Fase Presencia

The ability of protein A to bind to immunoglobulins without inhibiting the antigen- antibody reaction has resulted in its use in a wide range of analytical and preparative immunological techniques, and a more limited number of therapeutic applications (reviews: Goding, 1978; Langone, 1982a, 1982b).

1.1.5.1 Analytical and preparative immunological techniques

One of the most significant advances in immunological techniques was the formulation by Hjelm el al. (1972) o f a reliable, low cost method for purifying immunoglobulins from serum and ascites fluid, using affinity chromatography on protein A Sepharose. In this simple one step procedure, immunoglobulin can be recovered from the column using a variety of techniques including; lowering or raising the pH (Hjelm et al., 1972); the use of chaotropic buffers such as KSCN, (Kronvall, 1973), or electrophoretic elution (Morgan et al., 1978). The use of SpA-Sepharose columns provides not only rapid high performance affinity purification of IgG immunoglobulins per se, but also facilitates the isolation o f immunoglobulins for the subsequent purification of antigens. Furthermore, they may be employed in the analysis of crude biological samples for the presence o f monoclonal antibodies.

SpA has been labelled with many types of tracer, including radionucleotides, enzymes fluorescent probes and metals (Langone, 1982b). Labelled SpA is often used in direct binding assays where it replaces the second antibody. Examples of some of the uses of labelled SpA are listed below:

(A) The study of surface antigens on intact cells is greatly enhanced by the use of labelled SpA as an anti-5 reagent in place of the normal second antibody (anti-receptor antibody) (Goding, 1978). Its use avoids the problems of lack of specificity of antisera and the non-specific binding of the anti-receptor antibodies to the cells via Fc receptors, observed by Warner (1974).

(B) The quantitative determination o f antibodies and antigens either on the cell surface or in solution, for a wide variety of purposes:

(i) M onitoring th e production o f m onoclonal antibodies by lymphocyte hybridomas. In particular SpA labelled with * ^ I followed by autoradiography allows a rapid and inexpensive screening procedure for several hundred hybridoma cells

(Brown et al., 1979).

(ii) Evaluation o f polyclonal B cell activation by various stimulatory substances using the haemolytic plaque assay developed by Gronowicz et al. (1976).

(iii) The detection and quantification o f antigens and IgG antibodies on human lymphoid cells, following rosette formation between these cells and SpA (labelled with glutaraldehyde) coated erythrocytes (Ghetie et al., 1984).

(iv) Labelled SpA either in the form of l^2]p labelled S. aureus or H25]j can be used in an assay for the quantitation of circulating immune complexes (CIC). The CIC are precipitated from serum using polyethyleneglycol and the washed precipitate either incubated with labelled SpA (Stevens and Bridts, 1981) or the CIC precipitated, redissolved and absorbed to S. aureus (via SpA receptors) and stimulated by incubation with I125]j SpA, (Barkas, 1981). The la tte r m ethod relies on the availability of multiple SpA binding sites. This type of approach will not detect all complexes, but is superior to the alternative platelet aggregation test as it can be carried out accurately on older serum samples whereas stored platelets do not aggregate.

(C) SpA labelled with a heavy metal tracer (i.e. gold) can be used in conjunction with electron microscopy to visualise the localisation of a protein to a particular cellular compartment within sectioned cells (Bendayan, 1984; Baron, etal., 1989). The use of SpA in place of a second antibody offers several advantages; it requires only one tracer regardless of the antigen used, non-specific binding is lower, it is as sensitive as the classical assay method while having a quicker reaction time, it can be used in situations where the conditions necessary to introduce other forms of tracer may prove harmful to labile substances, and unlike many second antibodies whose activity is often severely affected by the different methods o f labelling, SpA retains a high degree of functional activity when labelled with a variety o f reagents.

The utility of SpA as an immunological tool has been considerably enhanced by the application of recombinant techniques. Using such technology gene fusions may be made composed o f DNA encoding SpA and sequences specifying the enzyme conjugate to act as the tracer. In vivo translation of the constructed hybrid "gene" by a bacterial cell results in a multifunctional chimaeric protein, containing the SpA IgG binding regions and the desired enzyme activity. Fusion proteins produced in this manner have several advantages over SpA-enzyme conjugates made by chemical means. Most notably a gene fusion system will yield a defined conjugate in terms of attachment site and molar ratio between carrier and peptide. The loss of enzyme activity often associated with the process of chemical conjugation will be avoided. Furthermore, when a synthetic peptide is being used as a tra c e r, it is easier to manufacture using oligodeoxynucleotide synthesis (which allows for rapid and efficient production of up to 150 bp, 50 a.a) than by peptide synthesis. The latter route relies on more complex methodology, which is both time consuming, and often fails as some peptide sequences are inherently difficult to synthesise.

