral antibody is IgG. Its structure is illustrated in Figure 12.5.

12.2.2 Monoclonal antibodies

It was noted in Section 12.2.1.1 that an activated B cell will subdivide to produce a clone of identical daughter cells each of which secretes identical antibodies. If a single activated B cell could be isolated and cloned then the resulting antibodies derived from a single clone of daughter cells is termed a monoclonal antiserum. Unfortunately it is not feasible to produce monclonal antobodies via this route as the quantities of antibodies thus obtained are limited since culture and growth of the cell line will rapidly result in cell death due to the mortality of B cells. It is therefore necessary to render activated B

Figure 12.5 Diagrammatic representation of a molecule of human IgG.

cells immortal and this is achieved by fusing the genetic material from the required B cells with a cancerous B cell from the same species since cancerous cells are immortal and the resulting hybrid cells should contain the genes for production of the required antibody and for immortality. Large scale tissue culture of the resulting fused cells should enable production and harvesting of monoclonal antibodies on commercial scales.

In practice mice or rats are used and the most common type of mouse used for monclonal antibody production is an inbred strain known as BALB/C. The cancerous B cells required for fusion are myeloma cells. Kohler and Milstein in 1975 demonstrated that mouse myeloma cells could be fused with B cells taken from the spleen of immunized mice and the resultant hybrids produced antibody. The technique is called somatic cell hybridization and the cell product termed a hybridoma (Figure 12.6).

The five major steps involved in monclonal antibody formation via this process are as follows:

(1) Immunization of the selected animals (usually BALB/C mice) with immunogen.

(2) Isolation of the spleen from a hyperimmunized animal and fusion of B cells from the spleen with myeloma cells from the same species.

(3) Culture of the resulting hydidoma cells in (HAT) medium.

(4) Selection of single clones of immortal cells secreting the required antibody.

(5) Scale up of the culture process to produce the required monclonal antibody.

The initial stage is to repeatedly inject the antigen into a group of about six animals, often in conjunction with immunostimulants such as Freund's adjuvant (complete for the first immunization followed by incomplete for subsequent ones). Immunizations take place at intervals of about a week and with successive immunizations there is an increased stimulation of B cell clones within the animal responding to the antigen. The presence of

SDCCi'iC intiDDdY tftt'SIOr

Figure 12.6 Principle of hybridoma formation.

SDCCi'iC intiDDdY tftt'SIOr

Figure 12.6 Principle of hybridoma formation.

high concentrations of antibody to the antigen can be demonstrated by taking blood samples from the animal and analysing the sample. In this way the animal giving the best response to the antigen can be identified.

A suitable source of these B cells is the spleen from which they are harvested after the animal showing the best response has been sacrificed. The fusion partners (the myeloma cells) are now well established cell lines and many of these lines have mutated to being non-secretors or better still, non-synthesisers of antibody. The latter is the ideal partner for the antigen-stimulated B cell. The cell fusion is normally carried out using large numbers of the two cell types, as the rate of successful fusion is low (about 1 in 105 cells). The inclusion of 50% polyethylene glycol (PEG, Mr 140— 4000) and about 5-10% dimethyl sulphoxide (DMSO) in the solution creates a favourable medium for membrane-membrane fusion between cells to occur.

Due to the low fusion rate and of the probability that fusion will occur between identical cells in addition to that between B cells and myeloma cells, the fused cells are transferred to a medium which only allows the required successful fusions to flourish. The standard technique is to use myeloma cells which have lost the capacity to synthesize hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT), one of two enzymes required for the eventual synthesis of DNA in the cell.

The other pathway leading to DNA synthesis is the salvage pathway and it can be blocked by addition of the chemical aminopterin to the cell culture medium. If the transferred cells from the fusion step are placed in a medium containing aminopterin (A) then only cells containing HGPRT can survive. The gene for this enzyme will be derived from the activated B cells and use of this pathway for DNA production is also encouraged by the presence of hypaxanthine (H) and thymidine (T) in the medium since these compounds are utilised in the synthesis by the enzyme. The medium is termed HAT medium due to the presence of these three additives in it.

Unfused cells and fused cells containing genetic material from only the B cells will not survive in the HAT medium as the unfused myeloma cells die due to inability to synthesize DNA while the other cells eventually die out. Hybridoma cells are able to proliferate as they contain the genes for immortality and for HGPRT.

As antigens contain many epitopes, it follows that the resulting hydridoma cells will secrete a number of antibodies corresponding to these epitopes. It is therefore necessary to screen for clones which produce antibodies to the required epitope. This is achieved in two steps; firstly single hydridoma cells are isolated and then these are screened to identify which are producing the required antibodies.

Two strategies can be used for cloning cells:

(a) Limiting dilution: the hydrid suspension is diluted so that the volume put into each culture well contains on average a single cell. No growth will occur in those wells that receive no cells and those wells that receive more than one cell may result in antibodies of two specificities being found. The clones that develop may be broken up and the dilution process and cloning repeated several times to ensure monoclonality.

(b) Solid medium: a solid medium based on agarose gel has been developed which permits the cells to divide on the surface to form visible colonies. These can be broken up and the process repeated to obtain pure clones of hydridoma cells which can then be transferred to a suitable culture medium to test for antibody production.

The clones are screened for antibody production by using a suitable assay technique which can initially detect the class of Ig being produced, since IgG or IgM are commonly produced. This test uses a second antibody reagent which specifically binds to IgG or IgM molecules and this binding interaction can be monitored. The next step is to determine whether any of the clones are secreting antibody to any of the epitopes assocaited with the antigen. Once again the use of a second antibody reagent is employed (see Figure 12.7). The test uses the antigen or epitope immobilised on a convenient surface such as the wells of microtitre plates. If the culture supernatant contains antibodies from the antigen then these will bind to the epitope on the surface. Addition of a labelled

Figure 12.7 Use of anti-antibodies to detect synthesis of antibody by hybridoma cells.

Figure 12.7 Use of anti-antibodies to detect synthesis of antibody by hybridoma cells.

antibody, which specifically binds to the class of antibody identified in the first screen, to the wells will result in the formation of a second antibody complex linked to the first murine antibody attached to the epitope on the surface. After washing the plate to remove excess second reagent, the presence of the bound second reagent in the complex can be determined from the presence of label in the well(s).

Once the clones secreting the required antibody have been identified those cells are transferred to large culture vessels where their propagation can take place and monclonal antibodies subsequently harvested. A variety of culture systems can be employed ranging from conventional animal cell culture in bottles to use of hollow fibre systems onto which cells adhere and through which the culture medium is pumped. The latter systems offer advantages since the secreted antibodies can be collected in the medium emerging from the fibres and higher yields of antibody can be obtained (g/l compared with mcg/l for conventional culture systems). Alternatively, the murine hydridoma can be propagated in BALB/C mice where the concentration in ascites fluid can reach 5-20 mg/ml. A diagrammatic representation of monoclonal antibody production is shown in Figure 12.8.

Monoclonal antibodies are chemically homogeneous but may not be truly monospecific as they can react with antigens that share an epitope or can react with epitopes that are structurally closely related.

Figure 12.8 Diagrammatic representation of monoclonal antibody production.

12.2.3 Application of monoclonal antibodies

There has been a dramatic increase in the use of monoclonal antibodies and the greatest expansion has been in the field of therapy where they have been used to target drugs to specific tissues. However they will continue to find an ever increasing role as analytical and diagnostic reagents due to their reproducible properties of specificity and affinity for specific antigens, and from the ability to produce them in commercial quantities.

12.2.3.1 Analytical

Because of their specificity, monoclonal antibodies are used extensively in the assay of serum and urine levels of hormones (e.g. detection of human chorionic gonadotrophin in the urine of pregnant women), drugs, enzymes, etc. They are also finding application in assays for environmental contaminants such as pesticides and dioxins, in soil, water and air samples. A wide range of immunoassay procedures has been developed which employ a variety of end points. Of particular importance are noncompetitive, nonisotopic assays based on fluorescence and spectrophotometric end points, the latter resulting from use of enzyme labels to rapidly convert substrates to coloured products. Such assays are used for therapeutic drug monitoring where fully automated assays provide results within seconds or minutes per sample.

12.2.3.2 Diagnostic

Monoclonal antibodies have been used to identify specific antigens associated with cell surfaces. This has led to the development of rapid tissue typing techniques prior to transplantation surgery, the classification of cells and sub-populations (particularly the T cell family), cell-cell interactions and differentiation, the biochemistry of cell surfaces, the classification of micro-organisms and the detection of tumour markers associated with certain types of tumour.

12.2.3.3 Therapeutic

One of the most exciting potential uses of monoclonal antibodies is in cancer. The imaging and location of metastases using radiolabelled antibodies has been used with some success, but the use of antibodies as targeting agents for drugs, plant and bacterial toxins is an area into which tremendous effort is being directed. Combinations of antibodies joined together through their Fc regions are also being investigated for therapeutic use. These so called bi-specific antibodies work by targeting the second piggy-backed antibody to the required site within the body, such as a cancer cell, where the targeting antibody binds. The accompanying antibody is specific to a second cell such as a T cell which it captures. The resulting accumulation of T cells on the surface of the cancer cell may result in its destruction through initiation of the immune response.