The subsequent purification of the recombinant SpA fusion proteins is readily achieved in a single affinity chromatographic step by capitalising on the IgG binding properties of the SpA moiety (Uhlen et al., 1984a). For some immunological procedures, such as acting as an antigen for antibody generation the eluted fusion protein is suitable for immediate use. Further processing may be required for direct structural or functional studies. In the former case, the SpA region does not interfere w ith the immune response. Indeed, Lowenadler et al. (1986; 1987), describes a novel method for obtaining specific antibodies against short peptides in which the repetitive structure of SpA acted as an adjuvant enhancing the immune response against the attached short peptide. The system described by Lowenadler et al. (1986; 1987) has also been used to express longer synthetic genes from which antibodies were successfully prepared. This should allow for the developm ent of antibodies directed against both linear and discontinuous epitopes, the latter accounting for a substantial fraction of the antibodies produced against a variety of native proteins (Berzofsky, 1985). It should also be noted

that the SpA- fusion protein used to induce the production of polyclonal and monoclonal antibodies can also be used for the screening of the resulting hybridoma (Valerie et al.,

1987).

The use of the purified fusion protein in structural and functional studies often requires the separation of the SpA affinity tail from the protein of interest. This separation can be achieved by introducing a specific chemical o r enzymatic cleavage site at the junction between the two proteins. Nilsson e t al. (1985b), used such a system to facilitate the production and purification of native human insulin-like growth factor (IGF-1). A synthetic IGF-1 gene was fused to SpA and then, using site directed mutagenesis, an acid labile Asp-Pro cleavage site introduced at the fusion point. Following recombinant production, the fusion protein was eluted from an affinity column and treated with 70% formic acid to cleave the Asp-Pro peptide bond. The mixture of the two cleaved proteins was then passed through another affinity column, where the SpA portion of the fusion and the non-cleaved materials bound to the IgG ligand and were retained, leaving the desired protein to be collected in the eluate.

To further enhance the use of spa in gene fusions a number of protein fusion vectors have been developed by Nilsson et al. (1985a, 1985b). These vectors contain DNA encoding the IgG binding region of SpA followed by a multiple cloning site containing 5 unique restriction sites for insertion of a foreign gene, allowing for rapid construction of the required gene fusion.

1.1.5.2 Therapeutic applications

The anti-tumour effects of some Gram-positive microorganism extracts were reported as early as 1906 (Coley, 1906). In more recent years it was observed that after surgery to remove lung cancer, patients who had a staphylococcal emphysema had a better survival rate than those who had an uncomplicated post-surgical period (Ruckdeschel et al., 1972, Takita, 1970). A potential role for SpA became more apparent when it was

demonstrated that circulating immunoglobulins appeared to act as blocking factors, inhibiting cell-mediated cytotoxicity against tumour cells (Baldwin et al., 1973; H ellstrom et al., 1969; H ellstrom and Hellstrom, 1970; Sjorgren et a l., 1971). Circulating immunoglobulin was also found in abnormally high levels in patients with acute immune diseases (Jones et al., 1986; Steel et al., 1974). Therefore, SpA appeared a logical host reagent for removing immune complexes from tumour-bearing serum by affinity binding extracorporeally, allowing the later return of the plasma to the patient. The only other method of removing the circulating immune complexes would be the removal of the plasma, followed by return of donor plasma, with the associated risk of contaminated blood products, or the use of a plasma replacement solution o f albumin and saline, which lacks essential plasma components. The fact that SpA has been shown to have a higher affinity for immunocomplexes than for free IgG allows for removal of the damaging immunocomplexes while allowing the plasma to retain a reasonable level of free IgG (Kessler, 1975). However, it soon became apparent that the immunological consequences of plasma therapy were far more complex, with many researchers noting the occurrence o f tumour regression following the passage of plasma volumes too small to achieve significant blocking factor removal (reviews: Fer and Oldham, 1985; Mackintosh et al., 1985; Jones et al., 1986 and Solal-Celigny, 1985). Romagnani et al. (1980), observed that serum CIC levels showed a similar decrease in dogs with mammary carcinoma (in both those exhibiting tumour regression and those not). Therefore, the removal of a significant portion of circulating CICS did not appear to be essential for tumouricidal effects; although there was no doubt that perfusion of patient serum over protein A bearing S. aureus, and over purified SpA, resulted in clinically significant responses. The possibility of direct activation o f hormonal antitumour effects through SpA interactions with sera from tumour bearing animals was suggested by Steele etal. (1974). Further experiments then ensued, which although supporting the activation of hormonal antitumour effects, cast doubt on the role of SpA in the interaction. These often contradict one another. For instance in in vitro

experiments with SpA treated plasma used against acute myeloid leukeamia blast cells (AML), it has been shown that (i) the plasma must come from AML patients to obtain

cytotoxicity and (ii) the treated sera is non-toxic to normal cells and that SAW (S. aureus, Wood 46) not bearing SpA did not induce toxicity (M iller et al., 1982). However, in in vivo studies on dogs undertaken by Gordon et al. (1983) on canine spontaneous tum our systems, it was shown that perfusion of plasma over SpA producing S. aureus cells, or over non-SpA bearing SAW, was equaily effective in causing regression o f tumours in 6 out of 9 dogs. If SpA is the active agent, direct infusion might elicit similar tumouricidal effects. While such cases have been reported (Ray and Bandyopadhyay, 1983) using relatively pure SpA, a series of animal studies by Cohen et al., (1984) showed no tumouricidal effects and it is generally accepted that the interaction of SpA with plasma in vivo results in no antitumour effects.

The role of SpA has been further questioned by the work of Sukumar et al. (1984). In these experiments, rats treated with plasma which had been exposed to inactivated CNBr-Sepharose showed comparable reductions in tumour size to those treated with plasma exposed to SpA covalently linked to Sepharose. This suggested that the presence o f SpA may not be the critical factor. Further clinical studies (reviewed by Fer and Oldham, 1985) to duplicate the very effective anti-cancer treatment using SpA described by Terman et al. (1981), have failed to elicit such dramatic results, even when an identical materials and methods procedure was followed (Fer et al., 1984). The only difference that could be traced was that the method used to produce the SpA by Pharmacia (supplied in both studies) had been modified to increase the yield and purity. This led to the suggestion (Fer et al., 1984) that other bioactive staphylococcal products may have previously co-purificd with protein A, and that these had been eliminated in the later Pharmacia product.

The rp le o f SpA in perfusion systems is, th erefo re, controversial w ith some experimenters indicating that perhaps another component from S. aureus is the active factor, while others go even further and suggest that no bacterial product is necessary for the tumouricidal effects seen. The only common denominator necessary for tumour

regression in all the studies being the need for plasmaphoresis of tumour-bearer plasma. Fer and Oldham (1985) have suggested that perhaps a broad range of materials may trigger certain mechanisms mediated by a plasma factor, and that it is only through a series of detailed and systematic studies that the role of plasma immunoadsorption, and thus the role of SpA in cancer therapy, if any, will become apparent.

Although the direct treatment o f cancer using SpA may not be effective, SpA plasmaphoresis can still be a vital part of cancer therapy. SpA can be used to counter some of the side effects associated with some standard chemotherapy treatments; such a use is reported by Zimmerman et al., (1982) for the treatment of a tumour associated syndrome similar to thrombotic thrombocytopenic purpura. This syndrome arose following a rapid response in a patient treated with the chemotherapeutic drugs, fluorouracil, doxorubicin and mitomycin. It was hypothesised that as a tumour is reduced by chemotherapy the pre-existing state of antigen excess decreases, allowing the formation of soluble antigen-antibody complexes. These complexes induce the release of platelet substances which have the potential to initiate intramuscular coagulation and, because they accumulate in the blood vessels, cause local vascular injury, as well as initiating the deposition of platelets and fibrin, leading to the above syndrome. Plasmaphoresis was used in 5 treatment stages to reduce the CIC level of 324 mg I '1 to within the normal range of 10 to 65 mg 1'1. This particular use for SpA may become more common as more effective chemotherapeutic drugs with greater potency are developed and a rapid response by the patient becomes a more common occurrence.

Plasmaphoresis using SpA-Sepharose can also be used as a procedure for removing high titre antibodies in short term therapeutic procedures. An example of such a case, where the role of SpA-Sepharose was vital, has been reported by Nilsson et al. (1981). A haemophiliac with a high titre of factor IX antibody required surgery for an invasive pseudotumour into the base o f his left elbow. Exchange plasmaphoresis w as not feasible because of the possibility of stimulating factor IX inhibitor, and the fact that

the patient had antibodies against tissue antigens. It was not possible to neutralise the anti-factor IX antibodies using a factor IX concentrate, as the commercially available products contain small amounts of activated coagulation products, which in the amount necessary to treat the above case could have lead to intravascular coagulation. Treatment of 6000 ml of the patients plasma to remove antibodies by extracorporeal adsorption of the plasma to SpA-Sepharose, followed by transfusion, decreased the antibody titer and total immunoglobulin content to 20% of the original values. This allowed the remaining antibodies to be neutralised by infusion of factor IX concentrate. Conventional substitution therapy, combined with immunosuppression to prevent further antibody synthesis, was then given. This procedure allowed the operadon to be performed without further complications.