Another theraputic application of monoclonal antibodies is as rescue agents where they are injected into the blood of a patient who has present in their body a potentially lethal amount of some toxic agent. This can be the result of a drug overdose, e.g digoxin, or can result from the bite of a spider or snake, or can result from bacterial toxins such as those associated with blood poisoning. The injected antibody rapidly binds to the toxic agent which is inactive in the resulting complex. In this form the neutralised toxin is eventually excreted from the body.

Vaccines derived from genetic engineering also promise to provide us with non pathogenic vectors which have been genetically modified to express on their surface one or more antigenic epitopes from viruses, bacteria and organisms which currently produce widespread illness and mortality such as measles, turberculosis, diphtheria, poliomyelitis and so on. Thus a single vaccination to a polyvaccine from such an engineered vector would be highly cost effective and safer to use. Alternatively, instead of using live recombinant vectors it is possible to produce and isolate recombinant surface antigen proteins for use as vaccines. Examples of recombinant vaccines include recombinant BCG, an avirulent bovine tubercle bacillus, and the recombinant surface antigen protein for hepatitis B.

12.2.3.4 Humanised antibodies and alternative production methods

It can be appreciated that if murine antibodies are repeatedly used as in vivo therapeutic agents in man then they will evoke an immune response as they are foreign proteins. In order to overcome this problem a variety of alternatives have been explored to make murine antibodies more like human antibodies, to produce human antibodies by genetic engineering, and by use of in vitro methods which do not involve animals at all.

It was noted in Section 12.2.1.3 that antibodies can be split by pepsin into two fragments, one of which retains the antibody binding capacity while the other corresponds to the constant region of the molecule. Digestion of the specific murine antibody and isolation of the Fab fragment can therefore be achieved. In a similar manner human IgG can be divided into its Fc and Fab fragments. It is posible to splice together the Fab fragment of the murine antibody with the isolated non-antigenic Fc fragment of the human IgG to produce a hydrid or chimaeric antibody which possesses the required specificity but with reduced antigenicity.

Since the amino acid sequences can be determined for both the Fc region of human Ig and the Fab region of the murine antibody, it is possible to synthesise the codon sequence corresponding to the entire chimaeric molecule. When the appropriate promoter and initiator codons have been added to the gene it is possible to insert it into an appropriate host via a plasmid or bacteriophage, with subsequent translation producing a genetically engineered human IgG of the required epitope specificity.

A more recent approach involves randomly synthesising codons within the Fab region spliced onto the codon sequence for the human Fc region. The specificty of the resulting antibody is noted and a library of codon sequences built up correlating sequence to specificty. In this way it is hoped that tailor made antibodies of required specificity will be synthesied via genetic engineering from the library information.

FURTHER READING

Brown, T.A. (1991) Essential Molecular Biology, Volume 1 A Practical Approach. Oxford: IRL Press.

Emery, A.E.H. (1984) An Introduction to Recombinant DNA. Chichester: John Wiley and Sons Ltd.

Fanger, M.W. and Guyre, P.M. (1991) Bispecific antibodies for targeted cytotoxicity. Tibtechnology 9, 375-380.

Holliger, P and Winter, G. (1993) Engineering bispecific antibodies. Current Opinion in Biotechnology 4, 446-449.

Hudson, L. and Hay, F.C. (1989) Practical Immunology, 3rd ed. Oxford: Blackwell Scientific Publications.

Hurle, M.R. and Gross, M. (1994) Protein engineering techniques for antibody humanisation. Current Opinion in Biotechnology 5, 428-433.

Lerner, R.A., Kang, A.S., Bain, J.D., Burton, D.R. and Barbas, C.F. (1992) Antibodies without immunisation. Science 258, 1313-1315.

Old, R.W. And Primrose, S.B. (1985) In Principles of Gene Manipulation, edited by N.G.Carr, J.L.Ingraham and S.C.Rittenberg. Oxford: Blackwell Scientific Publications.

Paliwal, S.K., Nadler, T.K. and Regnier, F.E. (1993) Rapid process monitoring in biotechnology. Tibtechnology 11, 95-101.

Pezzuto, J.M., Johnson, M.E. and Manasse, H.R. (1993) Biotechnology and Pharmacy New York: Chapman and Hall.

Tomlinson, E. (1992) Impact of new biologies on the medical and pharmaceutical sciences. Journal of Pharmacy and Pharmacology 44, Supplement 1, 147-149.

Walker, J.M. and Gingold, E.B. (1993) Molecular Biology and Biotechnology, 3rd edn. Cambridge: The Royal Society of Chemistry.

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